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Structure and Function of the nght Harvesting ChlorOphyll A/B Protein Complex: Investigations Using Reconstitution and Monoclonal Antibodies presented by Sylvia Catherine Darr has been accepted towards fulfillment of the requirements for Ph.D. degree in BiOChemiStry Mafia: Wm professoro Date 7 November 1985 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 a- ‘ V n:.«}- I") a; U MSU LIBRARIES m RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. '0' \j KL “Q" o’ STRUCTURE AND FUNCTION OF THE LIGHT HARVESTING CHLOROPHYLL A/B PROTEIN COMPLEX: INVESTIGATIONS USING RECONSTITUTION AND MONOCLONAL ANTIBODIES By Sylvia Catherine Darr A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1985 _—— h..) ~ur /~--?3 / a ;3 ABSTRACT STRUCTURE AND FUNCTION OF THE LIGHT HARVESTING CHLOROPHYLL A/B PROTEIN COMPLEX: INVESTIGATIONS USING RECONSTITUTION AND MONOCLONAL ANTIBODIES By Sylvia Catherine Darr The light harvesting chlorophyll a/b protein complex (LHC-Il) which serves photosystem II has been investigated using both reconstitution of isolated protein into LHC-deficient thylakoid membranes and character- ization of the individual polypeptides with monoclonal antibodies. In the reconstitution studies, LHC-II was isolated from Hordeum vulgare thylakoid membranes using the detergent octyl polyoxyethylene and reconstituted into partially-developed thylakoid membranes which lack LHC-II. Functional association of the reconstituted antenna complex with photosystem II was evaluated with three independent tech- niques: 1) room temperature chlorophyll fluorescence, 2) electron transport induced by light absorbed preferentially by chlorOphyll a or by chlorophyll b, and 3) chlorOphyll fluorescence Spectra at 770K. It was found that a three—fold excess of reconstituted LHC-II was required to achieve activity equivalent to the LHC-II activity gained during in vivg_membrane assembly. Monoclonal antibodies were used to investigate the structure of LHC-II polypeptides in Pisum sativum and Hordeum vulgare. A collection of seventeen monoclonal antibodies elicited against isolated LHC—II show Sylvia Catherine Darr six classes of binding specificity. Two classes of antibodies recognized single polypeptides (either the 28 or the 26 kD), thus establishing that these two polypeptides are not derived from a common precursor. Other classes of antibodies cross reacted with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light harvesting chlorOphyll a/b polypeptides of photosystem I (LHC-I). Binding studies showed that the unique antigenic determinants on the 26 and 28 kD polypeptides were surface exposed and that there are 27 capies of the 26 kD and 2 copies of the 28 kD polypeptides per 400 chloro- phylls. Western blots of thylakoid membranes which lack chlorophyll b (both the chlorina :2 mutant of barley and intermittent light-treated barley seedlings) showed that the 26 but not the 28 kD polypeptides were greatly reduced in number. A pr0posal that LHC-II be divided into internal and peripheral components containing the 28 and the 26 kD polypeptides, respectively, is discussed. To my parents ii ACKNOWLEGDEMENTS Thanks to Charlie Arntzen for his generosity and patience during my stay in his laboratory. Thanks also to the members of the Arntzen lab, both past and present, especially Cathy Chia, Jan Watson, Ellen Johnson, John Duesing, and Kit Steinback, for sharing their ideas and contributing to the camaraderie of the group. Thanks to my guidance committee: Shelagh Ferguson-Miller, Jack Holland, Gerry Babcock, Norman Good, and Mel Schindler. Thanks also to Mel for the gifts of OPOE. Thanks to Shauna and Chris Somerville and the members of their laboratories, who welcomed me into their groups and exposed me to a different (genetic) point of view. Finally, special thanks to those pepple whose encouragement and support have made my graduate years a pleasurable experience: Bridgette Barry, who never seemed to tire of hearing the thesis blues; my parents and brother, who continued to encourage me throughout these long years of school; and finally Mark, whose love and understanding have made it so much easier. iii TABLE OF CONTENTS List of Tables . . . . . . .i. List of Figures. . . . . . . . List of Abbreviations. . . . . CHAPTER 1. Literature review . Introduction. . . . . . . StrUCtUre 0f LHc-II o o o o o o o 0 Isolation of chlorOphyll-protein Polypeptide composition. . . . Pigment and lipid composition. Primary structure. . . . . . . Tertiary structure . Development of LHC-II . . Function of LHC-II. . . . Grana stacking . . . complexes Adaptation to light environment. . . . . Reversible regulation of energy transfer CHAPTER 2. Reconstitution of LHC-II into thylakoid membranes IntrOduction. O O O O O O O O O O O O O O I O O O O I O 0 Materials and Methods . . iv .vii o ‘0 (A) w TABLE OF CONTENTS Page List of Tables ....... I. . ............. . . . vi List of Figures. . . . . . ................... vii List of Abbreviations. . . . . ...... . ..... . . . . . ix CHAPTER 1. Literature review . . . . . . . . . . . . . . . . . . 1 Introduction ........................ 1 Structure of LHC-II . . . . . . . . . . . . . . . . . . . . 3 Isolation of chlorOphyll-protein complexes . . . . . . 3 Polypeptide composition. . . . . . . . . . . . . . . . 9 Pigment and lipid composition. . . . . . . . . . . . . 10 Primary structure. . .............. . . . 11 Tertiary structure . . . . . . . . . . . . . . . . . . 13 Development of LHC-II . . . . . . . . . . . . . . . . . . . 16 Function of LHC-II. . . . . . . . . . . . . . . . . . . . . 20 Grana stacking . . . . . . . . . . . . . . . . . . . . 21 Adaptation to light environment. . ......... . 24 Reversible regulation of energy transfer . . . . . . . 25 CHAPTER 2. Reconstitution of LHC-II into thylakoid membranes . . 29 Introduction. . . . . . . . . . . . . . . . . . . . . . . . 29 Materials and Methods . . . . . . . . . . . . . . . . . . . 31 iv Page Results . . . . . . . ...... . . . . . . . . . . . . . 41 Isolation of barley LHC-II in octyl polyoxyethylene. . 41 Comparison of reconstitution methods ......... 46 Characterization of membranes reconstituted by the freeze-thaw protocol using LHC-II isolated in OPOE. . . . . . . . . . . . . . . . . . . . . . . 54 Discussion ................ . ....... 66 Subunits of barley LHC-II isolated in DPDE . . . . . . 66 Reconstitution of LHC-II . . . . . . . . . ..... . 67 CHAPTER 3. Monoclonal antibodies to LHC-II . . . . . . . . . . . 75 Introduction. . . . . . . . . . . . . . . ....... . . 75 Materials and Methods . . . . . . . . . . . . . . . . . . . 77 Results . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Characterization of the antibody collection . . . . . 88 Further characterization of MLH1 and MLHZ ....... 101 Organization of LHC-II 12.!112 . . . . . . . . . . . .111 DevelOpment of the 28 and 26 kD polypeptides . . . . .125 DiSCUSSion. O O O O O O O O O O O O O O O O O O ...... 133 Common antigenic determinants between LHC-II and LHC-I O O O O O O O O O O O O O O O O O O O .134 Polypeptide diversity in LHC-II. . . . . . . . . . . .135 ChlorOphyll:protein ration in LHC-II . . . . . . . . .137 Structure of LHC-II in vi!g_ .............. 138 Appendix: Publications . . . . . . . . . . . . . . . . . . . . .143 Bib] iography O ..... O O O O O O O O O O O O O O O O C O O .144 LIST OF TABLES Page Summary of reconstitution techniques . . . . . . . . . . . . 48 ChlorOphyll fluorescence of reconstituted samples. . . . . . 55 Photosynthetic electron transport in reconstituted samples . 57 Characterization of LHC-II develOpment in vivo . . . . . . . 64 Comparison of half-rise time calculated for a two component exponential and that measured in reconstituted thy] aka-i ds 0 O O I O O O O O O O O O O O O O O O O O O 72 Summary of binding specificity of the monoclonal antibody c0] 1eCti on O O O O O O O O O O O O O O O O O O O O O O 89 vi IO. 11. 12. 13. 14. 15. 16. 17. LIST OF FIGURES 39$ SOS-PAGE of barley LHC-II . . . . . . . . . . . . . . . . . 40 Absorption spectra of LHC-II. . . . . . . . . . . . . . . . 43 ChlorOphyll fluorescence emission spectra of LHC-II . . . . 45 Diagrams of chlorOphyll fluorescence. . . . . . . . . . . . 51 Density gradient centrifugation of reconstituted samples. . 59 ChlorOphyll fluorescence emission spectra of reconstituted samp] es 0 O O O O O O O O O O O O O O O O O O O O O O 62 Standard curve of radial immunodiffusion assay. . . . . . . 81 Western blot of thylakoid membranes with class I and class II antibodies . . . . . . . . . . . . . . . . . 91 Western blot of isolated LHC-II with class I and class II antiDOdi es 0 O O O O O O O O O O O O O O O O O 0 O O 93 Western blot with class III and class IV antibodies . . . . 96 Western blot of thylakoid membranes with class V and class v1 antibOdies O O O I O I O O O O O O O O O O O O O I 98 Western blot of LHC-II and LHC-I with class V and class VI anti bOdi es 0 O O O I O O O O O O O O O O O I O I O O 100 Western blot of trypsin-treated LHC-II with class I and class II antibodies . . . . . . . . . . . . . . . . . 103 Western blot of trypsin-treated LHC-II with a class III antibOdy O O I O O O I O O I O O O O O O O O O I O O 105 Binding of antibodies MLH1 and MLH2 to intact and trypsin- treated thylakoid membranes . . . . . . . . . . . . . 108 Competitive binding of labeled and unlabeled antibody . . . 110 Scatchard plot of MLH1 binding to thylakoid membranes . . . 113 vii 18. 19. 20. 21. 22. 23. 24. 25. Competition between MLH1 and MLH2 . . . . . . . . . . . . . ImmunOprecipitation of LHC-II by antibodies MLH1 and MLH2 . Western blot of non-denaturing SDS-PAGE ...... . . . . Western blot of CPII* . . . . . . . ...... . . . . . . Western blot of CP29. . . . . . . . . . . . . . . . . . . . Western blot of mutant and wild type barley thylakoid membranes . . . . . . . ......... . ..... Western blot of thylakoid membranes isolated from IML-treated barley seedlings . . . . . . . . . . . . A model of the antenna chlorophyll-protein complexes 0f photosystem II C O O O O O O O O O O O O O O O O 0 viii 115 118 122 124 127 129 132 141 BSA CPI CPII * CPII CP29 DCPIP DPC ELISA max IML LHC-I LHC-II OPOE 680 P700 PAGE PBS PSI LIST OF ABBREVIATIONS Bovine serum albumin. ChlorOphyll-protein I, a PSI reaction center chlorOphyll-protein isolated in non-denaturing SDS-PAGE. (also LHCP3) The smallest LHC-II chlorOphyll protein isolated in non-denaturing SOS-PAGE. An oligomer of CPII (also LHCPI). A chlorophyll-protein containing chlorophylls a and b bound to a polypeptide of approximately 29 kD. 2,6-dichlorophenol indophenol, an electron acceptor of photosynthetic electron transport through photosystem II. Diphenyl carbazide, an electron donor to photosystem II. Enzyme-linked immunosorbant assay. Initial constant level of fluorescence. The maximal level of fluorescence. Intermittent light, 2 min light, 118 min dark cycles. The light harvesting chlorOphyll a/b protein complex serving photosystem I. The light harvesting chlorophyll a/b protein complex serving photosystem II. Octyl polyoxyethylene. The reaction center of photosystem II. The reaction center of photosystem I. Polyacrylamide gel electrophoresis. Phosphate-buffered saline. Photosystem 1. ix Photosystem II. The first quinone electron acceptor of photosystem II. The second quinone electron acceptor of photosystem 11. Sodium dodecylsulfate. CHAPTER 1 LITERATURE REVIEW Introduction The light reactions of photosynthesis are catalyzed by a series of membrane-bound pigment protein complexes in the chlorOplast thylakoid membrane. These proteins bind all the chlorophyll as well as various cofactors (hemes, quinones, pheOphytins, iron-sulfer centers, etc.) which mediate the redox reactions of the electron transport chain (Kaplan and Arntzen, 1982). The products of light-induced electron transport, NADPH and ATP, are used in the dark reactions of photosyn- thesis to drive the reduction of C02 to produce phosphorylated sugars. Thus the energy initially obtained from light is conserved in the synthesis of carbon-containing compounds. Most chlorOphyll in a photosynthetic organism does not directly participate in electron transport. Early experiments measuring photosynthetic reactions during short flashes of light showed that one oxygen molecule was evolved during each flash for every 2480 chlorophyll molecules in the sample (Emerson and Arnold, 1932). This observation led to the concept of photosynthetic unit, a group of pigments which 1 c00perate to produce an oxygen molecule (Park, 1965). The discovery of two types of reaction centers (see Emerson and Rabinowich, 1960) that operate in series (Hill and Bendall, 1960) coupled with the knowledge that 8 quanta were required for the evolution of each oxygen molecule (Emerson, 1958) allowed the photosynthetic unit to be defined as 400-600 chlorOphylls. This unit is envisioned to consist of the complete assemblage of pigments, enzymes, photosystem I, and photosystem II, needed to catalyze electron transfer from water to NADPH (Park, 1965). Antenna chlorophylls function by transferring absorbed excitation energy to a single chlorOphyll molecule (or a dimer of chlorophyll molecules, the special pair) in the reaction center. The reaction center (RC) chlorOphylls can transfer their excited state electron to a nearby acceptor, thus initiating photosynthetic electron transport. This system of linking many antenna chlorOphylls to a single reaction center chlorOphyll is a mechanism for increasing the efficiency of photosynthesis. It effectively feeds the energy absorbed by a large number of chlorOphylls to a single reaction center, thereby enhancing the turnover rate of photosynthesis under physiological illumination conditions (Glazer, 1983). Antenna pigments are bound to different types of protein complexes in the various classes of photosynthetic organisms (Glazer, 1983). The green photosynthetic bacteria (Chlorobiaceae and Chloroflexaceae), the cyanobacteria, and the red algae have extrinsic antenna pigment-protein complexes which bind to the surface of the membrane and transfer energy to the reaction centers intrinsic in the membrane. In green bacteria, the antenna bacteriochlorOphylls are contained in a sack-like structure called the chlorosome. Cyanobacteria and red algae have rod-like stacks of protein, termed phycobilisomes, which contain antenna phycobilin pigments. In contrast, the purple photosynthetic bacteria, green algae and higher plants have antenna pigment protein complexes which are intrinsic in the photosynthetic membrane. In the purple bacteria, repeating tetramers of’ a2 82 containing 4 or 6 bacteriochlorophylls and 2 or 4 carotenoid molecules form large pools of antenna pigment which transfer excitons to the reaction center (Drews, 1985). In higher plants and green algae, the antenna pigments can be divided into two groups: those associated primarily with either photosystem II or photosystem I. Fractionation of the chloroplast thylakoid membrane (see next section) has revealed that antenna pigments in higher plants and green algae are contained both within the reaction center complex itself, and also within specific antenna pigment-protein complexes, termed light harvesting complexes. The light harvesting complex which serves photosystem II is the subject of this thesis. It is the major chlorOphyll-binding complex in the thylakoid membrane, comprising 50% of the chlorophyll in the photosynthetic unit (Hiller and Goodchild, 1981). Structure of LHC-II Isolation of chlorOphyll-protein complexes. Beginning in the 1960's, the structure of chloroplast thylakoid membranes was investigated using detergents to disrupt lipid-protein interactions, thus surmounting the poor solubility of hydrophobic proteins in aqueous solutions. Anionic detergents such as soaium dodecyl sulfate (SDS) or sodium dodecylbenzene sulfate ($085), and nonionic detergents such as Triton X-100 and digitonin were used to solubilize the membranes (Boardman and Anderson, 1964; Vernon 32:31:: 1966). Anionic detergents, though generally harsher and more likely to destroy functional activity, provided the added benefit that solubilized components could be easily separated by gel electrOphoresis. In 1966, Ogawa and colleagues and later Thornber and colleagues (1967a) showed that two stable chlorOphyll- protein complexes could be produced by electrophoresis of SDS or SUBS-solubilized thylakoid membranes. These two complexes, termed chlorOphyll-protein I and II (CPI and CPII) in order of increasing mobility, contained approximately 75% of the chlorOphyll. The remaining chlorophyll ran at the front of the gel, did not comigrate with protein, and was assumed to be pigment released from denatured pigment-protein complexes. The chlorophyll a : chlorOphyll 6 ratio was significantly different between CPI and CPII which indicated that separate complexes with potentially different functions had been identified. Since then successive modifications of the solubilization and electrOphoresis procedures have revealed the presence of several more chlorOphyll-protein complexes. In general these adaptations involved minimizing the detergent : chlorophyll (or protein) ratio, decreasing the solubilization time, using low temperatures for solubili- zation and electrophoresis (4°C), and using a high pH PAGE system. Unfortunately the CP nomenclature was never standardized; as more chlorOphyll-proteins were identified, the nomenclature became rather confused. Oligomers of CPII, running at higher apparent molecular weights were identified and termed CPII-d (Hiller et.al., 1974), CPII—a (Remy et.al., 1977), or CPII* (Green and Camm, 1982). Concomitantly with these advances, research with plants containing no chlorOphyll b (either the chlorOphyll b-deficient mutant of barley, or intermittent light treated seedlings; see development section) indicated that CPII, which was enriched in chlorOphyll b, was absent. These plants still contained measurable amounts of photosystem II and photosystem I activity (Thornber and Highkin, 1974; Argyroudi-Akoyunoglou, 1971). Thus CPII could not correspond to a reaction center complex, and must be an antenna chlorOphyll protein complex. The name light harvesting chlorOphyll protein (LHCP) was introduced (Thornber and Highkin, 1974) and substituted for CPII in many nomenclatures. CPI was hypothesized to be the reaction center of photosystem 1. Several additional chlorophyll-protein bands were identified by Anderson and colleagues (1978): CPla, an oligomer of CPl; CPa, a chlorOphyll a containing complex thought to be the photosystem II reaction center (also called CPIV, Hayden and Hopkins, 1977); as well as 1 and LHCP2 (the monomer or smallest two Oligomers of LHCP, termed LHCP LHCP band was called LHCP3). They reported only 10% free chlorophyll in their preparation (Anderson et.al.,1978) which was a significant decrease compared to previous preparations. CPa was later fractionated into two chlorOphyll proteins through the use of additional detergents such as lithium dodecyl sulfate or octyl glucoside. These two fractions were termed CPIII and CPIV (Delepelaire and Chua, 1979) or CPa-1 and CPa-2 (Green et.al., 1982). They were also called CP47 and CP43 (Green and Camm, 1981) in an attempt to name the CP after the apparent molecular weight of its apOprotein. These two chlorophyll proteins were thought to represent the individual reaction center polypeptides of photosystem II. The best fractionation of thylakoid membrane proteins by SOS-nondenaturing gel electrophoresis may have been reached by Machold and colleagues (1979) who reported the isolation of ten chlorOphyll proteins from the thylakoids of barley. They also introauced a different nomenclature system (which will not be described here, for the sake of brevity). 3 or CPII) The light harvesting chlorophyll protein monomer (LHCP can be subfractionated into three chlorOphyll containing bands using various techniques of non—denaturing SDS-PAGE. Under some conditions, a different chlorOphyll-protein can be resolved running Just above LHCP3 in the gel. This band, termed CP29 (Camm and Green, 1980) contains slightly less chlorOphyll b than CPII. Its apoprotein is a single polypeptide of 23 kD in bean and 29 or 30 kD in spinach or barley (Machold and Meister, 1979; Camm and Green, 1980). The function of this 3 or CPII can also be subfrac- chlorOphyll-protein is unknown. LHCP tionated to yield two chlorophyll containing bands using octyl glucoside solubilization of membranes and SDS-PAGE (Green and Camm, 1982). These two bands each contain a single polypeptide and are thought to be subunits of CPII. Finally, it should be noted that the identity of LHCP Oligomers has also been investigated. Two Oligomers and a monomer of LHCP were reported originally (Anderson et.al., 1978; Markwell 33.31;,1978). Since then it has been shown that the smaller oligomer, LHCPZ, is actually the light harvesting chlorOphyll protein of photosystem I (Remy and Ambard-Bretteville, 1984). In summary, the work with nondenaturing SOS-PAGE has lead to the isolation of several chlorophyll-protein complexes from thylakoid membranes, and the develOpment of the CP nomenclature. Improvements in initial technique decreased the pr0portion of free chlorOphyll on the gel and lent credence to the hypothesis that all of the chlorOphyll in the membrane is bound to protein. The polypeptide apOproteins of the various CP's were identified using denaturing SDS-PAGE (see later discussion). The use of ionic detergents, however, tended to destroy the activity of isolated chlorOphyll-proteins. Therefore little progress was made using these techniques to identify the function of isolated structures. A parallel and complimentary approach to studying the structure of the thylakoid membrane was the use of nonionic detergents, which generally did not destroy the activity of isolated pigment-proteins. Early experiments solubilizing thylakoid membranes with either Triton X-100 or digitonin (Boardman and Anderson, 1964; Vernon 33:21:: 1966) paved the way for the separation of photosystem I and photosystem II and the characterization of their components. This approach was more structural in orientation; emphasis was placed from the beginning on identifying structural components and cofactors (see Arntzen, 1978). Electron microsc0py was often used to examine the membranous sheets and particles in detergent-derived fractions (Arntzen, 33:31., 1969; Vernon “32:31:, 1971). Thus efforts were made to link the biochemical identi- fication of detergent-derived preparations with the particles observed in the membranes by electron microsc0py. This approach lead to the concept of supramolecular "complexes" of polypeptides and cofactors in the thylakoid membranes, and provided the framework for our current understanding of the structure of thylakoid membranes. A complex of proteins containing light-harvesting or antenna chlorOphylls was first isolated from digitonin-solubilized membranes (Wessels and Borchert, 1975). It was termed the light-harvesting complex (LHC), had a low chlorOphyll a : chlorOphyll b ratio, and contained the polypeptides previously isolated in CPII (or LHCP3) (Arntzen and Ditto, 1976). Purified LHC, prepared using density gradient centrifugation of Triton x-100 solubilized membranes, was reported by Burke and colleagues in 1978. Since then LHC-II has been prepared from Triton X-100 soluoilized membranes using other methods to purify the protein, such as: rapid centrifugation through a sucrose cushion (Foyer and Hall, 1979), ion exchange column chromatography (Suss and Brecht,1980), partition in polymer phase systems (Albertsson and Andersson, 1981), and gel filtration (Suss, 1983). LHC isolated from SDS solubilized thylakoid membranes has also been reported (Lotshaw et.al., l982). Until 1980, LHC was the only known light-harvesting antenna complex in the membrane (Thornber, 1975). It was thought to contain all the chlorophyll b present in the thylakoid. This hypothesis was changed by Mullet and colleagues who fractionated isolated photosystem 1 particles (1980a). They found that four polypeptides which contained chlorOphyll b could be removed from PS1 by further solubilization with Triton X-100 without affecting the reaction center activity. This complex of chlorOphyll a and chlorOphyll b binding polypeptides was later isolated (Haworth 33:31., 1984; Kuang et.al., 1984; Lam.EE;31;' 1964). DevelOpmental studies using chlorOphyll b-deficient and PSI-deficient mutants confirmed the identity of these polypeptides (Mullet et.al., 1980b; Mullet 33.31;, 1981b). This discovery of an antenna pigment- -protein associated with photosystem I has led to the use of LHC-I to signify the LHC associated with PSI and LHC-11 for the LHC associated with photosystem 11 (see Kaplan and Arntzen, 1983). The reaction center terminology has also been clarified by the addition of the term "core complex“ to describe a reaction center with its intrinsic chlorOphylls and redox co-factors but without its light harvesting complex. Thus, for example, photosystem I is comprised of a PS1 core complex and its associated LHC-I. I shall use this terminology thrOughout this section and the rest of the thesis. Polypeptide composition. LHC-II is comprised of a group of polypeptides between 22 and 29 kD in size. The number of polypeptides identified has varied with the method used to isolate the complex, and the resolution of the SDS-PAGE used to visualize the polypeptides. Initially two polypeptides were observed when CPII was re-electrophoresed with denaturing gel electro- phoresis (Anderson and Levine, 1974; Kan and Thornber, 1976). Later three polypeptides were found in CPII from Chlamydomonas, lettuce, and barley (Bar-nun 33:31.,1977; Henriques and Park, l977; Machold 33:31.,1979). SDS-PAGE of LHC-II prepared with Triton X-100 also produced variable results. Of the species most studied: four polypep- tides have been found in pea LHC-II (McDonnel and Staehelin, 1980; Ryrie 33.21;, 1980; Mullet, 1983); three or four polypeptides in barley (McDonnel and Staehelin, 1980; Suss and Brecht, 1980; Ryrie and Fuad, 1983); and two, three, or four polypeptides in spinach (Foyer and Hall, 1979; Albertsson and Andersson, 1981; Ryrie and Fuad, 1982). The molecular weight of all of these polypeptides was between 29 and 22 kD. Several factors may be influencing the pattern and number of polypeptides observed in SDS-PAGE of isolated LHC-II and CPII. Among these are species-specific variation in the number of LHC-II IO polypeptides and the use of different PAGE systems with various concentrations of acrylamide. Since the polypeptides are relatively similar in size, they may be difficult to resolve except under the best of conditions, and different PAGE systems may produce different degrees of resolution. It is also possible that other factors such as post- translational modifications affect their mobility in some gel systems. It should be noted that the molecular weights cited in this thesis for LHC-II polypeptides are apparent molecular weights determined by SDS-PAGE. The absolute molecular weights for these hydrOphobic poly- peptides is likely to be somewhat larger. Pigment and lipid composition. CPII isolated on non-denaturing SDS-PAGE contained 3 chlorOphyll a's and 3 chlorOphyll b's per 28 kD polypeptide (Kan and Thornber, 1976). In addition, the complex also contained one carotenoid per polypeptide (Thornber et.al., 1967b). Three classes of carotenoids were present: neoxanthin, violaxanthin, and E3-carotene. These classes of pigments were found at similar ratios (7 chlorophylls per 26 kD poly- peptide) in LHC-II isolated using Triton X-100 (Ryrie gt43143 1980), with the exception that another carotenoid, lutein, was found to be the most prevalent, with only traces of B-carotene present. In contrast, using similar methods, others have found twice as many chlorOphylls per polypeptide: 13.4 chlorOphylls per 23 kD polypeptide,(Burke 33.21;, 1978); >10 per 25 kD polypeptide,(Li 1985); and 12 chlorOphylls per 24 kD polypeptide (Bar-nun.gt;al;,1977). This difference has yet to be resolved. One possible explanation is that Triton x-1oo in the preparation interferes with Lowry assays for protein, and that 11 differences in the amount of Triton X-100 in the purified LHC-II used for the assay produced the conflicting results. There is no evidence for a specific association of LHC-II with any of the major lipid classes in the thylakoid membrane (Ryrie 33:31:, 1980), but one fatty acid, trans 3-hexadecenoic acid, has been found to co-purify with CPII. (Tremolieres, 1981). It also appears in developing thylakoids concurrently with the LHC-II (Dubacq and Tremolieres, 1983). However, a specific role for this fatty acid is unknown. Recently a mutant of ArabiOOpsis which lacks this fatty acid was characterized and no changes in light harvesting activity in the isolated thylakoids were found (McCourt et.al., 1985). This suggests that if trans 3-hexa- decenoic acid has a specific role in LHC-II structure or function, it must be subtle. Primary structure. The polypeptides of LHC-11 are structurally similar to each other. The amino acid compositions of the three LHC-II polypeptides of Chlamydomonas are identical (Hoober et.al., 1980). A similar result was found for the 21.5 and 23 kD LHC-II polypeptides of the green alga Acetabularia (Apel, 1977). The analysis of peptide fragments produced by proteolysis of the polypeptides has revealed very few differences. Two dimensional electrOphoresis of tryptic fragments of each of the Acetabularia LHC-II polypeptides produced only two unique fragments (out of 16). This result has been extended to the LHC-II of pea, where Schmidt and colleagues have shown that polypeptides 15 and 16 (the 26 and 25.5 in my nomenclature) have similar sized proteolytic fragments 12 and also cross-react with a polyclonal antibody prepared against a purified Chlamydomonas LHC-11 polypeptide (Schmidt et.al., 1981). The gene encoding the most prevalent polypeptide in LHC-II, the 26 kD, has been cloned (Broglie et.al., 1981) and sequenced in pea (Coruzzi, et.al., 1983; Cashmore, 1984), petunia (Dunsmuir, et.al., 1983), Lemna gibba (Karlin-Neumann, et.al.,1985), and wheat (Lamppa, 33:31:, 1985). The gene is present in the nucleus in multiple c0pies which can be divided into 5 gene subfamilies (Coruzzi 33.31;, 1983; Dunsmuir, 93:31., 1983; Karlin-Neumann, 32:31:, 1985) which each appear to be expressed. In the case where a gene from each of the 5 subfamilies has been cloned and sequenced, no variation was observed in the total length of the coding sequence, thus indicating that all of the genes cooe for a single sized polypeptide (Dunsmuir, 1985). Some sequence variation was observed among the five genes sequenced, which translated to about 10 amino acid changes in the protein sequence derived from each clone. It is not known if these differences could alter the mobility of the polypeptides in SDS-PAGE. Thus it is not clear whether the cloned gene family codes for one or several of the polypeptides of LHC-II. Furthermore, it is not known if the differences in amino acid sequence could affect the function of the polypeptides in XIXE: Differential expression of the members of the LHC-II multigene family has been observed in develOping wheat leaves (Lamppa 33:31., 1985). A three-dimensional structure of the 26 kD polypeptide was pr0posed based on the mean hydrOphobicity of the amino acid sequence (Karlin- Neumann et.al., 1985). This model includes three membrane spanning 13 a-heliCes oriented with the amino terminus of the sequence on the stroma, or external, side of the thylakoid, and the carboxy terminus on the lumen, or internal, side of the membrane. The sites of pigment binding can not be deduced from the primary structure (Dunsmuir, 1985) nor from this model of the protein folding. Only two histidines, the theoretical ligand to chlorOphyll magnesium, are located within the hydrophobic region, while biochemical evidence indicates that 7 to 13 chlorOphylls are associated with each polypeptide. Tertiary structure of LHC-II. LHC-II functions in the thylakoid membrane by absorbing incident photons and transferring the energy to reaction centers of photosystem II. The mechanism of energy transfer through the antenna is generally considered to be Forster's inductive resonance (Pearlstein, 1982). This type of energy transfer is dependent on the distance and relative orientation between chromOphores. The rate of energy transfer is inversely proportional to the sixth power of the distance between the chromOphores. It follows that there must be precise limitations on the LHC-II apoprotein to maintain its associated array of pigments in the proper configuration. The orientation of pigments can be measured with linear dichroism, polarized absorption, and polarized fluorescence emission spectra. These techniques all indicate that the pigments of LHC-II are oriented in the thylakoid membrane (Breton, 1982). Mainte- nance of this orientation is accomplished by a non-covalent interaction between the polypeptide backbone and the chromOphores. As yet, however, little is known about the amino acid sequences involved in the pigment- 14 protein interaction, or the secondary or tertiary structure of the LHC-II ap0proteins. Pigment orientation and protein structure can be determined precisely using X-ray crystallography techniques. Application of these techniques to hydrOphobic membrane proteins, however, is hindered by the necessity of crystallizing the sample before X—ray analysis can be performed. Recently this problem was solved for the photosynthetic reaction center of Rhodopseudomonas viridis (Deisenhofer et.al., 1984). It is h0ped that similar techniques can be used to allow the crystalli- zation of other hydrOphobic membrane proteins such as LHC-II. In the meantime, it is useful to consider other antenna pigment-protein complexes for which detailed structures are known. Two extrinsic, hydrOphilic antenna pigment-proteins have been crystallized and studied: the c-phycocyanin biliprotein component of phycobilisomes in a blue- green alga; and the bacteriochlorophyll-a protein complex of a green photosynthetic bacterium. The c-phycocyanin component of the cyanobacterium Mastigocladus laminosus forms a series of disks in the phycobilisome (Glazer, 1983). Each disk is comprised of two proteinaceous rings which bind phyco- bilins, open chain tetrapyrolle pigments. x-ray analysis indicates that each ring is formed by three sets of o8 subunit pairs (i.e. (a8 )3) (Schirmer 33:31:, 1985). The pigments are bound in each subunit with their transition dipoles roughly perpendicular to the axis of the antenna rod. Parallel orientation of all the pigments leads to efficient energy transfer through the antenna. The three-fold symmetry, which is rather unusual in protein structure, apparently promotes 15 absorption of incident energy from more angles than does two-fold symmetry. The second antenna pigment-protein complex whose three dimensional structure is known is the bacteriochlorophyll a protein complex of the green photosynthetic bacterium Prosthecochlorus (Matthews, et.al., 1979). This complex serves as the link between intrinsic reaction center polypeptides and extrinsic antenna chlorosomes by binding them together and transferring energy absorbed in the chlorosome to the reaction center. Like the c-phycocyanin protein complex, the bacterio- chlorophyll a protein complex is a ring comprised of three subunits. Each subunit contains seven bacteriochlorophyll molecules which are wrapped by a single polypeptide formed into a 15-stranded B -pleated sheet. The pigments lie with their phytyl chains pointing toward the center of the subunit, creating a hydrophobic inner core. There is no direct overlap of the macrocycle rings, although ring substituents often lie close to each other. The magnesium of six of the seven bacterio- chlorophylls is noncovalently bound to the peptide backbone. Histidine side chains are the presumed ligands for five of these. The sixth is apparently a peptide nitrogen. This example illustrates the relation- ship between pigment orientation and protein structure in a chlorOphyll- binding antenna complex. However its application as a model for a hydrophobic antenna complex such as LHC-II may be limited. The subunits of the Prosthecochlorus bacteriochlorophyll a protein are approximately 39 kD and contain seven pigments, while the subunits of LHC-II are smaller, about 26 kD, and contain 6 to 13 chlor0phylls and a carotenoid. Furthermore, ultraviolet circular dichroism measurements of LHC-II (Nabedryk et.al., 1984) estimate that the protein is approximately 16 7 % a -helix and < 10 % E3-sheet. Thus it is unlikely that -sheet plays an important role in the structure of LHC-II. Preliminary descriptions of the three dimensional structure of LHC-II have been published using electron microscopic images of negative stained 2-dimensional crystalline aggregates. 8y tilting the sample and employing fourier transforms of the resulting images a 3-dimensional model may be reconstructed (Kuhlbrandt, 1984; Li, 1985). Like the other two antenna complexes described above, the structure of LHC-II was pr0posed to be a trimer of identical subunits, each of which are about 60 A long at the axis perpendicular to the membrane. The complex spans the membrane, but the distribution of protein is highly asymmetric with approximately 80% of the stain-excluding volume present in one leaflet of the membrane and exposed to one surface. The resolution of these studies is 16 A or greater, which is too large to distinguish individual chromophores. Therefore it is not known whether the chlorophylls are bound within the protein core between transmembrane helices, or externally to the protein in the lipid bilayer. Another issue which this model does not address is the role of the various LHC-II polypeptides. They cannot be distinguished at this resolution. Development of LHC-II The structural genes for LHC-II are located in the nucleus as demonstrated by both genetic experiments (Kung et.al.,1972) and treatment with protein synthesis inhibitors (Machold and Aurich, 1972; Cashmore,1976). The polypeptides are synthesized in the cytoplasm as 17 larger precursors which are cleaved upon uptake into the chloroplast , (Apel and Kl0ppstech, 1978; Schmidt 23:31:31981). In contrast to LHC-II, many other chloroplast polypeptides, including the reaction center polypeptides of photosystem II, are encoded by the chloroplast genome. Thus LHC-II and its major membrane associate, the PSII core complex, are under different genomic controls. The mechanisms which regulate the coordinated synthesis and assembly of organelle proteins by both the nucleus and the chlorOplast genome are relatively unknown at present, and represent an active area of research in plant molecular biology. Biosynthesis of LHC-II is regulated by light at two separate levels. Phytochrome, a photo-interconvertible pigment in higher plants, regulates the appearance of LHC-II mRNA. Etiolated plants exposed to a brief period of red light will begin to synthesize LHC-II mRNA after a short lag period. This induction can be prevented by subsequent exposure of the plant to far-red light (Apel, 1979; Tobin, 1981). However, LHC—II does not accumulate in the thylakoids after only a red light pulse; continuous illumination is necessary for further develOpment of the LHC-II (Apel,1979; Tobin, 1981). This is due to a second level of developmental control involving pigment synthesis in the chlorOplast. In angiosperms, the biosynthesis of chlorOphyll requires light for the reduction of protochlorophyllide to yield chlorophyllide (Castel- franco, 1983). In the absence of light, chlorOphyll is not synthesized and even plants with LHC-II mRNA do not accumulate LHC-II in the membrane (Apel and Kl0ppstech, 1980). Bennett (1981) has shown that in 18 the dark LHC-II ap0protein is present at low levels in thylakoid membranes, but is apparently turned over before it accumulates. This effect of darkness on the devel0pment of LHC-II has been further studied under conditions of intermittent illumination (2 mins light, 118 mins dark cycles). In 1971, it was observed that these conditions prevented the development of CPII (Argyroudi-Akoyunoglou 33:31:, 1971) while allowing the reaction center core complexes to develOp and become functional (Hiller et.al.,1973; Armond 33:31:,1976). Under these conditions, mRNA for LHC-II has accumulated to levels 50% of those in normal leaves and is present in polysomes detectable in run-off translation experiments (Cuming and Bennett, 1981; Viro and Kloppstech, 1982). No pools of untransported or unprocessed LHC-II were observed and it was concluded that LHC-II accumulation in the thylakoid was prevented by rapid turnover of newly incorporated sequences in the thylakoid membrane. In apparent disagreement, Slovin and Tobin (1982) looked for turnover of newly synthesized LHC-II in pulse-chase experiments with intermittent red light illumination of Lemna gibba. They found that measurable turnover was not great enough to account for the low levels of LHC-II ap0protein in the membrane and proposed instead that translation of mRNA was also a light-regulated step. Analysis of mutants which lack chlorOphyll b has further strengthened the observation that in the absence of chlorOphyll, LHC-II ap0proteins do not accumulate in the thylakoid membrane. Early experiments with a chlorOphyll b-deficient mutant of barley indicated that the chlorOplast membranes did not contain LHC-II or its major ap0proteins (Thornber and Highkin, 1974; Genge 32:31:,1974; Haldron and Anderson, 1979). The chlorophyll b binding antenna polypeptides of 19 photosystem I (LHC-I) were also observed to be absent (Mullet 32:31:, 1980b). However, more recent evidence indicates that the LHC-II ap0proteins are present at low levels (detectable by immunological techniques) in the membrane and in actively translating polysomes (Apel and Kl0ppstech, 1980; Ryrie, 1983a). Similar results have been observed in chlorOphyll b deficient mutants of pea (Schwarz and Kloppstech, 1982), Chlamydomonas (Michel et.al., 1983), clover (Markwell et.al., 1985), and maize (Miles 33.31;,1979). Bellemare and colleagues (1982) have demonstrated that the low levels of LHC-II ap0proteins in the barley mutant is not due to blocks in the synthesis of the LHC-II ap0proteins. Furthermore, they measured the transport of LHC-II polypeptides into the chlor0plast, processing, and insertion of them into the thylakoid membranes in_vitro in wild-type and mutant plastids. Both were equally capable of each of these processes. The most reasonable conclusion, then, is that in the absence of chlorophyll, especially chlorOphyll b, the LHC-II ap0proteins are unable to form a structure that is protected from in_viv2_proteolysis, and are rapidly turned over in the membrane. LHC-II also does not accumulate in the absence of carotenoids in developing plastids (Hayfield and Taylor, 1984). In both carotenoid deficient mutants and in seedlings treated with herbicides which suppress carotenoid synthesis, LHC-II ap0proteins are not visible in SDS-PAGE . However regulation of LHC-II develOpment in the absence of carotenoids is different than in the absence of chlorophyll b, since plastid deveTOpment in general is arrested at an early stage, and LHC-II mRNA fails to accumulate. 20 In most of the deveIOpmental studies described in this section, individual ap0proteins of LHC-II have not been discussed. This is because of confusion over exactly how many polypeptides constitute the whole complex. Where SDS-PAGE was used to describe the devel0pment of LHL-II, the predominant 26 k0 polypeptide, or the green CPII band was characterized (Machold and Aurich, 1972; Kung 33:31:, 1972; Hiller 33:31:31973; Armond §£:£!;31976)‘ In those cases where an antibody was used to identify LHC-II ap0proteins it was raised either to the whole complex (Apel,1979; Schwartz and Kloppstech, 1982; Ryrie, 1983a) or to the major 26 kD polypeptide (Bennett, 1981; Slovin and Tobin, 1982). In one well documented example, an antib0dy raised against a single LHC-II polypeptide of Chlamydomonas, cross-reacted with two other LHC-II polypeptides in Chlamydomonas, and also recognized two LHC-II polypep- tides in pea (Chua and Blomberg, l979). Therefore the exact identity of LHC-II polypeptides measured in any of the studies employing polyclonal antibodies probably cannot be determined, since the antibodies cross- react rather extensively. Function of LHC-II The basic function of LHC-II, as described earlier, is to transfer energy absorbed in antenna chlorOphylls to the reaction center core complex for use in photosynthesis. In addition, LHC-II plays important roles in the structure of the thylakoid membrane and in the regulation of energy distribution to the photosystems. These secondary functions of LHC-II will be described in this section. The role of LHC-II in 21 forming grana stacks in the chlorOplast is discussed first. Following that are two t0pics regarding regulation: adaptation to overall light intensity environment, and reversible regulation of energy distribution between PSI and P511. Grana stacking. The most striking characteristic of chlor0plasts when viewed in thin section by transmission electron microscopy is the adhesion between groups of thylakoid membranes. This adhesion forms stacks of membranes, not unlike stacks of pancakes, termed grana stacks. Each layer of a grana stack is interconnected with layers of other grana stacks via unappressed membranes, termed stroma membranes. Thus the inside of a chloroplast is filled with a three dimensional interlocking series of membranes appressed into grana stacks. (The structure is nicely illustrated in Figure 1 of Boardman 33:31:, 1978.) It has been well established through separation of grana and stroma membranes by detergents or mechanical means, and analysis of the two fractions by functional assays and freeze-fracture electron microscopy, that the two regions of the chlorOplast thylakoid are enriched in different protein complexes (see discussion in Arntzen, 1978; and Andersson and Anderson, 1980). The grana membranes contain mainly photosystem II and LHC-II, while the stroma membranes are enriched in photosystem I and the coupling factor. Several lines of evidence indicate that LHC-II mediates grana stacking. The appearance of LHC-II in the membranes of partially- develOped thylakoids correlates directly with the onset of grana formation (Armond, et.al., 1976). Furthermore, reconstitution of 22 purified LHC-II into artificial liposome membranes causes adhesion of those membranes in a way that mimics the thylakoid membrane. This reconstituted stacking responds normally to the cation environment (see below) (McDonnel and Staehelin, 1980; Mullet and Arntzen, 1980; Ryrie 35:31:, 1980). In contrast, Ryrie (1983b) has shown that reconstitution of another thylakoid pigment-protein complex, photosystem I, does not cause aggregation of the membranes. Finally, modification of the N-terminal segment of LHC-II disrupts the ability of membranes to stack. Proteolytic cleavage of the segment by mild trypsin treatment eliminates grana stacking in both thylakoid membranes (Jennings 32:31:,1978; Steinback 33:21., 1979) and liposomes reconstituted with purified LHC-II (McDonnel and Staehelin, 1980; Mullet and Arntzen, 1980). In 1966, Izawa and Good discovered that grana stacking of isolated thylakoids could be reduced by lowering the concentrations of monovalent cations in the medium from 100 mM to less than 10 mM (Izawa and Good, 1966). Similar effects could be observed with divalent cations by reducing their concentration from 10 mM to less than 1 mM. Elimination of the grana stacks is accompanied by a general mixing of the protein complexes in the membrane (Staehelin, 1976), which can be reversed by reincubation of the thylakoids with cations. Explanations for the mechanism of grana stacking based on electrostatic forces and the screening of bulk surface negative charges by cations have been proposed (see Barber, 1980, 1982). Nevertheless, in view of the specific effect LHC-II has on membranes reconstituted in_vitro, and considering the apparent requirement for the N-terminal segment of LHC-II before stacking can be induced, it is likely that LHC-II plays a Specific role in grana stacking. 23 Mullet and colleagues have hypothesized that LHC-II participates in a specific “contact mechanism” in stacking (Mullet, 32:31:,1981a). They believe that cations are required to provide enough surface charge neutralization to allow Opposing membranes to approach each other. Then the surface exposed N-terminal segment of LHC-II (which is positively charged) bridges the gap by binding to negatively charged regions on opposing LHC-11's. (see Kaplan and Arntzen, 1982, for more discussion) This would explain the observation from freeze-fracture images of stacked membranes that LHC—II units are lined up from one membrane to the next (McDonnel and Staehelin, 1980). Mutants which lack LHC-II have been used to analyze its role in grana stacking. The most commonly used mutant is the chlorOphyll b-deficient mutant of barley. The results obtained with this mutant have been somewhat confusing, however, because the mutant membranes, though lacking CPII (Thornber and Highkin, 1974), have extensive grana stacks. The apparent contradiction was resolved by the observation that grana in thylakoids isolated from the mutant unstacked at lower than normal cation concentrations, and contained a polypeptide which comigrated with the 23 k0 polypeptide of LHC—II (Burke 33421;, 1979). This observation was extended by Ryrie (1983a) who showed,using immunological techniques, that all the polypeptides of LHC-II are present at low levels in thylakoids of the mutant barley. Apparently this low level of LHC-II apoprotein is enough to allow grana stacking to form. Recently, Somerville (1986) has pointed out that several other mutants exist which apparently conflict with the grana stacking mOdel. Further analysis of these mutants potentially could have a large effect on current mooels of grana stacking. 24 Adaptation to light environment. Plants adapt their photosynthetic mechanism to different environments over both long and short time scales. At the biochemical level these adaptations generally correspond to alterations in the number of antenna pigment-protein complexes in the membrane. The light intensity which is available to drive photosynthesis varies over a wide range from full sunlight at the t0p of the canopy to deep shade at the forest floor. Many plant species (or ecotypes of widely ranging species) have adapted their gross morphology as well as their thylakoid membrane structure to function within a specific range of light intensities. “Shade“ plants generally have thin leaves that are rich in chlor0plasts, while "sun" plants tend to have thicker leaves, sometimes covered with a reflective coating, which contain fewer chlor0plasts (Boardman, 1977). At the biochemical level the chloroplasts of shade plants are enriched in chlorOphyll b relative to those from sun plants and have more extensive grana stacks (Bjorkman, 1981). This is caused by an enrichment in the amount of LHC-II in the thylakoid membranes (Fork and Govindjee, 1980). It is still somewhat controversial, however, whether this increase in LHC-II actually constitutes a change in the photosynthetic unit (i.e. chlorOphyll molecules per electron transport chain). Other researchers have found that P311 is also increased in the thylakoids of shade plants (Melis and Harvey, 1981). These differences have yet to be entirely resolved, partly because the current concept of photosynthetic unit size assumes a 1:1 ratio of reaction centers in the electron transport chain and does not recognize that PSII : PSI ratios may vary (Bjorkman, 1981). 25 The adaptations described above are relatively slow processes which involve permanent changes in the amount of LHC-II in the membrane in response to the average light intensity. Plants also have a mechanism to rapidly adapt to microclimate changes in the light quality of their environment. This mechanism does not require the synthesis and assembly of LHC-II in the membrane and is activated on a time scale of minutes rather than hours or days. A discussion of the rapid regulation of energy transfer follows. Reversible regulation of energy transfer. Photosystems II and I Operate in sequence to complete the transfer of an electron from water to NADPH. For Optimal efficiency it is essential that the two reaction centers catalyze electron transfer (undergo charge separation) at the same rate. The absorbance spectrum of the two photosystems are significantly different; PSI preferantially absorbs far red light ( > 690 nm) while PSII preferentially absorbs at wavelengths less than 680 nm, especially in the accessory pigment range of 440 to 650 nm. Thus under certain light conditions one of the reaction centers can be preferentially sensitized over the other, leading to an imbalance in the excitation of the photosystems. In 1969 Bonaventura and Meyers, and Murata independently discovered that a mechanism was present in algae to adjust to spectrally imbalanced light. Algae which had been previously exposed to long wavelengths of red light (light 1) exhibited high fluorescence when they were shifted to short wavelengths of red light (light 2), but the fluorescence level slowly decreased as the cells adapted to the light. The fluorescence level, or “State” of the pigment bed, was reversible. This was the first evidence 26 that dynamic "State“ changes were occurring in the photosynthetic pigment bed in response to chromatic shifts in incident light. Cation mediated energy transfer. For nearly a decade, the State changes described above were thought to be mediated by cation concentration of the chloroplast stroma (see Barber, 1976). Similar effects on fluorescence yield are observed in liEEE when thylakoids are reversibly stacked and unstacked by changes in cation concentration. Fluorescence is high while the thylakoids are stacked and low when they are unstacked. This is due to increased interaction Of LHC-II with PSI reaction centers in the random mix Of membrane proteins in the unstacked state (see Arntzen, 1978). It was generally assumed that similar mechanisms must be Operating in_vivg_(8arber, 1980). As Bennett has recently pointed out, however, it was never established that the cation concentration could be decreased enough in_vivg_to produce the observed changes in fluorescence. Furthermore, there was no mechanism to explain how light 1 or light 2 could reversibly control the concentration of cations to as large an extent as was necessary (Bennett, 33431;, 1984). An entirely different mechanism was prOposed in 1980 to explain light induced changes in energy transfer. This mechanism is described in the next section. Protein phosphorylation regulates energy transfer. In 1980, Bennett and colleagues reported that reversible phOSphorylation of LHC-II caused changes in the fluorescence emission of isolated chlorOplasts. It has since been shown that a redox-sensitive kinase phosphorylates LHC-II under conditions when the plastoquinone pool is reduced (Allen 33.31;, 1981). Phosphorylated LHC-II is released from association with P311 and is free to migrate into the stroma (Kyle et.al., 1983), where it may 27 interact with photosystem 1. Thus under light conditions where photosystem II is turning over more rapidly than photosystem I, and the plastoquinone pool becomes reduced, the membrane adjusts by dissociating LHC—II from photosystem II and increasing its association with photosystem I (Staehelin and Arntzen, 1983). The implications of this mechanism, whereby protein phosphorylation of LHC-II mediates the distribution of energy between PSI and P511, on the structure of LHC-II is not yet understood. Two populations of LHC-II have been hypothesized to exist in the membrane; one tightly bound to photosystem II and the other free to diffuse away when it is phosphorylated (Kyle 33:31:, 1983). What constitutes the bound or free LHC-II unit is not known. Two polypeptides of LHC-II are phOSphory- lated: the 26 and the 25.5 k0 proteins (Bennett, 1979; Bennett 33:31:) 1980). These two polypeptides appear phosphorylated equally in the Oligomers of LHC-II visible on non-denaturing gel electrOphoresis (Bennett EELELL’ 1980). In addition, both phOSphorylated polypeptides appear in the stroma regions of the thylakoid (Andersson 32.21;,l982). There is some evidence that their ratio may vary in the grana and the stroma (see Table III in Andersson 33:31:, 1982); the 24.5 k0 poly- peptide appears to be enriched relative to the 26 k0 polypeptide in the stroma of phosphorylated thylakoids, but this result is unconfirmed as yet. Furthermore, whether the 28 and the 23.5 k0 polypeptides are in both free and bound LHC-II is completely unknown. 28 The interrelated subjects of structure and function in the antenna pigment-protein complex, LHC-II, are the tOpic of this thesis. I have completed two research projects which address these subjects. The approaches have been quite different: reconstitution of LHC-II into thylakoid membranes, and the use of monoclonal antibodies to investigate the differences between LHC-II polypeptides. The results of these projects are reported in the next two chapters. CHAPTER 2 RECONSTITUTION OF LHC-II INTO THYLAKOID MEMBRANES Introduction The assay for biological activity Of electron transport components in the chlorOplast thylakoid membrane is straight forward: redox reactions can be measured by the use of artificial electron donors and acceptors coupled with various spectral or polarographic techniques. Combined use of these techniques can and has led to a detailed understanding of many of the energy coupling processes of photosyn- thesis. In contrast, analysis of the function of non-enzymatic (and non-redox) components in the thylakoid such as the antenna chlorophyll- proteins of photosystems II and I, has been much more difficult. The reaction that these proteins catalyze, the transfer of absorbed energy to the reaction center, is difficult to measure since it occurs on a picosecond time scale. Even measuring the amount of specific antenna chlorOphyll-proteins in thylakoids is difficult, since they generally contain no unique chlorOphylls or other easily measurable components. Therefore study of the function of antenna pigment-protein complexes 29 30 such as LHC-II has not proceeded as rapidly as other areas of photosynthesis. A partial solution to the difficulty of studying the antenna pigment-protein complex LHC-II, is to reconstitute it ln_vitro into membranes which contain other thylakoid membrane components but lack LHC-II. This technique allows the amount of LHC-II in the membrane to be controlled, permitting quantitative evaluation of its contribution to Specific processes. For example, the effect of total LHC-II levels in the membrane on the interaction of LHC-II and P511, or on the amount of energy transfer to photosystem I could be studied. This chapter describes the development and characterization of a method to reconstitute functional LHC-II into partially developed thylakoid membranes. Several methods were compared in order to find the most efficient reconstitution technique. In initial studies, LHC-II isolated in Triton X-100 was utilized. Later, a method to isolate barley LHC-II with the detergent octyl polyoxyethylene (OPOE) was develOped. This detergent has a much higher critical micelle concentration than Triton X-100, making it more dialyzable and therefore theoretically better for reconstitution than Triton X-100. Once an efficient reconstitution technique was identified, the resulting membranes were further characterized and compared to normal membranes containing LHC-II. The results are reported this chapter. 31 Materials and Methods Culture of barley and pea seedlings Hordeum vulgare var. Morex (barley; Michigan Seed Foundation, East Lansing MI) and Pisum sativum var. Progress #9 (pea; Ferry Morse Seed Company, Mountain View CA) were grown in growth chambers under 16 hr photOperiOO and ambient humidity. Day temperature was 21°C and night temperature was 18°C. Seeds were planted in vermiculite and watered regularly with half strength Hoagland's solution. Barley seedlings were used for preparation of thylakoid membranes and LHC-II 8 to 10 days after germination. Pea seedlings were used for preparation of LHC-II at 14 to 21 days. Intermittent light treatment of barley seedlings and isolation of thylakoid membranes Barley seedlings which were grown for seven days in complete darkness were exposed to intermittent light (IML) cycles of 2 min. light, 118 min. dark for 48 hours. Thylakoid membranes were isolated from these seedlings using a modification of the method Of Mills and Joy (1980). Fifty grams of leaf tissue was homogenized in 230 mls of a solution containing 50 mM Tricine-NaOH pH 7.8, 1 mM MgCl2, 2 mM EDTA (pH 7.8), 330 mM sorbitol, and 0.1% (w/v) BSA at 4° C. The homogenate was filtered through 12 layers of cheese cloth to remove cell fragments. Plastids were pelleted through 40% percoll (v/v) in the homogenization buffer by centrifugation at 4,0009 for 3 min. The pellet was then resuspended in a solution of low osmotic strength (10 mM Tricine-NaOH pH 7.8, 10 mM NaCl, 0.1% BSA) to lyse the envelope membrane of intact 32 chloroplasts, and centrifuged at 12,0009 for 10 min. The pellet was resuSpended in 10 mM Tricine-NaOH, pH 7.8, )0 mM NaCl, and 100 mM sorbitol. Any clumps of membranes remaining after resu5pension were broken up with a tissue homogenizer. Chlorophyll concentration was estimated using the method Of MacKinney (1941), and the chlorOphyll a : chlorOphyll b ratio was measured by the low temperature method Of Boardman and Thorn (1971), as modified by Haworth, 33:31; (1983). The membrane preparation was held in the dark on ice and used within 4 hours of isolation for reconstitution. These membranes were used for every reconstitution experiment discussed in this chapter, and are sometimes referred to as intact to distinguish them from thylakoid membranes which were fragmented by treatment with digitonin. Isolation of LHC-II from pea or barley using Triton X-100 LHC-II was isolated from the thylakoids of pea or barley using the method of Burke 33:21:, (1978) with the following modifications. The concentration of EDTA in the first washing step was decreased to 0.75 mM. Thylakoid membranes were resuspended to 0.8 mg chlorOphyll/ml and solubilized in 0.78% (w/v) Triton X—100. The Triton concentration on the sucrose density gradients was decreased to 0.02% (w/v). After centrifugation Of the sample in 0.1 to 1.0 M sucrose density gradients at 100,0009 for 16 hrs, the band near the tOp of the gradient which exhibited high chlorOphyll fluorescence was collected. The next step in the purification methOd is to aggregate the LHC-II by the addition of 5 mM MgClZ. For the purposes of reconstitution, however, this aggregation step was avoided, and the partially purified LHC-II solution was used directly. In some cases, the LHC-II preparation was stored by freezing 33 at -20°C. Upon thawing, no changes in the aggregation state of the protein (as revealed by low temperature fluorescence spectra) was ODSEY‘VEO . Isolation of LHC-II from barley using octyl polyoxyethylene. As an alternative to the use of Triton X-100 in reconstitution, we isolated LHC-II in octyl polyoxyethylene (OPOE). This detergent is a mixture Of 3 to 12 oxyethylene units per octane. The critical micelle concentration of the components Of the mixture range from 6.6 to 17.5 mM (Rosenbush 33:31:, 1981). The LHC-II isolation was based on that of Burke gt;al;_(1978). Chloroplasts were isolated by homogenizing 50 grams of barley leaf tissue in 300 mls of a solution containing 0.1 M Tricine-NaOH, pH 7.8, and 0.4 M sorbitol at 4°C. The homogenate was filtered through 8 layers of cheese cloth and centrifuged at 30009 for 10 min. The pellet was resuspended in 200 mls of a solution of 0.1 M sorbitol and 0.75 mM EDTA, pH 7.8, and centrifuged at 20,0009 for 5 min. The pellet was washed twice more by resuSpending in 0.1 M sorbitol and centrifuging at 20,0009. The final pellet was resuspended in distilled water containing 0.1 mM dithiothreitol. ChlorOphyll concentration was determined by the method of MacKinney (I941). The thylakoids were diluted to a final concentration of 0.8 m9 chlorOphyll/ml and octyl polyoxyethylene (OPOE) was added to a final concentration of 1.25% (w/v). This mixture was stirred at 210 C for 30 min in the dark. Unsolubilized membranes were removed by centrifuging the mixture at 41,0009 for 30 min. Eight mls of the supernatant was layered on the top of each of six 3.4 to 34% (w/v) linear sucrose density gradients (38 mls total gradient volume) 34 containing 0.25% OPOE (w/v) and centrifuged at 100,0009 in a swinging bucket rotor for 16 hours. After centrifugation the tubes were exposed to high intensity illumination from the side and a fraction Of approximately 5 mls in the top half of the gradient which showed high chlorOphyll fluorescence was collected. LHC-II was further purified by aggregation from this solution; MgCl2 was added to a final concentration of 5 mM, and the solution was stirred for 10 mins at 210 C in the dark. Quenching of the chlorOphyll fluorescence in the solution during stirring was observed and was interpreted as visible evidence of aggregation (Burke et;al;,l978). The aggregated LHC-II was then removed by centrifugation at 18,0009 for 10 min. The pellet of LHC-II was resuSpended in about 20 mls of a solution containing 10 mM Tricine-NaOH, pH 7.8, and 10 mM NaCl. Magnesium ions were removed by chelation; EDTA, pH 7.8, was added to a final concentration of 10 mM, and the solution was stirred for 10 min. at 40 C in the dark. LHC-II was recovered by centrifugation at 18,0009 for 10 min. The EDTA wash was repeated, and the final LHC-II pellet was resuspended in a solution containing 10 mM Tricine-NaOH, pH 7.8, and 10 mM NaCl. This purified, aggregated LHC-II was dispersed by diluting it to 0.5 mg chlorOphyll/ml, adding OPOE to a final concentration of 0.6% (w/v), and stirring for 10 min. at 210 C in the dark. Dispersion of the aggregated LHC-II was accompanied by a visually detectable increase in chlorOphyll fluorescence. This solution Of LHC-II, dispersed in OPOE, was used immediately for reconstitution into thylakoid membranes. 35 Dialysis-induced reconstitution of LHC-II into barley thylakoid membranes. LHC-II isolated with Triton X-100 or OPOE was reconstituted into barley thylakoid membranes using dialysis. Solubilized LHC-II and intact thylakoid membranes isolated from IML-treated seedlings were mixed so that the ratio of LHC-II chlorOphyll : thylakoid chlorOphyll was 2:1. The mixture was dialyzed in a solution of 10 mM Tricine-NaOH, pH 7.8, 10 mM NaCl, and 100 mM sorbitol for 16 hrs at 4°. Control preparations of LHC-II and thylakoid membranes were diluted with a volume of 10 mM Tricine-NaOH, pH 7.8, 10 mM NaCl, equivalent to the membranes of LHC-II added for reconstitution, respectively, and dialyzed as above . Reconstitution of LHC-II isolated in Triton X-100 into digitonin solubilized barley thylakoids In order to facilitate dialysis-induced reconstitution, intact thylakoid membranes were broken up into vesicles by digitonin solubilization. Thylakoid membranes (125 ug/ml) isolated from IML-treated barley seedlings were mixed with 0.25% (w/v) digitonin and incubated for 15 min on ice. Unsolubilized membranes were removed by centrifugation at 13,0009 for 2 minutes. Generally 70 to 80% of the thylakoid chlorOphyll-proteins remained in the solubilized fraction. This solution was mixed with the partially purified Triton X-100 preparation of LHC-II in the ratio of 1:1 or 2:1 LHC-II chlorOphyll : thylakoid chlorOphyll, and dialyzed for 16 hours at 40 in a solution of 10 mM Tricine-NaOH, pH 7.8, 10 mM NaCl. During this dialysis 36 chlorophyll in the sample decreased by 10 to 20%. Control samples were prepared by diluting LHC-II or solubilized thylakoids with apprOpriate volume of 0.25% digitonin or 0.78% Triton solutions, respectively, and dialyzing. Freeze-thaw induced reconstitution of LHC-II isolated in OPOE into intact barley thylakoid membranes. The technique of rapid freeze and thaw has been used previously for reconstitution of LHC-II into liposome vesicle membranes (Ryrie 33:31:) 1980; Mullet and Arntzen, 1980; McDonnel and Staehelin, 1980; Murphey, 1984) or into IML-treated thylakoid membranes (DQY.EE;21;’ 1984). These previous reports all utilized LHC-II in Triton X-100. Since dialysis- induced reconstitution had proved to be rather ineffective, the freeze- thaw method of Ryrie 35421;.(1980) was utilized for reconstitution of LHC-II isolated in OPOE into intact barley thylakoids. Immediately before reconstitution, the LHC-II solution was sonicated for 30 seconds in a bath sonicator. Equal volumes of LHC-II and thylakoid membranes were mixed so that the ratio Of LHC-II chloro- phyll:thylakoid chlorOphyll was 2:1 or 1:1. The solution was mixed thoroughly and rapidly frozen by immersing the tube containing the solution in liquid nitrogen. The mixture was allowed to thaw slowly in a 210 C water bath and was further sonicated for 30 seconds. Control samples were prepared for all measurements by diluting LHC-II or thylakoid membranes with the apprOpriate solution to the same final volume as the reconstitution mixture and freeze-thawing and sonicating the sample as described above. 37 Measurement of ChlorOphyll Fluorescence Transients ChlorOphyll fluorescence was measured with a photodiode positioned at 900 relative to an intense blue actinic beam, provided by an incandescent bulb with the light filtered through a Corning Filter no. 4-96 (as described previously in Paterson and Arntzen, 1982). The photodiode was protected from scattered blue light by a red cutoff filter (Corning Filter no. 2-64). Total chlorOphyll (LHC—II plus the thylakoid membranes) in the two ml reaction volume was always less that 10 ug, with thylakoid chlorOphyll held at 2.5 ug. The reaction mixture also contained 10 UN 3(3,4-dichlorOphenyl), 1-dimethyl urea (DCMU) and 10 mM NH40H. The voltage output of the photodiOde was monitored on a Nicollet model 206 digital oscillosc0pe. The half-time of the variable fluorescence induction transient was determined from recorded traces. Measurement of Photosynthetic Electron Transport Photosystem II-dependent electron transport activity was measured using 0.03 mM 2,6-dichlor0phenol indOphenol (DCPIP) as electron acceptor and 1.0 mM diphenyl carbazide (DPC) as electron donor in a solution of 50 mM Na-phosphate, pH 6.8, 10 mM NaCl, 5 mM MQCl 100 mM 2. sorbitol. The reaction mixture also contained the uncouplers NH4Cl (1 mM) and gramicidin (10'7 M). The reaction was monitored Spectrophotometrically at 580 nm as described previously (Darr.EE;El;' 1981). Actinic light was filtered through narrow band pass filters at 650 i 6 nm and 684 :.5 nm, preferentially exciting chlorophyll b and chlorOphyll a, respectively. In some samples, 10 uM DCMU was added to block PSII-dependent electron transport. 38 Density gradient centrifugation Reconstituted membranes and control samples were layered on tOp of 34 to 68% (w/v) sucrose density gradients (containing 10 mM Tricine-NaOH, pH 7.8, and 10 mM NaCl) and centrifuged at 250,0009 for 20 hours in a swinging bucket rotor. The gradients were fractionated from the tOp into 20 drOp samples. The chlorOphyll concentration of each fraction was estimated by measuring absorbance at 675 nm. Absorption Spectra from 620 to 720 nm were recorded for fractions of interest. Polyacrylamide gel electrOphoresis Polyacrylamide gel electrOphoresis was performed using the method of Laemmli (1970). Gradient gels of 11 to 17% acrylamide were run at 4°C as described previously (Steinback et.al.,1979). Measurement of chlorophyll fluorescence emission Spectra Chlorophyll fluorescence emission spectra were recorded with a SLM model 4800 spectrofluorometer linked to a HP 85 desktOp computer. Samples containing 10 ug chlorOphyll/ml were prepared in 66% glycerol and frozen in liquid N immediately prior to analysis. Excitation was 2 at 440 nm. Emission was measured from 650 to 800 nms. The data were not corrected for instrument response. 39 Figure 1. SDS-PAGE of barley LHC-II. LHC-II was purified from barley thylakoid membranes using the detergent OPOE. Lane A: barley thylakoids (10 ug chlorophyll); lane 8: LHC-II (1.5 ug chlorophyll). 41 Results Isolation of barley LHC-II in octyl polyoxyethylene. The LHC-II prepared from barley thylakoids using OPOE contained three polypeptides of 27, 25, and 24 k0 (Figure 1, lane 8). A 22 k0 polypeptide was also present as a minor constituent. The relative amount of this protein varied between preparations. In contrast, LHC-II isolated from pea thylakoids with either OPOE or Triton X-100 contains 4 polypeptides of 28, 26, 25.5, and 24.5 k0 (see Chapter 3). Absorption spectra of barley thylakoids and LHC-II derived from barley thylakoids with OPOE are shown in Figure 2. The chlorophyll a : chlorophyll b ratio in isolated LHC-II is 1.15. The large amount of chlorOphyll b in the preparation is reflected by the absorption at 655 and 475 nms (Figure 28). The red absorption maximum shifts slightly when aggregated LHC-II is dispersed by treatment with EDTA and 0.6% OPOE (Figure 2C). ChlorOphyll fluorescence spectra measured at 77°K of similar samples are shown in Figure 3. The lack of a fluorescence peak at 735 nm in the isolated, aggregated LHC-II (Figure 38) indicates that the sample was free of contaminating photosystem I (Butler, 1977). This LHC-II from barley had two strong emission maxima at 681 and 700 nm. The amount of fluorescence emission at 700 nm in low temperature chlorOphyll fluorescence spectra was sensitive to the aggregation state of the pigment protein complex. Aggregated LHC-II, isolated with Triton X-100, has been shown to display enhanced fluorescence at 695-700 nm relative to detergent solubilized LHC-II (Mullet and Arntzen, I980; Ryrie 33:31:, 1980). This is confirmed by the data shown in Figure 3, panels 8 and C for LHC-II isolated in OPOE. In addition, there is a 42 Figure 2. Absorption spectra of LHC-II. LHC-II was prepared from barley thylakoids using the detergent OPOE. A: barley thylakoid membranes (10 ug chlorOphyll/ml); B: isolated LHC-II aggregated by Mg+2 treatment (50 ug chlorOphyll/ml); C: isolated LHC-II aggregated as in B, and dispersed by EDTA washes and addition of 0.6% OPOE (10 ug chlorOphyll/ml). A 681 674 Absorbance n J l I 1 1 L 400 450 500 550 600 650 700 750 Wavelength (nm) 44 Figure 3. ChlorOphyll fluorescence emission spectra of LHC-II. Fluorescence emission spectra of barley LHC-II isolated with the detergent OPOE were recorded at 770K. The same samples as in Figure 2 were used. A: barley thylakoid membranes; 8: isolated, aggregated LHC-II; C: isolated, dispersed LHC-II. Fluorescence Intensity (relative units) 45 685 A l l L l 1 681 700 B J l l J l 681 C 650 675 700 725 750 775 800 Wavelength (nm) 4b species-specific effect on the relative amount of fluorescence at 700 nm. LHC-II isolated from barley thylakoid membranes with either OPOE (Figure 3) or Triton X-100 (Day.EE;El;’ 1984) shows increased 700 nm fluorescence upon aggregation as compared to LHC-II isolated from pea thylakoid membranes (Mullet and Arntzen, 1980; Ryrie 32:31:, 1980). The relatively low amount of 700 nm fluorescence which remained in the barley LHC-II after dispersion in 0.6% OPOE (Figure 3C) could be entirely eliminated by the addition of 1% (w/v) Triton X-100 (data not shown). Comparison of reconstitution methods In order to study the function of LHC-II after reconstitution into a thylakoid membrane, it was important to maximize the efficiency Of reconstitution and also to insure that all LHC-II inserted into the membrane was native. Most reports of reconstitution of LHC-II previously published had shown that more than half the protein was present in semi-crystalline aggregates (see for example: McDonnel and Staehelin, 1980). Since we were interested in studying the functional interaction of LHC-II with photosystem II after reconstitution, it was essential that aggregation of the LHC-II be avoided. TO this end several reconstitution techniques were surveyed in order to find one which yielded the most efficient incorporation of native LHC-II without aggregation. The first method we tried was fusion of phospholipid vesicles containing LHC-II to thylakoid membranes. In order to avoid aggregation Of LHC-II after reconstitution, we eliminated any aggregation steps in the isolation of LHC-II. Specifically, we avoided the use of cations to 47 aggregate the LHC-II during isolation, and the use of Biobeads 5M2 to remove excess detergent before reconstitution. Instead, we utilized a partially purified preparation of LHC-II which was still dispersed in Triton X-100 (see methods). The dispersed LHC-II was incorporated into phosphatidyl choline vesicles in a solution of 10 mM Tricine-NaOH, pH 7.8, 10 mM NaCl, using a single freeze-thaw. However preliminary examination of the vesicles with freeze-fracture electron micrOSCOpy revealed that aggregated protein was present. Furthermore, these patches of aggregated protein were still present after the vesicles had been fused to thylakoid membranes (data not shown). It seemed likely that the use of phosphatidyl choline liposomes had caused aggregation of LHC—II after reconstitution. Thylakoid membranes are 91 mole % mono- and digalactosyl diglycerides (Quinn and Williams, 1978). Phosphatidyl choline, one of the few charged lipids in the membrane constitutes only 3 mole % of the total. Thus the surface charge density Of phOSpholipid vesicles is quite different from the native thylakoid membrane. We hypothesized that LHC-II in the reconstituted membrane was aggregating in response to the more highly charged lipid environment. Recently these observations have been confirmed by Sprague 33:31:.(1985) who reported that both prior aggregation state and the type of lipid in the liposome affect the amount of aggregated LHC-II present in membranes after reconstitution. Thus using phosphatidyl choline vesicles as carrier membranes for LHC-II reconstitution was not practical. Next, several methods for reconstituting LHC-II directly into thylakoid membranes were tested. These methods are compared in Table 1. The function of LHC-II in reconstituted membranes was estimated and compared in Table I by measuring induction kinetics of room temperature 48 .Ae mpnmh mum. ms N\H ~\H NH m_ mm:_puomm apmceo umcwmcm a_p=$ soc; umum_omp mvpoxmpxzp do u ask . u pogucou an» ac ammucwucma o no cummmcaxmm\fiu pocucou new cmu=u_um:oumg cmwzuoa wucmgmempn us» my :o_uu=uog «mongoose; .mcm; umucmmucn m_ muwoxe_xzu .ogpcou ucm mumu=p_m:oumg mo mucwpmcecu mucoumogospm mo mms_a omngupm: ..__»:aogo—=u An. muwoxopxzu u Huaozm .HHN no: mpcmcoasou we owumc och .mmcp—umwm xupcon umpmogaImzH soc» vmaupomm monogaeos u_oxapxgp ;p_; ascends _mtm>om ween: cab=3_»m=oumc we: uoao Lo ooH-x copptb Legged ep_z wage—om? 3H-o=4 ceaIx coupgh S N3 c.o + m.~m m.H + m.m¢ mem»P~_e agape? can .m I. .. ea~._wn=_om ood-x =OB_LP S RN ~.N + o.~N ~.m + H.Nm mwms_epu =_=ouwape «an .e .. I. ea~_._a=.om ooHIX eop_ch w mm o.~ + H.0N m.o + o.om mwmapu_e eceobwm_e »a_zen .m I I 8.5 S NN m.H + m.~m m.~ + ~.m¢ upmapa_e buep=_ »u_caa .N I I 8.5 w em o.~ + ~.e~ m.H + ¢.mm zagb-m~aacc buuue_ aa.taa .H we.~\fip we .N\Hp muwoxe_»ga =o_bu=emt mo_oxepsgo mewoxe_»gu bcaebamcu supcen “H-914 ucmucmm cmu=u_um:oumm pogucou copusuwumcoumm wmummcmnumw mucocoasou .mmzawcsumu Pucm>Om mcwma “H-014 saw: umu=u_um:ouoc mu_oxmp»:u =_.~\Hu. was?» mm_gI$_m= mucmummco=_w —P»;aogo_;u wesumcmqsmu Eco; mo compcogsou .mm=c_csuou :o_a=a?umcoumg mo xumsssm H a_aap 49 chlorOphyll fluorescence emission transients. In the presence of DCMU, which blocks PSII electron transport from the primary quinone, QA’ to the secondary quinone, 08’ the rate of fluorescence increase to its maximum level is proportional to the intensity of the exciting light and to the size of the antenna associated with the reaction center (see Figure 4, upper diagram). As more antenna chlorOphyll is connected to each reaction center, the rate of exciton migration into that center is increased, and fluorescence reaches its maximum level more rapidly than it does in reaction centers with less functional antenna (see Figure 4, lower diagram). The half-rise time (tI/Z) for fluorescence in the presence of DCMU was measured for each reconstitution technique. A decreased t1/2 in the reconstituted sample vs. the control is a measure of the function of LHC-II in the reconstituted membranes. The first technique to be tried was dialysis of LHC-II directly into the membranes. LHC-II isolated in Triton X-100 was mixed with thylakoid membranes isolated from IML-treated barley seedlings in the ratio of 2:1 LHC-II:thylakoid (by chlorOphyll) and dialyzed. After 16 hrs, a small decrease in the t1/2 of fluorescence (Table 1, line 5) was observed. We concluded that the amount of activity Observed in these membranes was probably limited by the efficiency of reconstitution. Previously Ryrie 33:31; (1980) had found that dialysis did not efficiently drive the reconstitution of aggregated LHC-II into liposome vesicles. To further stimulate the reconstitution of LHC-II, the thylakoid membranes were broken up into smaller vesicles by digitonin solubili- zation before the addition of LHC-II and dialysis. This disruption should have destabilized the membranes and allowed greater insertion of 50 Figure 4. Diagrams of chlorOphyll fluorescence. TOp: cartoon illustrating electron transport through photosystem II, the block caused by DCMU, and resulting fluorescence. Botton: Room temperature chlorOphyll fluorescence transients from control and reconstituted thylakoid membranes. F0 and Fmax are indicated. For ease of comparison, the two transients have been drawn with equal F0; the reconstituted sample “+LHC“ normally would have a higher F0. Fluorescence Intensity M ( LHC ) I \\ I DCMU fluorescence max +LHC -LHC M_ I/ !———-l 100ms Light on 52 the protein. In fact, the tI/2 was somewhat more reduced in these preparations (Table 1, rows 3 and 4). However, this method was not practical for further stuoy of the thylakoid membranes. The mixture of Triton and digitonin was too harsh; 80% of the P511 electron transport activity (measured by reduction of DCPIP in the presence of the electron donor DPC) was lost even in control samples, and degradation of chlorOphyll-proteins was apparent after dialysis (data not shown). Thus, it appeared that dialysis was not effective at driving the reconstitution of LHC-II into thylakoid membranes. However it was possible that the use of a more dialyzable detergent might force greater incorporation of protein. To test this supposition, LHC-II which had been isolated in the detergent octyl polyoxyethylene (see the first section in this chapter) was dialyzed with intact thylakoid membranes. OPOE has similar structure and solubilizing properties to Triton X-100, but its critical micelle concentration is significantly larger ( >6mM for OPOE vs. 0.3 mM for Triton x-100; Rosenbush 32:31:,1981; Hjelmeland and Cranbach, 1984). During dialysis, OPOE micelles should be removed from solution more rapidly than Triton X-100 micelles and therefore should force the protein to seek a different hydrophobic environment by inserting into the membrane more rapidly. However, the fluorescence of membranes which had been dialyzed for 16 hrs with LHC-II in OPOE, displayed very little enhancement of LHC-II activity relative to those dialyzed with LHC-II in Triton X-100. (Table 1, line 2 vs. line 5). The OPOE had apparently dialyzed out of solution, since the LHC-II control sample had clearly aggregated (data not shown). It was concluded that the limitation was probably not removal of the detergent from the solution. 53 It is interesting to note that no difference was observed between the dicot (pea) and the monocot (barley) LHC-II in the dialysis-mediated reconstitution systems (Table 1, lines 2-5). LHC-II from the two species are different in both polypeptide composition and fluorescence emission characteristics (see previous section in this chapter). Apparently, in spite of these differences, the structure of LHC-II in the two plant groups is conserved well enough for the activity of the two types of LHC-II to be indistinguishable by this method of measuring LHC-II/PSII association. While the dialysis experiments were in progress, Day and colleagues (1984) had reported the reconstitution of LHC-II into thylakoid membranes using a rapid freeze and thaw treatment followed by a brief sonication. They used LHC-II that had been isolated in Triton X-100, then aggregated by the addition of cations and exposure to Biobeads-5M2. As one would expect, they found that after reconstitution, the LHC-II was aggregated in large semi-crystalline patches in the membrane. However, they also found that the LHC-II was functioning in these membranes; cation-induced thylakoid stacking and cation regulation Of energy distribution was demonstrated. We mOdified the technique of Day 33:31; (1984) to minimize aggregation of LHC-II in the membranes. Using the detergent OPOE, LHC-II can be aggregated by addition of cations during its isolation and then dispersed again by the addition of more OPOE after the cations have been removed. Thus a highly purified, and yet dispersed, LHC-II preparation can be used for reconstitution. Triton x-100 has not previously been used to disperse LHC-II prior to reconstitution, because the detergent is so difficult to remove from solution, and because 54 excess Triton X-100 can have detrimental effects on the rest of the chlorophyll-proteins in the membrane. OPOE, with its high critical micelle concentration is more easily removed from solution, and apparently less detrimental to the rest of the membrane when present in low concentrations. The result of reconstituting LHC-II dispersed in OPOE with thylakoid membranes isolated from IML-treated barley is shown in line 1 of Table 1. The membranes exhibited a fluorescence half-rise time that was 50% of the control. This is twice as much activity as was Observed by any of the other techniques we tried. The rest of this chapter describes a more thorough characterization of membranes reconstituted with this technique. Characterization of membranes reconstituted by the freeze—thaw protocol using LHC-II isolated in OPOE. ChlorOphyll fluorescence transients. A more extensive analysis of chlorOphyll fluorescence transients exhibited by reconstituted membranes is shown in Table 2. Reconstitution in the ratio Of 2:1, LHC-II:thylakoids (by chlorOphyll) decreased the t /2 of reconstituted 1 thylakoids 54% relative to the control thylakoids. The decrease in t1/2 was dependent on the relative amount of LHC-II, since a 1:1 reconstitution decreased the tI/Z by 25% relative to the control. Mixing LHC-II with thylakoid membranes without reconstitution treatment did not change the observed t1/2’ Electron transport activity. Another method of assessing the function of antenna chlorOphylls in the thylakoid is to measure electron transport activity induced by light preferentially absorbed by either 55 Table 2 ChlorOphyll fluorescence of reconstituted samples. Half time of room temperature chlorOphyll fluorescence transients of thylakoids recon- stituted with LHC-II dispersed in OPOE. Reconstituted Ratio of Thylakoid mixture Unreconstituted LHC:Thylakoid control t , ms mixture t ms 1/2 t ms -------..--_- -112:---- 12-992592§21 ----1£§: ...... 1:1 35.4 _+_ 1.9 26.6 1 1.5 36.4 I 5.3 'k (25%) 2:1 35.4 _+_ 1.9 16.2 _+_ 2.6 36.4 _+_ 5.3 * (54%) * Values in parentheses are % decrease in half-rise time between reconstituted membrane and the control expressed as % Of the control value. Room temperature chlorOphyll fluorescence transients of thylakoids reconstituted with LHC-II dispersed in OPOE by a freeze-thaw treatment. One ml reaction volume contained either 5 ug chlorOphyll (1:1) or 7.5 ug chlorOphyll (2:1). The reaction mixture contained 10 uM DCMU. Unreconstituted mixture is LHC-II and thylakoid membrane control samples mixed immediately prior to measurement, without freeze-thaw treatment. 56 chlorOphyll b or chlorOphyll a. The P311 core complex contains predominantly chlorophyll a, while LHC-II contains approximately equivalent amounts of chlorOphyll a and chlorOphyll b (Kaplan and Arntzen,1982). Thus PSII reaction centers served by associated LHC-II will catalyze prOportionally higher rates of electron transport when exposed to 650 nm light (preferentially absorbed by chlorOphyll b) than will reaction centers without LHC-II. Measurements of electron transport in reconstituted and control membranes in the presence of nonsaturating intensities of 650 and 684 nm light are shown in Table 3. The ratio Of electron transport in light preferentially absorbed by chlorophyll b (650 nm) to that in light preferentially absorbed by chlorOphyll a (684 nm) increased with reconstitution of the LHC-II. This indicates that the reconstituted LHC-II is functionally associated with the P511 core complex. Simply mixing LHC-II with thylakoids in equivalent amounts immediately prior to assay did not increase this ratio from the control membrane value (data not shown). Density gradient centrifugation. To test whether LHC-II was physically inserted into the thylakoid membranes upon reconstitution, density gradient centrifugation was carried out with all samples. Thylakoids isolated from IML-treated barley seedlings reached a density equilibrium at 1.131 g/cm3 while LHC-II equilibrated at 1.171 g/cm3 (Figure 5A). Reconstitution treatment of the LHC-II and thylakoid mixture produced a single broad chlorOphyll containing band (Figure SB) with an equilibrium density equal to that of the thylakoids in Figure 5A. Apparently all the LHC-II had become inserted into the membrane fraction. Although the band was not symmetrical across the gradient, it was homogeneous in composition when analyzed by absorption Spectra; 57 Table 3 Photosynthetic electron transport in reconstituted samples. Electron transport induced by 650 or 684 nm light in thylakoid membranes reconstituted with LHC-II. LHC:thylakOid 650 nm 684 nm 650/684 * * 0:1 (thylakoid 8.9 + 1.4 18.9 + 1.3 0.47 control ) _ - 'k * 1:1 12.8 1.0'5 15.3 :_0.4 0.84 'k * 2:1 14.3 :_0.8 15.7 :_1.2 0.91 t - - umoles DCPIP(m9 chlorOphyll) 1(hour) 1 PSII-dependent electron transport was measured in membranes freeze-thaw reconstituted with LHC-II which was dispersed in OPOE. DPC (1.0 mM) was used as electron donor and DCPIP (0.03 mM) as electron acceptor. Further details of the measurement technique are descirbed in the methods section of this chapter. ChlorOphyll concentrations were the same as in Table 2. 58 Figure 5. Density gradient centrifugation of reconstituted samples. Freeze-thaw reconstituted samples were separated by centrifugation on sucrose density gradients. A675 was measured for each 20 drop-fracti on - A: Open circles, thylakoid membrane control (25 ug chlorophyll); clo sed circles, LHC-II control (50 ug chlorophyll); squares, solution density of the samples. 8: Open circles, Freeze-thaw reconstituted mixture 2 z 1 of LHC-II and thylakoids (75 ug chlorophyll); closed circles, unreconstituted mixture Of LHC-II (50 ug) and thylakoids (25 ug). 59 LO” 1.o-» ‘A675 0.5m A l l I I fi j fl j 5 Fraction Number j J— ‘_ 10 01.180 ->L15O I-L12O (mun/5) MIsueO 6O i.e., the chlorOphyll composition was constant in all fractions of the peak (data not shown). This broad distribution pattern may be the result of formation Of heterogeneously sized vesicles upon reconsti- tution treatment. When thylakoids and LHC-II were mixed without a freeze-thaw treatment and immediately layered on tOp of a sucrose density gradient no apparent reconstitution was observed, as indicated by the separation of the sample into two chlorOphyll containing bands (Figure 58). Spectral analysis of the samples recovered in the upper and lower portions of the gradient showed no differences from the IML-treated thylakoids or LHC-II run separately in Figure 58. ZZP‘§_ChlorOphyll fluorescence emission spectra. ChlorOphyll fluorescence emission spectra at 770 K of freeze-thaw reconstituted membranes, LHC-II, and thylakoid controls are shown in Figure 6. Thylakoids from IML-treated seedlings exhibit fluorescence spectra that differ from those exhibited by fully developed thylakoids (compare Figure 3A with Figure 6A) because Of a general decrease in the amount Of long wavelength emission and a 5-7 nm blue shift Of the 735 nm peak. This has been discussed previously by Mullet 33:31; (1980b). Spectra from control LHC-II samples that were exposed to a freeze-thaw treatment in the absence of thylakoid membranes (see methods) are shown in Figure 6B. The 700 and 68l nm emissions are nearly equal. Before exposure to reconstitution treatment, the fluorescence spectrum of this sample resembled that in Figure 36 with relatively lower 700 nm emission. The increase in the 700 nm component indicates that the LHC-II control aggregated in response to conditions simulating reconstitution (see the discussion of Figure 3B earlier in this section). The fluorescence spectrum of the thylakoids reconstituted with LHC-II (2:1, LHC-II: 61 Figure 6. Chlorophyll fluorescence emission spectra of reconstituteci samples. Chlorophyll fluorescence emission spectra of freeze-thaw reconstituted samples were recorded at 770K. A: control thylakoid membranes from IML-treated seedlings diluted and subjected to reconstitution treatment in the absence of added LHC-II. 8: control LHC-II diluted and subjected to reconstitution treatment in the absence of added thylakoids. C: Reconstituted mixture of LHC-II and thylako ‘3 d5 ’ 2:1 by chlorophyll. Fluorescence Intensity (relative units) 62 685 650 684 675 760 755 750 Wavelength (nm) 775 800 63 thylakoid, by chlorOphyll) is shown in Figure 6C. The emission maximum Shifted from 685 nm in the thylakoids to approximately 684 nm, and a short wavelength shoulder was present. This may be interpreted as evidence that some Of the reconstituted LHC-II remains partially disconnected from PSII, and retained its original 68l nm emission (see Figure 3C). However, the lack of a large 700 nm component in these Spectra indicated that LHC-II was not self-aggregated after reconsti- tution. This result is quite different than that reported by Day and colleagues (1984). Low temperature fluorescence emission spectra of their reconstituted membranes displayed a large 700 nm emission component. Thus we concluded that the use of OPOE had reduced the self-aggregation of LHC-II in the reconstituted membranes. Greening of intermittent light-treated seedlings. When intermittent light-treated barley seedlings are placed in continuous illumination, chlorophyll biosynthesis is no longer limited, and functional LHC-II accumulates in the thylakoid membranes (Argyroudi- Akoyunoglou 33:31:,1971; Armond et.al;1977). This in_!ixg develOpment of LHC-II provides a natural control with which to compare the reconsti- tution system. Table 4 presents the measurements of chlorophyll fluorescence transients and the ratios of 650 and 684 nm light-induced electron transport rates over the course of 24 hours of greening. The data for fluorescence half-rise times for develOping membranes in this table can be compared with the values for reconstituted membranes in Table 2. The sample reconstituted in a ratio of 2:1 gave an equivalent change in the t1/2 percent decrease as that expected at about 16 hours Of greening in_vivg, assuming that the greening between 12 and 24 hrs was linear. Comparing the electron transport data for the same 64 Table 4 Characterization of LHC-II development in vivo. LHC-II activity in thylakoids isolated from IML-treated seEdlings greened under continuous illumination. Hrs of ChlorOphyll Fluorescence DCPIP continuous a/b induction reduction illumination ratio t1/2,ms 650/684 (% decrease) . 1 4 0 12.45 :_2.0 30.2 0.48 4 5.63 10.41 22.8 (25%) 0.51 8 3.602 16.5 (45%) 0.54 12 3.552 14.8 (51%) 0.80 24 3.232 12.0 (60%) 0.93 fully3 3.132 11.5 (62%) 0.94 greened 1measured by the method Of Boardman and Thorne (1971) measured by the method of MacKinney (1941) greenhouse-grown barley seedlings #wN normalized to Table 2 Thylakoids were isolated from IML-treated barley seedlings which had been exposed to as much as 24 hrs of continuous light. Fluorescence transients and electron transport were measured as described previously. Chlorophyll concentration was 2.5 ug/ml in both assays. 65 preparation in Table 3 with that for the develOping membranes in Table 4 indicates that the 2:1 reconstitution gave a ratio of 650/684 induced electron transport observed for membranes after 22 hrs of greenininn .2112: However, the chlorOphyll a : chlorOphyll b ratio in the reconstituted thylakoids was 1.85, much lower than that in the greened thylakoids at the 12 or 24 hour time point. Assuming that all the chlorOphyll b in the greening membrane is present as LHC-II and using chlorOphyll a : chlorOphyll b ratios for LHC-II and thylakoids from IML-treated seedlings as 1.15 and 12, respectively, the ratio of LHC—II : thylakoid chlorophyll after 18 hours of greening (assumed chlorophyll a:chlorOphyll b = 3.39) would be 0.65:1 (calculations derived from the data of Table 4). Thus three times more LHC-II (i.e. 2:1 vs. 0.65:1) was needed in the reconstitution system than in greening in 3132 to achieve Similar levels of antenna function. If one could directly measure the number of LHC-II in thylakoid membranes which had been greened for 18 hours, the resulting ratio of LHC-II chlorophyll to thylakoid chlorophyll would probably be less than the value calculated above. Not all of the chlorophyll b incorporated into the membrane is bound to LHC-II. The antenna complex of photo- system I contains a small fraction of the chlorOphyll b in the membrane, and is also develOping in continuous light. However, as a first approximation, it is clear that at least three times more LHC-II is required to achieve equal activity in reconstituted membranes compared to those develOping normally. 66 Discussion Subunits of barley LHC-II isolated in OPOE A method to isolate LHC-II from barley thylakoid membranes with the detergent octyl polyoxyethylene (OPOE) was developed. The resulting LHC-II contained three major polypeptides Of 27, 25, and 24 kD, with the 25 kD polypeptide present in the largest amount (Figure 1). A similar polypeptide pattern is found for barley LHC-II using Triton X-100 (McDonnel and Staehelin, 1980; Day et.al.,1984). In contrast LHC-II isolated from pea thylakoids with Triton X-100 or OPOE contained 4 polypeptides between 28 and 24.5 kD in apparent molecular weight. Whether barley LHC-II actually contains one less polypeptide than pea is a controversial point. Ryrie and Fuad (1982), using the second dimension gel system of O'Farrell (1975), have identified four polypeptides of 28.5, 25.0, 23.5, and 23 k0 in barley LHC-II. However, it is possible that the extra band present in these authors preparation is a degradation proouct. The pea LHC-II presented in the same figure looks as if it were partially digested with a protease such as trypsin. It is possible that this is the result of general degradation, and that the barley LHC-II has suffered a similar fate. In addition, Machold (1980) has shown that the major 25 kD band Of barley LHC-II can be resolved into a doublet of polypeptides if 6M urea is included in a gel prepared with reduced cross-linking reagent. This splitting of the 25 kD polypeptide would also proauce four LHC-II polypeptides. However, the special conditions necessary to obtain the Splitting in this case suggest that the difference between the two members of the 25 kD doublet is not simply the size of the polypeptide, but rather some other factor 67 that influences the polypeptides' mobility when the sieving properties of the gel are reduced. Our OPOE preparation of LHC-II also contained a 22 kD polypeptide which was present in small amounts that were variable between preparations. This polypeptide has not been reported in preparations which apply the methoo of Burke 33:31 (1978) to barley thylakoids (i.e., solubilization in Triton X-100 and density gradient centrifugation; McDonnel and Staehelin, 1980; Day 33:31:,1984). However, a polypeptide of 22.5 kD has been reported in LHC-II isolated by ion exchange chromatography of Triton x-100 solubilized barley thylakoid membranes. (Suss and Brecht, 1980). The identity of this polypeptide and why it is absent from the Triton x-100 preparation method of Burke is not known. The light harvesting chlorOphyll a/b protein complex of photosystem I (LHC-I) has been shown to contain polypeptides of approximately 22.5 kD (Mullet 33:31.,1980; Lam 33:31:,1984). It is possible that this antenna polypeptide copurifies with LHC-II under some conditions. An alternative explanation is that the 22 kD polypeptide is produced by degradation of the 27 k0 polypeptide. The 27 kD protein is missing in the preparation of Suss and Brecht (1980). Reconstitution of LHC-II This chapter describes a survey of several methods used for reconstitution of LHC-II into chlorOplast thylakoid membranes. The function of LHC-II in the membranes was evaluated as an indication of both the efficiency of reconstitution and the amount of damage caused the membranes by the reconstitution treatment. Previously it had been shown many times that LHC-II tended to aggregate after reconstitution 68 into either liposome or thylakoid membranes (McDonnel and Staehelin, 1980; Mullet and Arntzen, 1980; Ryrie 33:31:,1980; Day 53:31:31984; Murphey 35:31.,1974). Since we were interested in identifying a reconstitution technique which would allow study of functional interaction of LHC-II with other chlorophyll-protein complexes in the membrane, we were particularly interested in finding reconstitution conditions which did not cause self-aggregation of LHC-II. The most efficient reconstitution system (see Table I) was based on that of Day and colleagues (1984). As originally reported, this technique utilized highly aggregated LHC-II (aggregated during isolation by addition of cations, then further aggregated by rapid removal of the detergent) in a freeze-thaw induced reconstitution with thylakoid membranes. Self-aggregation of the LHC-II was found in the resulting membranes as indicated by low temperature fluorescence emission and as observed directly in freeze-fracture electron microscopy (Day 33:31:, 1984). We modified this technique by using LHC-II that had been isolated and dispersed in the detergent octyl polyoxyethylene (OPOE). It appears to have been successful, since no evidence of aggregation was observed in the low temperature fluorescence spectra of the reconstituted thylakoids. Previous studies. Two other studies of the reconstitution of LHC-II with photosystem II have been reported previously. These reports (Larkum and Anderson,1982; Murphey 33:31:,l984) described the reconsti- tution of LHC-II and photosystem II with the same intent as my work: to establish a system in which the interaction of LHC-II and photosystem II can be studied. Both groups reported that their reconstituted LHC-II was actively associating with photosystem II. In comparison to the 69 reconstitution of LHC-II into thylakoids from IML-treated seedlings, however, these two other approaches had several drawbacks. Murphey and colleagues (1984) reported reconstituting LHC-II with purified PSII particles in soybean phosphatidyl choline liposomes. The photosystem II preparation which they used, however, was originally develOped for oxygen evolution studies (Berthold 33:31:,l981), and contained substantial quantities of functional LHC-II. Therefore, the enhanced antenna activity that was observed may not be the primary association between LHC-II and PSII, but rather the addition of reconstituted LHC-II into an already functioning pool of antenna. Furthermore, the use of phosphatidyl choline vesicles for reconstitution tends to cause self aggregation of LHC-II (Sprague 33:31:,1985; see previous mention of this publication in the results section). The problem of PSII preparations that already contain LHC-II was avoided by Larkum and Anderson (1982). They used a highly purified PSII preparation for reconstitution with LHC-II and P51 in liposomes prepared from chlorOplast diacyl lipids. However, the amount of purified PSII available in their studies was limiting so that their analysis of functional interactions of LHC-II and PSII core complexes was restricted to fluorescence emission and excitation spectra. The use of intermittent-light treatment to prepare membranes for reconstitution circumvents the above difficulties. Thylakoids from IML-treated seedlings contain no functional LHC-II and only low levels of the ap0proteins. Furthermore, the quantity of membranes available from a single preparation is not limiting in most cases. An additional benefit from using these membranes for reconstitution is that they 7O preserve many of the normal lipid and intrinsic protein characteristics of the chloroplast thylakoid. Room temperature fluorescence assays. The comparison of reconstitution methods described in Table 1 relies on room temperature fluorescence kinetics to measure relative antenna function. The use of t1/2’ the time required for fluorescence to reach half its maximal level, is most informative when the variable fluorescence transient is a single-component exponential. In that case, the half-rise time, tI/2’ is inversely prOportional to the rate constant for the fluorescence rise. In two component, or biphasic, exponentials, the half-rise time is not as useful, since it is not prOportional to either of the rate constants. If addition of LHC-II to the thylakoid membranes simply added a second component with a large rate constant, then the composite fluorescence transient would show an increase in initial Slope. Applying the simple measurement of half-rise time to this two component exponential would be misleading since no change in the fluorescence emission of the PSII would have occurred. Theoretically, the possibility of LHC-II adding a second fast component to the variable fluorescence can be ruled out. Only reaction centers contribute to variable fluorescence (Zankel and Kok, 1972). A preparation of LHC-II, or any antenna chlorophyll-protein, should yield only invariable fluorescence, F0. However, I have observed a small amount of variable fluorescence in the Triton X-100 preparations of LHC-II. This indicates that the partially purified LHC-II was prooably contaminated with a small amount of photosystem II. The variable fluorescence was always less than 2% of the total (variable + 71 invariable) fluorescence and the half-rise time was less than 10 ms. It was not observed in any of the LHC-II preparations made with OPOE. In order to test whether the addition of LHC-II and creation of a two component exponential could account for the decreased half-rise times observed in the reconstituted samples, I have modeled the fluorescence transients and compared the calculated tI/Z with that measured for the sample. Using the following equation, the half-rise time of a two-component exponential was calculated. F(t) = Fmax - Alexp(-k1t) - Azexp(-k2t) where: F(t) = fluorescence at time t Fmax = maximal fluorescence level An = the proportional contribution of each component calculated from the contribution its variable fluorescence would make to the total composite variable fluorescence. kn = the rate constant of each individual component calculated from the measured t1/2 in control samples. The results of this calculation are shown in Table 5. The tl/2 values for control thylakoids and LHC-II are shown for each of two individual experiments, as well as the t1/2 calculated for a mixture of those components. In both cases, the calculated t1/2 was larger than the observed tl/Z' Thus a simple mixture of the LHC-II and thylakoid fluorescence transients producing a two component exponential could not 72 .m:_p umcwe on» cw :zogm :o_ueeenmca mg» 50% uwcsmeme was» cog» mucmumocozpw opnmwgo> mmmp puzzweom we: :o_pegeamgn Lepauwpgeq mmsu muw>gmmno mucmummcospm m_aepee> ea pesoEe asp muuo_mmc m=__ euro, 8:» cp HHqum< to; m=_e> zo_ apm>vpepmg use .uxmu esp cw crosm m:o_ue=am mg» mcpma mmPQEem pogucou HHIQ:4 wee uwoxepxgp gee «new mg» soc» cwaepaugeu we: mafia mmPgImpez .~\Hu —oep=ou ms» op emceneou ~\Hu so mmemguou ucmugma k. “a “NV As ~.mv r r o.~ + m.m~ ~.~m em.o ~.o + m.mm A5 ea. As “.mV 5 a. 4.3 + m.- o.e~ mm.o m.o + e.- Ammemgwwau xv .mwemgwwau fiv < ~\Hu cmgsmemz uwumpsu—eu e_oxa_»gh no.0 m.o + m.m 35 QNHQm < N\Hb HH-u=5 ku=u_umcoumc we» cw umczmeme use Pe_u:m=oaxw ucmcoasou 03» a com umuopaupeu we?» mmngepe; m «pack epoxepsgp "33-815 ca o_pea .me_oxepsep mo com_ceaeou 73 account for the decreased half-rise time observed in the reconstituted samples. Measurement of the t1/2 of a mixture of control LHC-II and thylakoid membranes confirmed this result (data not shown). Activity of reconstituted LHC-II. By comparing the light-harvesting properties of the freeze-thaw reconstituted preparations with membranes develOping in vivo, we have determined that about three times more LHC-II must be reconstituted in_vitro to achieve activity equal to that in the samples greened for approximately 19 hours in continuous light. If the reconstitution technique is randomizing the orientation of LHC-II insertion, then one would expect to need two times more LHC-II in the membranes to equal the 12.!113 activity. The apparent requirement for even more LHC-II in the membrane could have several explanations. If aggregated LHC-II were not thoroughly dispersed in OPOE before reconstitution, then part of the complex could be self-aggregated in the membrane and unable to interact with photosystem 11 core complexes. The fluorescence emission Spectra do not support this possibility. There is no 700 nm emission peak in the spectrum of the reconstituted membranes (Figure 6C). A fluorescence emission peak at this wavelength in purified LHC-II is generally considered to be a diagnostic for self-aggregation (Mullet and Arntzen, 1980; Ryrie 33:31:,1980). However the sensitivity of this assay is not known; it is possible that small amounts of aggregation would not produce a detectable emission peak at 700 nm. In contrast to these data, the fluorescence emission spectrum published by Day 222212.11984) of thylakoids reconstituted with aggregated LHC-II clearly shows a 700 nm emission peak. 74 Another possible explanation for the reduced activity of LHC-II in the reconstituted membrane is that some factor necessary for the interaction of LHC-II and photosystem II is limiting in partially develOped thylakoid membranes. For example, even though the lipid composition is identical between IML-treated and fully developed thylakoids, some lipids have not accumulated to the levels observed in fully develOped membranes (MacKender,1979). The 3-trans hexadecenoic fatty acid, which may play a role in the structure of LHC-II (McCourt 33:31:, 1985; Dubacq and Tremolieres, 1983), is present at reduced levels in IML-treated thylakoids compared to fully developed thylakoids. (Alternatively, other factors such as linker polypeptides which allow the recognition of PSII by LHC-II may be absent or limiting in the membrane. This would block efficient function of LHC-II by preventing its association with the complex. The presence of a 681 nm shoulder on the fluorescence emission spectrum in Figure 6C supports these proposals since it indicates that some LHC-II is fluorescing at its normal emission wavelength rather than transferring energy to PSII. There is some precedent for a requirement for specific proteins to mediate the association of antenna pigment-protein complexes: in cyanobacteria, phycobilisome assembly is mediated by unpigmented, linker polypeptides of 27 to 33 k0 (Glazer,1983). However,there is no evidence for the existence of a similar LHC-II/PSII linker protein at present. CHAPTER 3 MONOCLONAL ANTIBODIES T0 LHC-II Introduction Advancements in the isolation of LHC-II coupled with improved PAGE separation of thylakoid proteins have emphasized the existence of multiple polypeptides in LHC-II. The function of each individual polypeptide in the complex is unknown, as is the origin of size differences. The various molecular weights of the polypeptides in the complex could correspond to post-translational processing of a single gene proouct, since several of the polypeptides have been shown to be similar in amino acid composition, 2-dimensional tryptic fingerprints, and partial proteolytic digests (Apel, 1977; Hoober 32:21:, 1980). Alternatively, the various size classes of LHC-II polypeptides could correspond to the products of different genes. This is supported by the work of Schmidt and collegues who have immunOprecipitated two related polypeptides from in_vitrg_translation prooucts of isolated mRNA (Schmidt, 53:31:,1981). Finally, an additional level of complexity has been revealed by the discovery of several gene families for the 26 k0 polypeptide (Coruzzi, et.al.,1983). It is possible that heterogeneity 75 76 exists in the primary structure of the proteins within a single Size class. This combination of a group of similar polypeptides of several sizes and the potential for low levels of heterogeneity within each size class makes distinguishing and identifying the individual polypeptides of LHC-II a difficult task. In order to provide a tool for the identification and study of Specific LHC-II polypeptides, we have prepared monoclonal antibodies which react with the isolated complex. The use of monoclonal antibodies provides a sensitive technique for differentiating proteins. The antibodies consist Of a single immunoglobulin molecule which will recognize and bind with a single antigenic determinant. This allows polypeptides which share general similarities in structure to be easily distinguished if they contain regions of unique primary structure. This chapter describes the characterization of a collection of monoclonal antibodies to LHC-II, the use of two of the antibodies to study the structure and develOpment 0f LHC-II in Xi!22 and presents a mooel for the structure of the antenna polypeptides of photosystem II. 77 Materials and Methoos Culture of barley and pea seedlings Barley, peas, and the chlorina f2 mutant of barley were grown under the conditions described in Chapter 2. ChlorOplasts were isolated from these tissues by homogenizing 100 grams of shoot or leaf tissue in 200 mls of a solution of 50 mM Tricine-NaOH, pH 7.8, 10 mM NaCl, 400 mM sorbitol and 5 mM MgCl The 2. slurry was filtered through 12 layers of cheese cloth to remove cell fragments and centrifuged at 15009 for 10 mins. The pellet was resuspended in a solution of 10 mM Tricine-NaOH, pH 7.8, and 10 mM NaCl, and centrifuged at 10,0009 for 5 mins. The resulting pellet of chlorOplast thylakoid membranes was resuspended in 10 mM Tricine-NaOH, pH 7.8, 100 mM sorbitol, 10 mM NaCl, and 5 mM MgCl2. Intermittent light treatment of barley seedlings and isolation of thylakoids from the treated seedlings followed the procedures described in Chapter 2. Isolation of LHC-II LHC-II was isolated from thylakoids of Pisum sativum var. Progress #9 using the method of Burke et.al.(1979), described in Chapter 2. Triton x-100 was used to solubilize the membranes. Preparation of Monoclonal Antibodies Three 8 week old female balb/c mice were injected with 100 ug of purified LHC-II protein subcutaneously in 200 ul of a 50% (v/v) complete Freunds adjuvant solution. Twenty-seven days later they were injected 78 intraperitoneally (ip) with 100 ug of LHC-II in 150 ul phosphate-buffered saline (PBS, 10 mM NaH2P0 pH 7.3, 150 mM NaCl). 4, Four days later 50 ul samples of serum were collected from each mouse and tested for the presence of antibodies Specific for LHC-II using enzyme linked immunosorbent assay (ELISA). All three mice exhibited a good immune response to the antigen. The intraperitoneal injection was repeated on days 42 and 53 after the initial injection. Spleen cells were harvested three days after the final injection. The cells were fused to mouse myeloma Sp2-A9/0 cells in a ratio of 5:1 using 35% (w/v) PEG 1000 and 5% (v/v) DMSO (Galfre and Milstein, 1981). Twelve days after fusion, the hybridoma cultures were screened for the production of antibodies specific to isolated LHC-II using ELISA. Of 1522 initial colonies, thirteen were selected and cloned by limiting dilution. Approximately 100 clones of each colony were tested in ELISA for production of antibooies specific to LHC. Six clones of each group of 100 were selected and screened for Specificity to LHC polypeptides using western blot analysis. Of these, fourteen clones were finally selected and divided into six classes according to their binding specificity for LHC-II polypeptides in western blot. At least one member of each class was cloned a second time by limiting dilution to verify that it was truly monoclonal. Western blot analysis of six of the resulting clones from each line showed no variation in the polypeptide binding pattern. In a separate set of experiments, 5 hybridoma lines secreting antibooies specific for LHC-II were identified in a collection of hybridoma colonies prepared from mice injected with barley leaf cell membrane fractions. These lines were fused and cloned twice as described above. 79 Hybridoma cell culture was carried out using the methods described by Galfre and Milstein (I981). Antibody subclasses were identified using a Hyclone mouse mono- clonal sub-isotyping kit (Hyclone Laboratories, Logan Utah ). Ascites proouction To produce large quantities of monoclonal antibooies, ascites tumors were induced. Fourteen eight week old male balb/c mice were injected ip with 1 ml of pristane (2, 6, 10, 14 -tetramethylpentadecane). This injection was repeated 7 days later. On day 14, the mice were injected ip with 3 x 106 hybridoma cells in 0.3 ml of serum-free culture media. Seven mice were used for each cell line. Ascites tumors developed in approximately one week and the fluid was collected on alternate days for 8 to 12 days. Fluid produced by mice injected with the same cell line was pooled and frozen at -200 C until further use. Upon thawing, the fluid was centrifuged at 100,0009 for 30 mins to remove aggregated material. The supernatant volume was measured and then diluted 1:1 with cold phosphate buffered saline (10 mM NaP04, pH 7.3, 150 mM NaCl ;PBS.) Cold saturated (NH4)ZSO4 was added with stirring to yield 50% saturation, and the mixture was stirred on ice for 45 mins. Precipitated protein was collected by centrifugation at 10,0009 for 30 minutes. The pellet was resuspended with a small volume of a solution of 20 mM Tris-NaOH pH 7.4, 10 mM NaCl and dialyzed against 3 changes of the same solution for 24 hours. The dialyzed solution was aliquoted into single use vials and stored at -200 C. The concentration of antibody was measured using a Serotec radial immunodiffusion plate and Serotec antibody standard solutions (Serotec, Bicester, England). A 80 Figure 7. Standard curve of radial immunodiffusion assay. The concentration of antibody in partially purified ascites fluid was measured using this technique. The assay was linear over a five-fold concentration of antibody. r2 (mm)2 80 7O 6O 50- 4O 30 20 1O 81 I I I I I i l l i 0.2 0.4 016 0:8 lgG (mg/ml) 1.0 1.2 82 standard curve for this assay is shown in Figure 7. The assay was linear for antibody concentrations up to 1.2 mg/ml. 35 The antibodies were labeled using Amersham S labeling reagent (SLR, 1 mCi/ml, Amersham Corporation, Arlington Heights, IL). ELISA Enzyme-linked immunosorbent assay (ELISA) was done using a streptavidin HyBRL Screen Kit. (Bethesda Research Laboratories, Gaithersburg, MD) A Nunc 96 micro-well Immuno Plate I F (Gibco Labs, Grand Island NY) was coated with 0.75 09 of LHC-II protein in 50 ul of 15 mM NaHCO3, pH 9.2, per well by Incubation at 4° c for 8 hrs. The rest of the assay was carried out following the kit's instructions. Briefly, the remaining protein binding sites were blocked by adding 3% BSA to the well. Antibooy, either in spent cell culture fluid or diluted ascites fluid was added and allowed to react with LHC-II bound to the plate. Free antibody was then removed and the remaining bound antibOOy detected by adding goat anti-mouse immunoglobulin which was linked to biotin. This second antibody was allowed to bind the mouse antibody, and again unbound antibody was removed. Next streptavidin-linked horseradish peroxidase was added, and allowed to bind to the biotin. Finally the enzyme was detected colorimetrically in a 0.1 M Na-citrate buffered solution, pH 4.5, containing 0.03% (v/v) hydrogen peroxide, and 1 mM 2,2'-azino-di-(3 ethylbenzthiozoline sulfonic acid) diammonium salt. The reaction was quantified by measuring absorbance at 405 nm of each well using a Biotek EIA reader. 83 PAGE and Western Blots Polyacrylamide gel electrOphoresis (PAGE) was done using the method of Laemmli (1971). A gradient of acrylamide from 11 to 17% (w/v) was used to increase resolution. Protein samples in sample buffer ( 2% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, 0.0625 M Tris-HCl pH 6.8, and 0.01% (w/v) bromphenol blue) were boiled for 3 minutes prior to loading on the gel to thoroughly dissociate the chlorophyll from the protein. Eight ug of thylakoid chlorOphyll, 1.5 ug of LHC chlorOphyll, and 7.5 ug of P51 chlorOphyll were loaded in 1 cm lanes. A portion of each gel was cut and stained with coomassie blue (0.1% (w/v) coomassie brilliant blue R-250, 7% (v/v) acetic acid, 50% (v/v) methanol), and destained in a solution of 7% (v/v) acetic acid, 2% (v/v) methanol and 3% (v/v) glycerol. Western blots were prepared using the method of Towbin st; al;_ (1979). The gel was sandwiched next to a sheet of nitrocellulose and immersed in 192 mM glycine, 25 mM Tris-NaOH, pH 8.3, 20% (v/v) methanol. The protein was electrophoresed out of the gel onto the surface of the nitrocellulose filter (60 V, 6 hours). After electrOphoresis all remaining protein binding sites of the nitrocellulose were blocked by soaking the sheet in a solution of 1% BSA (w/v), 20 mM Tris-NaOH, pH 7.4, 150 mM NaCl, for 6 hrs. The western blot development procedure was modified from the methOO of Tsang st; 31; (1983). The monoclonal antibody solution (spent culture fluid, or partially purified ascites solution) was incubated with the nitrocellulose in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.3% (v/v) tween-20, and 1% BSA for 2 hours at 370 C. The blot was them washed in the same solution without BSA and antibody 4 times for 15 minutes at 250 C with continuous shaking. Alkaline 84 phosphate-conjugated goat anti-mouse immunoglobulin (Cappel Scientific, Malvern PA) was diluted 1:500 in the Tris/salt/Tween solution and incubated with the nitrocellulose for 2 hours at 250 C. The blots were develOped using the methOO Of Leary et;al;_(1983). Unbound conjugated antibody was removed by washing the blots 3 times for 15 mins with 100 mM Tris-NaOH, pH 7.5, 100 mM NaCl, 2 mM MgCl2 , 0.3% (V/v) tween-20 (AP 7.5 solution) and twice for 15 minutes with 100 mM Tris-NaOH, pH 9.5, 100 mM NaCl, 5 mM MgCl2 (AP 9.5 solution). For color development the blots were incubated with a solution of 0.33 mg/ml nitro blue tetrazolium, 0.17 mg/ml 5-br0mo-4 chlorO-3-indolyl phOSphate (Sigma Chemical Co., St. Louis MO) in AP 9.5. The bromochloroindolyl phosphate was first dissolved in dimethylformamide (20 ul/mg) before it was added to the aqueous nitro blue tetrazolium solution. The color reaction was allowed to proceed 10-15 minutes in the dark. The reaction was stOpped (by washing the blots first for 10-15 minutes in a solution of 10 mM Tris-NaOH, pH 7.5, and 1 mM EDTA, and then for 10-15 mins in a solution of 20 mM Tris-NaOH, pH 9.5, and 5 mM EDTA for 10-15 minutes. The blots were dried between sheets of blotting paper and photographed. Binding assays Unstacked thylakoid membranes were prepared from Pisum sativum var. Progress # 9 by homogenizing 50 grams of shoot tissue in a chilled solution of 50 mM Tricine-NaOH, pH 7.8, 400 mM sorbitol, 10 mM NaCl. The slurry was filtered through 12 layers of cheese cloth and centrifuged at 15009 for 10 minutes. The pellet was resuSpended in a low osmotic solution of 10 mM Tricine-NaOH, pH 7.8, and 10 mM NaCl to lyse any remaining intact chlorOplasts. The thylakoid membranes were pelleted 85 from this solution by centrifugation at 10,0009 for 5 minutes. The final pellet was resuspended in 10 mM Tricine-NaOH, pH 7.8, 10 mM NaCl, 100 mM sorbitol and held on ice until use in the binding assay. ChlorOphyll concentrations were measured by the method of MacKinney (1941). For the binding assay, unstacked thylakoid membranes were diluted into PBS containing 100 mM sorbitol and 0.25% (w/v) BSA. The solution was divided into 0.5 ml aliquots in 1.5 ml Eppindorf tubes and labeled antibody was added immediately. The antib00y was incubated with the membranes for 2 hours at 37° C on a rotary shaker. After this incubation, the membranes were pelleted by centrifugation for 2 minutes in a microfuge and the supernatant was removed by aspiration. The membranes were washed three times by resuspension in the PBS/sorbitol/ BSA solution and re-centrifugation. The final pellet was resuSpended in 100 ul of PBS, added to 5 mls of scintillation cocktail, and the radioactivity measured in a Beckman scintillation counter. Isolation of Photosystem I Photosystem I was prepared from Pisum sativum thylakoid membranes using the method of Mullet et;_al;_(1980a). For western blots, photosystem I protein was solubilized and boiled as described above. Trypsin treatment Isolated LHC-II was proteolytically digested by incubating LHC-II (LHC-II chlorOphyll concentration 300 ug/ml) with I ug/ml trypsin (Bovine pancreas type III, Sigma Chemical Co.) in 100 mm Tricine-NaOH, 86 pH 7.8, 100 mM sorbitol, 10 mM NaCl, 5 mM MgCl at 25° C. The reaction 2 was stopped at various time points by adding an aliquot of the trypsinized LHC-II solution to gel electrophoresis sample buffer and heating it to 1000 C. For the binding assays, pea thylakoid membranes (100 ug membrane chlorOphyll/ml) were incubated with 0.25 ug/ml trypsin at 25° C for 20 minutes as above. The thylakoids were then washed three times in resuspension solution before use in the binding assay. Non-denaturing gel electrophoresis Non-denaturing gel electrOphOresis was performed by the method of Camm and Green (1980). Pea thylakoid membranes were solubilized in 30 mM octylgluCOpyranoside at a detergentzchlorOphyll ratio of 40:1 (w/w). After 10 minutes of solubilization at 4° C, approximately 8 ug of chlorOphyll was loaded in each lane of the gel, and electrOphoresed at 12 mA for 3 to 4 hours at 4° C. Preparation of the polyacrylamide gel and the electrOphoresis buffers followed the method of Kirchanski and Park (1976). The protein in the gel was transferred directly to nitrocellulose for western blot analysis, or the pigmented bands were cut from the gel and re-electrOphoresed in the denaturing gel system of Laemmli (1970) as described earlier. The denaturing gel was either stained or the protein was transferred to nitrocellulose for a western blot. ImmunOprecipitation Pea thylakoid membranes were labeled in vivo by feeding 100 uCi 35S-methionine into each of three shoots cut from 10 day old seedlings. 87 The Shoots were then held in distilled H20 for 24 hrs under fluorescent lights. ChlorOplasts were isolated and diluted to 300 ug chlorOphyll/ml in a solution of 10 mM Tricine-NaOH, pH 7.8, 10 mM NaCl, 100 mM sorbitol, and 0.25% (w/v) BSA. The membranes were solubilized by incubation with stirring in 0.5% Triton X-100 for 30 mins at 25° C. Unsolubilized material was removed by centrifugation at 43,0009 for 30 mins. Solubilized thylakoids were stored at -20°C until use. Three percent of the original label was recovered as solubilized thylakoid proteins. For immunOprecipitation, solubilized thylakoids (80 ug of thylakoid chlorophyll) was incubated with 20 mg of MLH1 19G. The salt and Triton X-100 concentrations were held at 150 mM and 0.5% (w/v), respectively by adding the required amount of a 5 fold concentrated PBS solution or a 20% Triton X-100 solution. The mixture was incubated at 37° C for 2 hrs. For antiboay MLH2, solubilized thylakoids (800 ug of thylakoid chlorOphyll) were incubated with 17 mg 190. Three hundred ul of rabbit anti-mouse immunoglobulin "Immunobeads" (Biorad, Richmond CA) were added and incubated 2 hrs at 37° C with rotary shaking. Control samples were prepared using labeled thylakoids and immunobeads without the addition of antibooy. The beads were pelleted by centrifuging 3 mins in a clinical centrifuge and washed by resuspension and re-centrifugation three times in PBS containing 0.5% Triton. After washing, the precipitated proteins were solubilized in sample buffer and electro- phoresed using the conditions described earlier. The gels were dried and exposed to X-ray film. 88 Results Characterization of the antibody collection Seventeen monoclonal antibodies Specific for LHC-II have been identified and cloned. Their binding to chloroplast thylakoid proteins has been characterized by western blot analysis. On the basis of which polypeptides they recognize, the antibooies have been divided into six classes which are summarized in Table 6. Three additional monoclonal antibooies failed to bind LHC-II polypeptides in western blots even though they produced strong reactions with the Triton X-lOO-solubilized LHC-II in ELISA. We assumed that these antibooies were binding confor- mationally determined regions of the LHC-II; they were not further characterized. Antibooies which reacted on western blots with LHC-II showed two types of reactions: a response to a Single polypeptide or reaction with multiple polypeptides. Figure 8 shows an example of two single-protein specific antibodies, designated MLH1 and MLH2, which react with the 26 and 28 kD proteins of the thylakoid, respectively. To verify that the antibodies were reacting with authentic LHC-II polypeptides, western blots of purified LHC-II preparations are Shown in Figure 9. The coomassie stained proteins (Figure 80 and Figure 9a) showed four polypeptides in the LHC-II preparation, with apparent molecular weights of 28, 26, 25.5, and 24.5 kD. The monoclonal antibody of class I reacted strongly with the 26 k0 polypeptide, the dominant component of purified LHC-II, while the class II antibOdy (MLH2) reacted solely with the minor 28 kD polypeptide. Lane c of Fig. 9 demonstrates that these two proteins can be clearly resolved in the western blot using a mixture 89 mcpv:.a o: u II mc_uama»_oa on auoapace mo mcwu:_n n x :owuuo—Fou xuonwpce x x x x x x x x x I- x x x .- -I - - x x x x -I - - x x x -- II II II II II II x II II II II II x II m.H~ m.m~ em m.e~ m.m~ mm mm H-815 HHqus m HHH mgwanE we .02 mmmpu pecopuocos as» $0 xuwumeOOQm mcwu:_n we xgmsE=m 0 «Pack Figure 8. Western blot of thylakoid membranes with class I and class II antibodies. Pea thylakoid membranes were reacted with the class I antibody MLH1 and the class II antibody MLH2. Lanes a-c: western blot reacted with MLH2 (a); a mixture of MLH2 and MLHI (b); and MLHI (c). Lanes d-e: coomassie stained PAGE of thylakoid membranes (0); and purified LHC-II (e). c de b -68 kD “In. - . ~ .I .. m 2... I - . .. m w a“ q, a. M . . . I. . -12.5 .1, 4., g . m 92 Figure 9. Western blot of isolated LHC-II with class I and class II antibodies. Class I and class II antibodies were reacted with isolated LHC-II from pea thylakoid membranes. Lane a: coomassie stained PAGE of isolated LHC-II. Lanes b-e: western blots reacted with MLH1(b); a mixture of MLH1 and MLH2 (c); MLH2 (d); and a mouse polyclonal antibody to LHC-II (e). The 28 and 26 k0 polypeptides are clearly resolved in the western blot of purified LHC-II. m.¢N , m.mN/ . I 31' I I l Dme muons 94 of MLH1 and MLH2. As a control, polyclonal mouse antibodies elicited agai nst the LHC-II complex were used in Fig. 9, lane e to demonstrate that all four stained LHC-II polypeptides are present on the nitro- cel l ulose filter. The next two classes of antibodies recognized more than one polypeptide of LHC-II. A western blot of these antibodies, classes III and IV, respectively, is shown in Figure 10. Both purified pea LHC-II and pea thylakoid membranes are shown. MLHB, a class IV antibody, had a lower binding affinity as judged by the intensity of the color reaction in the western blot. Two members of class IV also recognized an unidentified polypeptide of approximately 20 kD in thylakoid membranes (data not shown). This polypeptide is not present in photosystem I preparations. It may be a breakdown product of LHC-II, or perhaps the 21 k0 (vicia) or 22.5 ((0 (Hordeum) polypeptide reported to co-purify With LHC-II by Suss and Brecht (1980). The final two classes comprised antibodies which recognized mu‘tiple LHC-II polypeptides as well as the polypeptides of LHC-I (the antenna pigment-protein of photosystem I). A western blot of whole thy] akoids reacted with these antibodies, classes V and VI, is Shown in F19. 11. The reactions with purified LHC-II and PSI are shown in Figure 12 . (F19. Some discrimination between the PSI polypeptides is present as well 12, lane e, where 2 LHC-1 polypeptides are bound and lane h, where th r‘ee ”15-1 polypeptides are recognized) The anti00dies of classes V aha V1 also tended to weakly bind various other thylakoid polypeptides Wh‘iCh do not appear to be associated with the light harvesting Comp] exes. We believe that this weak binding was non-specific since it (I ecreased rapidly upon dilution of the antibody. 95 Figure 10. Western blot of class III and class IV antibodies. Antibodies were reacted with both pea thylakoid membranes and isolated LHC-II from pea. Lanes a, c, e: thylakoid membranes; lanes b, d, f: isolated LHC-II. Lanes a, b: stained PAGE; lanes c, d: western blot with the class 111 antibody MLH5; lanes e, f: western blot with the class IV antibody MLHB. . .. ..3I an; _ .. _. _ _ 5 w 4 a __r__ a :5 125 MLI-I5 MLHB ' PAGE 97 Figure 11. Western blot of thylakoid membranes with class V and class VI antibodies. Class V and class VI antibodies were reacted with pea thylakoid membranes. Lane a: stained PAGE of thylakoid membranes; Lanes b-c: western blots with the class V antibody MLH10 (b); and the class VI antibody MLH12 (c). Note that these classes of antibodies bind more than the four LHC-II polypeptides in the membrane. I L! MLHIO MLH12 W 1....la ‘- 5" ’HI. 5“ m . D .u. . _ . .. I . a a a w . _ «I . . . . _. . . r . . PIE 8 6 p 5 4 9 2 12.5 99 Figure 12. Western blot of LHC-II and LHC-I with class V and class VI antibodies. A closeup of the LHC-II and LHC-I region of the western blots. Lanes a,d,g: thylakoid membranes; lanes b,e,h: isolated photosystem I polypeptides; lanes c,f,i: isolated LHC-II. Lanes a-c: stained PAGE; lanes d-i western blots reacted with the class V antibody MLHIO (d-f); and the class VI antibody MLH12 (g-i). Note that the PSI preparation used in lane h was contaminated with LHC-II so that both types of polypeptides appear in the lane. The molecular weights of the three LHC-1 polypeptides recognized by the class V and VI antibodies were 24, 23.5 and 21.5 kD. _IIIIINwIJEIIJ WIIIOPIJEIIJ filllmOI.P .902.— .mn— J>Ih .70....— -m& .33.... 101 Of the fourteen antiboaies characterized, Six were 1901 subtype, one was 19620, one was 1983, and six were 19M. Both IgG and IgM were present in each class that contained more than one member (data not shown ) - Further characterization of MLH1 and MLH2 Identification of binding sites. The location of the binding site for cl ass I and class II antibodies was determined using western blots of proteolytically digested LHC-II and binding assays with intact thylakoi ds. Triton X-100-solubilized LHC-II was digested with trypsin prior to gel electrOphoresis and western blotting. This treatment removes a 1-2 kD fragment from the amino terminus of the 26 kD poly- peptide which is normally exposed on the stroma surface of the thylakoid (Steinback fl, 1979; Mullet, 1983). It also digests at least a 3 k0 fragment from the 28 k0 polypeptide (Figure 13a). Western blots with either MLHI or MLH2 (Figure 13b and c) show that antibody binding is eliminated by this trypsin action. We conclude that the binding site for both these antibooies is located on the surface exposed trypsin- cleavable fragment. This fragment is not visible on the blot since the 99] SYStem did not resolve peptides of less that 8 k0. Polypeptide fragments of 17.5 and 15 kD, released by trypsin treatment, were not reactive wi th either antibooy. 1" Coritrast, a western blot with the class 111 antibody MLH3 showed that trypsin treatment did not eliminate antibody binding. This class 0f antibom‘ es binds the 26, 25.5, and 24.5 k0 polypeptides of LHC-11- After Proteolysis, the 26 kD polypeptide migrates with an apparent molecular Weight of 24 k0. (Figure 14a). The western blot (Figure 140) 102 Figure 13. Western blot of trypsin-treated LHC-II with class I and class II antibodies. A time course from 0 to 20 minutes of trypsin digestion of isolated LHC-II is shown. Panel a: stained PAGE; Panels b-c: western blot with the class 11 antibody MLH2 (panel b); and the class I antibody MLHI (panel c). In both cases the antigenic determinant is removed by trypsin digestion. 104 Figure 14. Western blot of trypsin-treated LHC-II with a class III antibody. A time course from O to 20 mins. of trypsin digestion of isolated LHC—II is shown as in Figure 13. Panel a: stained PAGE; Panel b: western blot with the class 111 antibody, MLH3. In this case, the antigenic determinant was not removed by trypsin treatment. 106 shows that MLH3 binds this 24 kD band after trypsin treatment. The fate of the 24.5 and 25.5 kD polypeptides, and whether they are digested by the trypsin cannot be determined in these gels. I have assumed that they are unaffected and comigrate with the dominant 24 kD proteolytic fragment. The antibody continued to bind the 24 kD polypeptide remai ning after proteolysis. Thus the determinant which this antibody binds is not present on the trypsin cleavable N-terminal segment of the 26 kD polypeptide. However, this data doesn't exclude the possibility that the determinant is surface exposed in a different segment of the protei n. In order to confirm the location of the trypsin-cleavable fragments 3°S-labeled of the 26 and 28 kD polypeptides, binding assays using anti body and intact thylakoid membranes were performed. Figure 15 shows the bi nding of antibooies MLH1 and MLH2 to thylakoid membranes. Sonication of the thylakoids for 3 mins with a probe tip sonicator to randomi ze membrane orientation, did not increase the maximum level of bindi ng (data not shown). Competition with cold antibody decreased the amount of label bound to the membrane (Figure 16). Mi ld trypsin treatment of the intact membranes reduced the antibody bound to about 10% of the saturated value for MLH2, and about 25% for MLHI (Figure 15). Under these conditions all the LHC-II in the membranes had been digested (western blot data not shown). The antibOGy binding which remained after trypsin treatment was not Saturable and increased in proportion to the amount of antibody present. He concluded that this binding was non-specific, and did not represent the appearance of new binding Sites. Since this sort of linear binding was "0t Visible in the saturated region of the non-trypsinized 107 Figure 15. Binding of antibodies MLH1 and MLH2 to intact and trypsin- treated thylakoid membranes. Antibodies were labeled with 35S-methionine. Panel a: binding of MLH2 to 4 ug of intact (Open circles) or trypsin treated (closed circles) thylakoids; panel b: binding of MLH1 to 1 ug of intact (Open circles) or trypsin-treated (closed circles) thylakoids. The specific activity of MLH1 was 1385 i 145 dpm/ug IgG; and of MLH2 was 822 :_50 dpm/ug IgG. 108 3O 20 10 555m «5.2 a: 119 MLH2 200 150 100 .ug MLH1 5'0 _ 5 4 3 2 1 225 ES 0: 6.. 109 Figure 16. Competitive binding of labeled and unlabeled antibody. The binding of labeled antibody was measured in the presence of excess unlabeled antibody. Panel a: MLH2 binding to 4 ug of thylakoid membranes (by chlorOphyll); panel b: MLH1 binding to 1 ug of thylakoid membranes (by chlorOphyll). Specific activity was the same as in Figure 15. The amount of labeled antibody added in both cases was enough to bind approximately 75% of the total binding sites. 1.8 ug of labeled MLH2 (a), and 5.3 ug of labeled MLH1 (b) was added to all samples. Specific activity of labeled antibody was as in Figure 15. pg MLH2 Bound 119 MLH1 Bound 110 2.0 a 1.5 1.0- 4 A J 200 300 .ug MLH1 100 111 membranes, it was likely that the trypsin treatment had increased non-Specific binding of antibOOy. Perhaps proteolysis, by cleaving surface exposed segments of the proteins, had altered the surface charge characteristics of the membrane enough to change the degree of non-Specific antibooy binding. Quantification of MLH1 and MLH2 binding sites. Scatchard plots of the binding data indicated that MLH1 bound 0.068 Sites per chlorOphyll molecule (Figure 17) and antibody MLH2 reacted with 0.0051 binding sites per chlorophyll (plot not Shown). Using a photosynthetic unit size of 400 chlorOphylls (Hiller and Goodchild, 1981), there were 27 binding sites for MLH1 and 2 for MLH2 per photosynthetic unit. Assuming that equilibrium binding saturated all sites in both cases, and that only one binding Site is present on each polypeptide, then this result is equivalent to the number of 26 and 28 kD polypeptides present per photosystem II reaction center. The binding constants, KD, were 7.4 X 10"“ M for MLH1 and 1.9 x 10'9 M for MLH2. Q‘ganization of LHC-II in vivo The organization of LHC-II i_n vivo was investigated with antibodies MLH1 and MLH2 in the experiments described in this section. Bi ndifl interference assay. If the 28 and the 26 kD polypeptides are subunits of a single pigment-protein complex in the membrane, then binding 01’ monoclonal antibooy to one of the polypeptides might block antibody bi nding to the other polypeptide. This possibility was tested by ”Milo"? ng the amount of labeled antibody bound in the presence of various Ccncentrations of the other unlabeled antibody; the results are shown in Figure 18 (Open circles). Here labeled MLH2 bound to the 112 Figure 17: Scatchard plot of MLH1 binding to thylakoid membranes. Pea thylakoid membranes (one 09 of membrane chlorophyll) were used for the binding assay. The specific activity of MLH1 was given in Figure 15. Ing/Ing 113 5.0 . 10.0 15.0 [lng] x 108 114 Figure 18. Competition between MLH1 and MLH2. Binding of labeled MLH2 (Open circles) or MLH1 (closed circles) to thylakoid membranes (4 ug of thylakoid chlorOphyll) was measured in the presence of excess MLH1. The abscissa shows the amount of unlabeled MLH1 added to each sample. 1.9 ug of labeled MLH2 or 10 ug of labeled MLH1 were present in each sample. The specific activity of labeled antibody was given in Figure 15. 115 2.0 to _erm wocsa I 5 0 1 . 5. O . 05 1.5 2.0 1.0 mg MLH1 12' .1 256m :32 5: 116 thylakoids was measured in the presence of unlabeled MLH1. No inter- ference was observed between the two antibodies. As a control, the amount of labeled MLH1 able to bind in the presence of the same concentrations of unlabeled MLH1 was measured (Figure 18, closed circles). Similar results were Obtained for the reciprocal experiment: labeled MLH1 competing with excess labeled MLH2 (data not shown). ImmunOprecipitation. Since no direct competition or interference was observed between antibodies binding the 26 and the 28 kD poly- peptides of LHC-II, it was possible that the two polypeptides were not even associated in the same structure in the membrane. This possibility was tested by imnunOprecipitating LHC-II from labeled thylakoid membranes which had been solubilized in Triton X-100. This detergent was chosen because it is known that low concentrations do not affect imunOprecipitation reactions (Knecht and Dimond, 1981; Gabay and Schwartz, 1982), and also because it does not the native structure of LHC-II (i.e., LHC-II can be isolated from Triton X-100 solubilized membranes with no apparent damage to its chlorOphyll fluorescence or absorption spectrum; Haworth et.al., 1982). An autoradiogram of the immunOprecipitated products is shown in Figure 19. Ten times more th.Ylakoids were used when imunOprecipitating with MLH2. Both anti boaies precipitate the 26 k0 polypeptide, as well as the 25.5 and 24-5 k0 polypeptides, although the smaller two polypeptides are not well reso'l ved. Unfortunately the 28 kD polypeptide is not visible in either immunOprecipitation. Since it is present in the membranes in quant‘i ties 10-fold lower than the 26 k0 polypeptide, perhaps this is not unexpected, since the specific activity of the labeled thylakoids was ”the" Iow. The use of fluorographic reagents such as sodium salicylate 117 Figure 19. ImmunOprecipitation of LHC-II by antibodies MLH1 and MLH2. Labeled thyalkoid membranes were solubilized in 0.5 % Triton X-100 and the LHC-II in the solution was immunOprecipitated with either MLH1 or MLH2. The ratio of antibody to thylakoid in the immunoprecipitation mixture is described in the methods. Lanes a and d: labeled thylakoid membranes (300,000 dpm): lanes b and c: immunoprecipitation with MLH1 (b) and MLH2 (c); lane e: control sample immunOprecipitated in the absence of monoclonal antibody. The Specific activity of the labeled thylakoid membranes was 10207 cpm/ug of thylakoid chlorophyll. THYL MLH1 MLH2 THYL CON 119 or Enlightening from NEN, increased the sensitivity of the autoradio- gram, but also blurred the bands and prevented their resolution. Nevertheless, given the Specificity of MLH2 in western blot and in binding assay, it is likely that the 26 kD polypeptide which immuno- precipitated with MLH2 did so because it was associated with the 28 kD polypeptide in the Triton X-100 solution. This indicates that the polypeptides are in contact with each other in the thylakoid membrane. 0f the other polypeptides apparently contaminating the immuno- precipitated products, the high molecular weight OI and 8 subunits of coupling factor are the most prominent. The labeled band running just * below these polypeptides in the MLH1 lane may be CPII , the oligomer of CPII (see next section). Non-denaturing PAGE. To further investigate the organization of the LHC-II complex 2 vivo, thylakoid membranes were fractionated using non-denaturing PAGE. As was described in Chapter 1, these techniques have been used extensively to isolate LHC-II from thylakoid membranes. CtilcarOphyll-containing bands in the gel are presumed to maintain some degree of their native structure (Thornber, fl, 1979). Since CPII (as LHC-II isolated in non-denaturing gel electrOphoresis is termed; Thornber and Highkin, 1974) and its higher molecular weight "oligomer", CPII * are known to contain more than one polypeptide (see for example: Anderson and Levine, 1974), this technique is another method in which associ ation between the 26 and the 28 k0 polypeptides can be assessed. T'Iiee non-denaturing PAGE system of Camm and Green (1980) was used to PFEpare the pea thylakoid membranes. This system was chosen because it yie] as a third putative LHC-II chlorophyll-protein, CP29. When re— ' electrophoresed under denaturing conditions, CP29 produces a Single 120 po1ypeptide with apparent molecular weight of 23 k0 in bean (Machold and Mei ster, 1979), 29 kD in spinach, and 30 kD in barley (Cam and Green, 1980). Using antibooies MLH1 and MLH2 it was possible to determine whether the the 26 and the 28 kD polypeptides of LHC-II are both present in CP29 and CPII. Figure 20 (lane a) shows a non-denaturing gel of pea thylakoid membranes, photographed without staining. This gel contained the three LHC—II related chlorOphyll-protein complexes, CPII*, CPII, and CP29. If this gel is blotted directly to nitrocellulose and reacted with antibody MLH2 , a single band is labeled which does not co-mi grate with any of the chlorOphyll-protein bands (Figure 20, lane c). Thus, at this resolu- tion, all of the 28 kD polypeptide appears to be present in a band runni ng above the pigmented bands. A similar blot using antibody MLH1 produces labeling of all three LHC-II chlorOphyll-proteins as well as another band running below that recognized by MLH2 (Figure 20, lane b). To confirm these observations, CPII* and CP29 were cut from the non-denaturing gel, and the protein was re-electrophoresed under denaturi ng conditions. CPII* was chosen for re-electrOphoresis because it is ‘l ess likely than CPII to be contaminated with co-migrating denatured LHC-11 polypeptides. CPII* produced 6 major polypeptides in denaturing PAGE (Figure 21, lane a). The largest three did not react "it“ any of the antibodies and are most likely comi grating polypeptides which are not a part of the chlorOphyll-protein complex. The other three POIypeptides were identified in western blots with antibodies MLH1 and MLH3 as the 26, 25.5, and 24.5 kD polypeptides of LHC-II. The 28 kD DOIyDEpt-i 06 was not visible on the stained gel and only barely d . etectab‘l e in the western blot. 121 Figure 20. Western blot of non-denaturing SDS-PAGE. Lane a: unstained PAGE; lanes b-c: western blot with antibody MLH1 (b); and antibody MLH2 (c). Lane a was photographed without coomassie staining; the dark bands are chlorOphyll-containing protein complexes. The dots on the left Side of lanes b and c mark where the chlorophyll-containing bands are on the blot. CP29 (erroneously labeled 29) did not appear to be recognized by either antibody. a b c CPI-ii“! it - 1... .- CP || — H . ‘f"*“?'~w- . ‘ ‘ one- - P CPII- ‘31 .. ~ = I..-) \ MLH1 MLH2 123 * * Figure 21. Western blot of CPII . CPII was cut from non-denaturing PAGE and re-electrOphoresed under denaturing conditions. Lane a: stained PAGE; lanes b-d: western blots with antibodies MLH2 (b); MLH1 (c); and the class 111 antibody MLH3 (d). 12.5- phéé MLH2 MLH1 1111.113 125 When CP29 was extracted and re-electrOphoresed (Figure 22), the 26 k0 and the 24.5 k0 polypeptides were visible in the stained gel. Western blots using antibodies MLH1 and MLH2 showed that the 28 k0 polypeptide, while not visible by staining, was present in the sample (Figure 22, lane c). Judging from its staining in the western blot, it was present in reduced amount relative to the 26 kD polypeptide than in isolated LHC-II. (Compare Figure 22, lanes b and c with Figure 9 lanes 0 and d) It is possible that the small amount of 28 k0 polypeptide present in CP29 is due to contamination of the region cut from the gel with denatured 28 kD protein running above. Thus it is unlikely that the 28 kD polypeptide of LHC-II is the apoprotein of CP29 reported previously (Camm and Green, 1980; Machold and Meister, 1979). The 24.5 kD polypeptide (Figure 22, lane a) which did not react with either antibody is probably the apoprotein identified earlier. [JewelOpment of the 28 and 26 k0 polypeptides The chlorOphyll b deficient (chlorina f2) mutant of barley is icriought to contain no functional LHC-II (Boardman and Highkin, 1966; 1’riornber and Highkin, 1974) but to contain low levels of the LHC-II éip>0proteins (Ryrie, 1983a). A preparation of mutant barley thylakoids was analyzed by western blot using antibodies MLH1 and MLH2 (Figure 23 ) . The 26 k0 polypeptide which is greatly reduced in the membranes as iriciicated by the coomassie blue staining (lane 2) was only weakly labeled by the MLH1 antibody (lane 5). In contrast, the 28 k0 POIypeptide showed approximately equal intensity in the blot of both The amount mutant and wild-type thylakoids (Figure 23, lanes 7 and 8). Of membranes which was loaded on the gel was not normalized between the 126 Figure 22. Western blot of CP29. CP29 was cut from non-denaturing PAGE and re-electrOphoresed under denaturing conditions. Lane a: stained PAGE; lanes b-c: western blots with antibody MLH1 (b) and MLH2 (c). 12.5- .“... . .....I ‘ 1 PAGE MLH1 MLH2 128 Figure 23. Western blot of mutant and wild type barley thylakoid membranes. The thylakoids of a chlorophyll b-deficient mutant of barley were reacted with antibodies MLH1 and MLH2 and compared with wild type barley thylakoids. Lanes 1-3: stained PAGE of wild type barley thylakoids (1); mutant thylakoids (2); and isolated barley LHC-II (3). Lanes 4-8: western blots of wild type barley reacted with MLH1 (4); mutant thylakoids reacted with MLH1 (5); wild type membranes reacted with a mixture of MLH1 and MLH2 (6); mutant membrane reacted with MLH2 (7); and wild type membranes reacted with MLH2 (8). MLH1 MLH2 uilo-ty intens‘ compar' Howeve is ver to the hands 7) ve' hembr thyia is in the L turn' meht be e Howe pro- chl The (AI acI pig LHI We Se 130 wild-type and mutant samples by any parameter other than relative intensity of non-LHC-II stained bands. Therefore, quantitative comparisons between mutant and wild-type membranes are not possible. However, it is clear that the ratio of the 26 to the 28 k0 polypeptides is very much reduced in the mutant (Figure 23, lanes 5 and 7) relative to that ratio in the wild-type membranes (lanes 4 and 8). The stained bands in the 15 to 20 k0 range (most visible in Figure 23, lanes 6 and 7) were observed in all blots of mutant or IML-treated thylakoid membranes (see below and Figure 24) and sometimes in the control thylakoid membranes. The identity Of these proteins is not known. It is intriguing to hypothesize that they are in_!ixg_breakdown products of the LHC-II polypeptides. It is known that the LHC-II polypeptides are turning over rapidly in these two types of membranes (see the develop- ment section of Chapter I) and one might expect that the membranes would be enriched with partially digested fragments of the polypeptides. However, such fragments have not as yet been identified. Intermittent light (IML) treatment of etiolated barley seedlings produces an arrested developmental state which is similar to that of the chlorOphyll b-less mutant of barley. (See Chapter 1 for discussion.) The chlorophyll a : chlorophyll b ratio of these thylakoids is high (Argyroudi-Akoyunoglou, 33:31; 1971). The membranes are functionally active but have a reduced photosynthetic unit size due to the absence of pigmented LHC-II and LHC-I complexes (Hiller, 33:31:, 1973; Armond gt;al;, 1977; Mullet 33:31:, 19800). They also have low levels of some LHC-II ap0proteins in their membranes (Cuming and Bennett, 1981). Western blots of thylakoid membranes isolated from IML-treated barley seedlings yielded results that are qualitatively similar to those of the 131 Figure 24. Western blot of thylakoid membranes isolated from IML-treated barley seedlings. Thylakoid membranes were isolated from the partially developed IML-treated barley seedlings, from IML-treated seedlings which had been allowed to green under continuous light for 4 hours, and from fully green control seedlings. The membranes were reacted with antibodies MLH1 and MLH2. Lanes 1-3: stained PAGE of fully greened control thylakoid membranes (1); IML-treated thylakoids (2); and 4 hours greened thylakoids (3). Lanes 4-9: western blots of control thylakoids reacted with MLH2 (4); IML-treated thylakoids reacted with MLH2 (5); 4 hours greened thylakoids reacted with MLH2 (6); control thylakoids reacted with MLH1 (7); IMl-treated thylakoids reacted with MLH1 (8); 4 hours greened thylakoids reacted with MLH1. Note that lane 6 was cut Slightly on a diagonal and therefore overlaps both halfs of the blot. 123 456789 68 kD- :1: 2'5 45- 3 33: 12.5- E l I l I—MLH2-‘—MLH1—‘ 133 chlorina f2 mutant. The ratio of the 26 kD to the 28 kD polypeptide was much reduced in the IML membranes relative to that in the control membranes (Fig. 24, lanes 7 and 4 vs. lanes 8 and 5). When IML-treated seedlings are placed in continuous light, the synthesis of chlorOphyll b is no longer suppressed and LHC-II develOpment begins (Argyroudi- Akoyunoglou, 23:31; 1971). In western blots of thylakoids from IML-treated seedlings that were exposed to 4 hours of continuous illumination, the 26 kD polypeptide increased while the 28 kD poly- peptide appears to have remained relatively constant (Fig. 24, lanes 9 and 6). Thus the 26 and the 28 K0 polypeptides respond differently to the lack of chlorOphyll b in both of these types of membranes. The 26 k0 polypeptide is reduced, while the 28 is apparently unaffected. Discussion Chapter 3 has described the isolation and characterization of a collection of monoclonal antibodies to LHC-II. This is the first collection of antibodies to be used to investigate the structure of LHC-II in the chlorOplast thylakoid. The antibodies can be divided into six classes which each identify a different determinant in the LHC-II polypeptides. Many of these antibody binding sites were present in more than one polypeptide. An unexpected result was the discovery of antibodies which cross-reacted with both LHC-II and LHC-I, the antenna polypeptides associated with photosystem 1. Furthermore the antibodies which recognized unique sites on individual polypeptides allowed several 134 conclusions about polypeptide diversity to be drawn. These antibodies were also used to measure the LHC-II polypeptides in the membrane and to investigate the structure and develOpment of the LHC-II polypeptides in vivo. 0n the basis of these data, a model for the structure of the antenna chlorOphyll-proteins of photosystem II is prOposed. Common antigenic determinants between LHC-II and LHC-I The monoclonal antibodies in classes V and VI reacted with polypeptides of both LHC-II and LHC-I. This reveals a previously unrecognized similarity between these polypeptides. Some antigenic determinants are identical in the two groups of polypeptides. However, this limited data does not allow any further conclusions as to the extent or origin of the similarities between polypeptides to be drawn. This observation may not be surprising since both groups of polypeptides have similar functions in binding antenna pigments and transferring energy to reaction centers. In previous studies, polyclonal antibodies to LHC-II have not been observed to cross-react with LHC-I polypeptides (see for example, Ryrie, 1983a). Possible explanations are that the common determinant is only weakly antigenic and/or antibodies binding to this region diSplay uniformly low avidity. Both these cases would probably be overlooked in a polyclonal antibody preparation because the most prevalent antibodies and those with the greatest affinity tend to dominate the result. However, the large number of class V and VI monoclonal antibodies isolated in this collection ( 1/3 of the total number) does not support the possibility that the common determinant in not antigenic. 0n the other hand, the monoclonal antibodies of classes V and VI in general si pr Sc am 06' ('91 be 6p; men COW Deg Droc were 135 display reduced specific binding in western blots compared to the other monoclonal antibodies described in this chapter. This may be the result of low avidity. Polypeptide diversity in LHC-II The collection of monoclonal antibodies defined both common and unique regions of the LHC-II polypeptides. Previously Apel (1977) had shown that two polypeptides of LHC-II in the green alga Acetabularia had similar amino acid compositions and two-dimensional tryptic finger prints. Related results were obtained by Hoober et;_al;'(1980) and Schmidt gt; 31; (l98l) working with the thylakoid membranes of Chlamy- domonas and pea respectively. The results described here extend these data to all four LHC-II polypeptides. The antibooies of classes III and IV clearly show that some regions of the polypeptides are identical among the LHC-II polypeptides. At least 4 common sets of antigenic determinants are present since antibOOy classes III, IV, V, and VI each recognize a different subset of the antenna polypeptides (see Table 6). It is possible that additional comIIon regions have been identified, because whether or not all members of each class bind exactly the same epitOpe has not been determined (For example, Class 111 contains 4 members which could each be binding a different determinant that is common between the same four polypeptides.) Monoclonal antibodies of classes I and 11 clearly discriminate between the 28 and 26 kD polypeptides of LHC-II (Table 6, Figures 8, and 9). It has been hypothesized that the 28 kD polypeptide is a partially processed precursor of the 26 kD polypeptide (Mullet, 1983). If this were the case, then all regions of the 26 kD polypeptide should repeat the dete How pre pro MLH pos pol ace p05 wh' pr! ()1 LH pr 136 the primary sequence of the 28 kD polypeptide, and any antigenic determinant present in the 26 should also be present in the 28 kD. However, MLH1 binds an epitOpe of the 26 k0 polypeptide that is not present in the 28 kD polypeptide. Thus the 26 can not be a product of proteolytic processing of the 28 kD polypeptide. It is possible that MLH1 recognizes a portion of the 26 kD polypeptide that has been post-translationally modified after it was processed from the 28 k0 polypeptide. Modifications such as glycosylation, phosphorylation, or acetylation could create a new antigenic determinant. However, this possibility seems unlikely, especially in light of classes III and V, which recognize other regions of the 26 kD polypeptide that are not present in the 28. These studies did not identify any antibodies which could discriminate the 25.5 and 24.5 kD proteins from the 26 k0 polypeptide of LHC-II. A tempting interpretation of these data is that the three proteins are derived from a single common precursor. However the evidence of Schmidt 33:21; (1981) that mRNA for both the 26 and the 25.5 kD polypeptides (polypeptides 15 and 16 in their nomenclature) can be translated in vitro, indicates that the 26 and the 25.5 are not the result of processing from a common polypeptide precursor, but are transcribed from separate mRNA's. It is possible that these two polypeptides have identical sequences, which would make them indistin- guishable by immunological methods. However, Schmidt and colleagues (1981) also demonstrated that the smaller polypeptide produced three uniquely sized fragments in partial proteolytic digests. There are several explanations for why an antibody to one of these unique regions was not identified in this collection. The most probable is that the 137 number of specific antibodies isolated was too small to have identified every unique region of the four polypeptides. Furthermore some regions may be less antigenic than others, making antibodies specific for those regions more difficult to obtain. The origin of the 24.5 polypeptide has not been determined. It is possible that it is a processing product from either the 26 or the 25.5 kD polypeptide. This set Of antibodies cannot distinguish the two possibilities. ChlorOphyllzProtein Ratios in LHC-II Quantitation of the 26 and 28 k0 polypeptides using radioactively labeled MLH1 and MLH2 (Figure 17) yielded the result of 27 and 2 polypeptides per 400 chlorOphylls, reSpectively. Assuming that 50% of the chlorOphyll in the photosynthetic unit is bound to LHC-II (Hiller and Goodchild, 1981), then 200 chlorOphylls must be distributed among the four polypeptides of the complex. If one excludes the three minor polypeptides (28, 25.5, and 24.5 k0), then 7.4 chlorOphylls must be bound to each 26 k0 polypeptide. Including the other LHC-II polypep- tides in this calculation would decrease this estimation of the chlorOphyllzprotein ratio. AS discussed in Chapter 1, some controversy is present in the literature over the number of chlorOphylls bound to isolated LHC-II. My results agree quite well with the values measured in CPII by Kan and Thornber (1976) and in LHC-II isolated in Triton x-100 by Ryrie 33:31; (1980). In order to reconcile my data with the larger chlorOphyll: protein ratio of 13 measured by others ( Bar-nun 33:31, 1977; Burke et.al. 1978; Li, 1985) the proportion of chlorOphylls assumed to bind 138 LHC-II in the photosynthetic unit would have to be increased to 88% . The PSII core complex is generally thought to contain about 40 chloro- phylls per P680’ and the PSI reaction center (i.e., core complex plus LHC-I) is thought to contain about 110 chlorophylls. (Kaplan and Arntzen, 1982). If LHC-II is accounting for 88% of the chlorOphyll in a membrane contianing 150 reaction center chlorophylls, then the photosynthetic unit size in those membranes must be 1250 chlorOphylls. It is unlikely that these membranes prepared from pea seedlings grown under standard conditions would contain such a large photosynthetic unit. Structure of LHC-II in vivo Several lines of evidence described in this chapter support a hypothesis that the 26 and the 28 kD polypeptides belong to separate, but linked, structural units in the thylakoid membrane. The two polypeptides can be isolated together (along with the 25.5 and 24.5 k0 polypeptides) in LHC-II solubilized in Triton X-100 (Figure 9). They can also be separated as in non-denaturing SDS-PAGE, producing CPII, a chlorOphyll-protein complex which does not contain the 28 k0 polypeptide (Figure 21). The two polypeptides respond differently to the absence of chlorOphyll b (Figures 23 and 24). In both the chlorOphyll b-deficient mutant of barley and in partially developed thylakoid membranes, the 28 kD polypeptide appeared to be invariant , while the 26 kD polypeptide was strongly affected. Apparently the 28 k0, like reaction center polypeptides, does not need chlorophyll b to stabilize it in the membrane. Independent develOpment of the two LHC-II polypeptides is 139 good evidence that they comprise separate structures in the thylakoid membrane. The concept of separate, but linked polypeptides in the antenna of photosystem II is illustrated by a two component antenna model such as that presented in Figure 25. In this mooel, the 28 kD polypeptide would comprise an internal or permanent antenna, while the 26 kD polypeptide would be contained in the peripheral or variable antenna. The 25.5 and 24.5 k0 polypeptides are assumed to also be in the variable antenna. A similar mooel dividing the antenna of photosystem II into two components has been prOposed previously, based on data from non-denaturing SDS-PAGE (Green and Cam, 1981). The organization proposed in the model for LHC-II shown in Figure 25 is similar to the structure of antenna chlorophyll proteins in the Rhodospirillaceae class of photosynthetic bacteria (Drews, 1985). These bacteria contain a single type of reaction center which is connected to antenna proteins containing carotenoids and bacteriochlorophyll. Two classes of antenna complexes transfer absorbed energy to the reaction center: the peripheral complex, 8800-850; and the internal complex, 8870. Each reaction center is associated with 8870 antenna complexes which in turn are connected to a “lake" of 8800-850 antenna complexes The ratio of components in the photosynthetic membrane is variable, depending on the history of the culture, but adaptive changes in the photosynthetic unit size occur by changes in the number of peripheral antenna complexes. The internal antenna remains in relatively constant prOportion to the reaction centers at all times. The peripheral and internal antenna components proposed for LHC-II are analogous to the 8800-850 and 8870. The internal antenna is small 140 Figure 25. A model of the antenna chlorophyll-protein complexes of photosystem II. This model divides the antenna into two components: an inner, tightly bound antenna, the 28 k0 polypeptide; and a peripheral, or variable antenna, the LHC-II. 141 Photosystem II p 28 kD polypeptide ® LHC-ll 142 and mainly serves as a linker between photosystem II and the rest of the antenna proteins. The variable antenna contains the bulk of the antenna pigments and is equivalent to the LHC-II whose structure was studied by fourier transform electron microscopy (Kuhlbrandt, 1984; and Li, 1985). The variable antenna can change in size relative to photosystem II in response to short and long term environmental factors. Some of the complexes of the variable antenna, perhaps the most external, can be phOSphorylated to regulate their association to PSII. I prOpose that the name LHC-II be restricted to the variable complex in the antenna. APPENDIX APPENDIX Publications Abstracts I. Darr, S.C. and C.J. Arntzen. 1983. Abstract #753. Reconstitution of LHC-PSII interaction. Plant Physiol. 725: 132. 2. Darr, S.C. and C.J. Arntzen. 1984. Abstract #60. Reconstitution of membrane protein assembly in the chlorOplast thylakoid. J. Cell Biol. 99: 17a. 3. Darr, S.C., S.C. Somerville, and C.J. Arntzen. 1985. Abstract #269. Structure of the light harvesting chlorophyll protein complex. J. Cell Biol. 101: 72a. Otherpublications 1. Nabedryk, E., P. Biaudet, S. Darr, C. Arntzen, J. Breton. 1984. Conformation and orientation of chlorophyll-proteins in photosystem I by circular dichroism and polarized infrared spectroscopies. Biochim. Biophys. Acta 767:-640-647. 2. Arntzen, C.J., S.C. Darr, J.E. Mullet, K.E. Steinback, K. Pfister. 1982. Polypeptide determinants of plastoquinone function in photosystem II of chloroplasts. in: Function of quinones in energy conserving systems. ed: 8. Trumpower. Academic Press, New York. pp: 443-452. 3. Darr, S.C., V. Souza Machado, and C.J. Arntzen. 1981. Uniparental inheritance of a chlorOplast photosystem II polypeptide controlling herbicide binding. Biochim. Biophys. Acta 634: 219-228. 143 BIBLIOGRAPHY BIBLIOGRAPHY Albertsson, P.-A., and B. Andersson. 1981. Separation Of membrane components by partition in detergent containing polymer phase systems: isolation of the light harvesting chlorOphyll a/b protein. J. Chromatog. 215: 131-141. 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