5.2-... litiifi 1. Id Mflfli C. I: 133...!!- 2.51.933!!! '52....31. - Ira. S 4x . 139%!!! I 1.950.... fluff. out. . z , it: ‘33 ‘i .3... III... It“? . = l {.5 .345; :. . .3 3.3.29... $1....«Wnnmg . w? .yfihxgg .z . hvw‘umm Jan inauuué its: . $23.... .muué. .a. “filial fig. dnumumrwltfifilivltmnRfltfil I . t I: 3-1.9. 1 Ir .. flaunts: . ‘4“...9’: 2.1.3! L. 9‘3 22."..5115... 5.3.... iii! . 3235.-.... .2 EggLIF. .1 4-351», ‘1‘ 3!!!n.!...3...5.....u .. I63: t 1...“. ....m%§ 3.2%....r {it .3. g}, I .1]! ?c€nfi3ubum..uhfl in... .v! .6 .- ll’. FR»?! 5"- <3 5! I. thuov-t. " 3§.£lnl..§ammh HF...“ ll’nil tits-1N. 5‘. y: L! .8 This is to certify that the dissertation entitled Reversibly Glycosylated Polypeptides presented by Ivan J. Delgado Orlic has been accepted towards fulfillment of the requirements for Ph.D. degree in Genetics Major professor C Date 0 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE ' DATE DUE 6/01 c-JCIRC/DateDue.p65-p.15 REVERSIBLY GLYCOSYLATED POLYPEPTIDES By Ivan J. Delgado Orlic A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Program 2002 ABSTRACT REVERSIBLY GLYCOSYLATED POLYPEPT IDES By Ivan J Delgado Orlic The reversibly glycosylated polypeptides (RGP) are a family of four plant- specific proteins in Arabidopsis that have been implicated in cell wall biosynthesis. The main reasons for this hypothesis are that RGPs react with nucleotide sugars and are localized in the Golgi apparatus. Yet to date no direct evidence exists for the involvement of RGPs in the synthesis of cell wall components. To address this question, biochemical, genetic, and molecular biological approaches were taken. Arabidopsis RGPs were detected both in the Golgi apparatus and the cytoplasm. AtRGPI and AtRGP2 are expressed in all tissues, and their respective proteins glycosylate in the presence of UDP-sugars, while AtRGP3 and AtRGP4 are only expressed in siliques, and their respective proteins do not glycosylate. AtRGPl and AtRGP2 are functionally redundant and essential for plant development because an Arabidopsis homozygous T- DNA insertion mutant in both genes is lethal. The results obtained can be interpreted as follows: RGPs are involved in the transport of nucleotide sugars from the cytoplasm to the Golgi apparatus, where RGPs may deliver their cargo to the Golgi lumen. To my family iii ACKNOWLEDGEMENTS There are so many people to thank that I am afraid I will miss more than one. To those that are not listed here, thank you. Thanks to Natasha, Ken, Hans, and Jonathan for an interesting and educational Ph.D. experience. To the people in Natasha’s lab: thank you Alex for getting me started; to Maor and Glenn for your example; to Harley for the laughs and your friendship. To Mike and Sybil for bringing the lab together; to Cindy for making my job with the lab clone collection a bearable one; and to Enrique and Rodrigo for all the good times. To the rest, thanks for all the little things. To the people in the PRL: to John Scott-Craig and Curtis for your willingness to share your expertise; to Mark, Miguel, and Jeff for your RNA expertise; and to Nikki, Oscar, Sigrun, Scott, Eric, and Jon, for the fun times. To Therese and Sue for all the sequencing and the fun talks. To Franz de Bruijn for your knowledge and the way in which you shared it. To the people outside the PRL: to Clayton and his lab for the fun times and for making me feel part of their group. To all the people I played soccer with, too many to name but eternally appreciated. To Sharon for her friendship. To Heiko, Christoph Benning, Ariel Orellana, Petra, Klaus Palme, Tony Bacic, Debby Delmer, Alan White, Sylvia de Pater, Bernie Epel, and Dirk Warnecke for their time, collaboration, and help in various aspects of this project. I owe them all a mountain of thanks. Finally, to the main reason why I was able to finish my doctorate, my wife, Susan, for her love and support, and our family, for making it so easy to know what really matters. iv TABLE OF CONTENTS LIST OF TABLES .................................................... viii LIST OF FIGURES ..................................................... ix LIST OF ABREVIATIONS ............................................... x CHAPTER I Introduction. Reversibly glycosylated polypeptides ............................. 1 Introduction ..................................................... 2 References ...................................................... 13 CHAPTER II Reversibly glycosylated polypeptide-1 ..................................... 17 Abstract ........................................................ 18 Introduction ..................................................... 19 Materials and Methods ............................................ 22 Results ........................................................ 32 Identification of an Arabidopsis cDNA Encoding an AtRGPl Protein . 32 Distribution of RGPl Transcript and Protein ..................... 38 AtRGPl is Soluble and Membrane Associated ................... 43 Glycosylation of AtRGPl .................................... 49 Discussion ...................................................... 53 References ...................................................... 57 CHAPTER III — Characterization of the four AtRGPs of Arabidopsis ............. 63 Abstract ........................................................ 63 Introduction ..................................................... 65 Materials and Methods ............................................ 70 Results ......................................................... 77 A multi- gene family of distinct Arabidopsis proteins ................ 77 Expression analysis of the AtRGPs ............................ 80 Four AtRGPs and two distinct activities ......................... 83 An AtRGPI/AtRGPZ double T-DNA insertion mutant may be lethal . . 86 Discussion ...................................................... 96 References ..................................................... 101 CHAPTER IV — Future work: proposed experiments to elucidate RGP function . . . . 105 Characterization of AtRGP mutants ................................. 106 Identification of proteins that interact with RGP ....................... 106 Elucidation of the three dimensional structure of RGP ................... 108 RGP Localization ............................................... 109 References ..................................................... 1 10 APPENDIX — Observations: experimental observations with unclear explanations . . 111 Materials and Methods ........................................... 112 vi Appendix 1: RGPs are a plant-specific multi-gene family ................ 119 Appendix 2: localization of His-T7-RGP1 and HA-AtRGP2 .............. 121 Appendix 3: phenoscreen of AtRGP mutants .......................... 124 References ..................................................... l 29 vii LIST OF TABLES Table 4.1 - Summary of results from experiments presented in this thesis ......... 107 Table A.3.1 - The RGPs genes so far identified .............................. 120 viii LIST OF FIGURES Figure 2.1 - Predicted protein sequence of AtRGPl (GenBank accession no. AF013627) and comparison with other RGP proteins .................................... 33 Figure 2.2 - Homologs of AtRGPl from other species ......................... 39 Figure 2.3 - AtRGPl RNA and protein are highest in suspension-cultured cells and roots from whole plants ...................................................... 41 Figure 2.4 - AtRGPl is soluble and membrane associated ....................... 44 Figure 2.5 - Membrane localization of AtRGPl ............................... 47 Figure 2.6 - AtRGPl is reversibly autoglycosylated ........................... 51 Figure 3.1 A - Chromosomal location of the different AtRGP genes ............... 78 Figure 3.1 B - Gene structure of the four AtRGP genes ......................... 79 Figure 3.2 - Sequence comparison of the AtRGP proteins predicted from their cDNAs 81 Figure 3.3 - Expression profiles of the various AtRGP genes .................... 84 Figure 3.4 - AtRGP] and AtRGP2 auto-glycosylate ........................... 87 Figure 3.5 - AtRGP3 and AtRGP4 cleave UDP-Glc to glucose ................... 89 Figure 3.6 - T-DNA insertion mutants in AtRGP] and AtRGP2 ................... 93 Figure 3.7 - In seach of a double atrgpI-Ilatrng-I mutant ..................... 94 Figure A.2.l - Over-expression and localization of His-T7-AtRGPl and HA-AtRGPl in Arabidopsis plants ..................................................... 123 Figure A.3.l - Arabidopsis growth stages .................................. 125 Figure A.3.2 - Growth stage progression and detection of phenotypic differences between wild type and Athp mutants ............................................ 127 ix LIST OF ABREVIATIONS RGP - Reversibly Glycosylated Polypeptide RLP - RGP - like protein ER - Endoplasmic reticulum PM - Plasma membrane SpsA - Bacillus subtilis Spore Coat Polysaccharide Biosynthesis Protein AcsAB - Acetobacter xylinum cellulose synthase gene HCA - Hydrophobic cluster analysis CW - cell wall RT-PCR - reverse transcriptase polymerase chain reaction STD - standard deviations DF - degrees of freedom CHAPTER I INTRODUCTION REVERSIBLY GLYCOSYLATED POLYPEPTIDES REVERSIBLY GLYCOSYLATED POLYPEPTIDES The reversibly glycosylated polypeptide (RGP) has been implicated in cell wall biosynthesis (Dhugga et al., 1991). RGP is a 41-kD protein that reacts with UDP-sugars and has been localized in the Golgi apparatus (Dhugga et al., 1997), the site of cell wall polysaccharide synthesis (N ebenfiihr and Staehelin, 2001). The first gene encoding an RGP was isolated by Dhugga et al. (1994). Zhaohong Wang, under the supervision of Dr. de Rocher, identified the first Arabidopsis RGP EST (Wang, 1995) and obtained the first RGP cDNA (Newman et al., 1994). This Arabidopsis RGP was used to identify other Arabidopsis RGPS (Delgado et al., 1998), and the characterization of these genes was the aim of this project. The name RGP comes from the observation that this protein glucosylates in the presence of uridine- diphosphate glucose (UDP-Glc), and releases this sugar in the presence of an excess of different UDP-sugars (Dhugga et al., 1997). Reversible glycosylation RGP is defined by two separate activities. First, in the presence of UDP-[”C]Glc, RGP self-glucosylates. And second, when an excess of unlabeled UDP-Glc or UDP is added to a reaction between UDP-['4C]Glc and RGP, no RGP self-glucosylation can be detected (Dhugga etal., 1991). The reversible self-glycosylation of RGP is the biochemical definition of this protein, and by this definition RGP has been characterized in mung bean (Franz, 1976; Read et al., 1986), pea (Dhugga et al., 1997), Arabidopsis (Delgado et al., 1998), potato (Bocca et al., 1999), and nasturtium (Faik et al., 2000). Another definition for RGP, one that does not require a biochemical analysis, takes advantage of the fact that RGPs are highly conserved proteins in plants (Delgado et al., 1998). All RGPs identified to date are 4l-kD proteins that are at least 68% identical to each other (see Appendix 1). Although multiple RGPs have been identified in Arabidopsis (Delgado et al., 1998) and potato (Bocca et al., 1999) based on their sequence identity, to date no biochemical characterization of the multiple RGPs found in a single plant species has been performed. The RGPs identified in Arabidopsis based on sequence similarity have been analyzed for their self-glycosylation potential in the presence of UDP-sugars (see chapter 3). The glycosylation of RGP has been demonstrated in the presence of UDP-Glc, UDP-Xyl, and UDP-Gal, which suggests that RGP might be involved in the synthesis of polysaccharides such as xyloglucan (Dhugga et al., 1991). The extent of RGP self- glycosylation in the presence of UDP-Glc, UDP-Xyl, and UDP-Gal (Dhugga et al., 1997) is in about the same ratio as the relative proportions of these sugars in xyloglucan (Hayashi et al., 1989). Our understanding of RGP self-glycosylation is hampered by the limited availability of radiolabeled UDP-sugars. The presence of UDP-sugars in plants other than UDP-Glc, UDP-Xyl, and UDP-Gal, makes it necessary to study RGP self- glycosylation in the presence of other UDP-sugars. The study of RGP glycosylation in the presence of UDP-sugars such as UDP-Man, UDP-Gch, UDP-Rhm, and UDP-Ara, would provide a better understanding of which UDP-sugars RGP can use as substrates for self-glycosylation. The nature of the glucose-protein bond between RGP and glucose has been determined by Singh et a1. (1995). In an effort to isolate the protein primer for starch synthesis, also known as amylogenin, Singh et al. (1995) isolated a 41-kDa sweet corn protein that self-glucosylated in the presence of UDP-Glc. When the glucosylated corn protein was purified, digested with trypsin and the tryptic peptides sequenced, a single glucose was found covalently attached to an arginine via a B-linkage. Dhugga et a1. (1997) found that the sequence of the tryptic peptides of the corn protein, which amounted to over 40% of the total estimated corn protein sequence (Singh et al., 1995), showed over 84% sequence identity with the pea RGP. This led Dhugga et a1. (1997) to rename the corn “amylogenin” as the corn RGP. Likewise, the full-length clone of the corn RGP] (accession U89897) is almost 100% identical to the sequenced tryptic peptides obtained by Singh et al (1995). These observations indicate that the corn self- glucosylating protein is an RGP homologue. The equivalent arginine shown to be glucosylated in the corn RGP (Singh etal., 1995) is also present in the pea RGP (Dhugga et al., 1997) and every other RGP so far identified (Delgado et al., 1998). Therefore, it has been presumed that this arginine is the site of glucosylation of the RGPs (Dhugga et al., 1997). The requirement for self-glucosylation of the equivalent arginine in the Arabidopsis RGP] was studied (see chapter 3). The glycosylation of RGP is well understood, but not the reversibility of this reaction. When RGP glucosylates in the presence of UDP-[”C]Glc, the label can be chased out from RGP by adding an excess of UMP, UDP, UTP, or any UDP-sugar with the possible exception of UDP-Man (Dhugga et al., 1991). Although the glycosylation of the pea RGP was not inhibited by UDP-Man (Dhugga et al., 1991), the glycosylation of the nasturtium RGP was inhibited by UDP-Man (Faik et al., 2000). Such conflicting results make it hard to determine which UDP-sugars are substrates for RGPs. Almost every study so far only characterized the reversible self-glucosylation of RGP by the displacement of Glc from glucosylated RGP using various UDP-sugars (Dhugga et al., 1991; Dhugga et al., 1997; Delgado et al., 1998; Bocca et al., 1999). To date, the displacement of Gal from galactosylated RGP has only been studied in nasturtium (Faik et al., 2000). Not surprisingly, the reversible self-galactosylation of the nasturtium RGP was different from the reversible self-glucosylation of RGP in other plants. The displacement of Gal from galactosylated nasturtium RGP takes place in the presence of UDP-Man and various UDP-uronic acids such as UDP-Gch and UDP-GalA (Faik et al., 2000). At present, it is unclear whether these differences are a results of differences in the reversibility of self-glucosylation and self-galactosylation of an RGP in a given plant, or a result of differences in the reversible self-glycosylation of RGPs in different plants. Nevertheless, ADP-Glc and GDP-Man were unable to chase the sugar from glucosylated pea RGP, suggesting that RGP is not involved in the synthesis of either starch or glycoproteins (Dhugga et al., 1991). The reversible self-glucosylation of one of the Arabidopsis RGPs, AtRGPl, was studied (see chapter 2). Non-processive glycosyltransferase Saxena et a1. (1995) identified a motif in the Acetobacter xylinum cellulose synthase gene (AcsAB) that was conserved in plant glycosyltransferases. They named this motif “domain A”, and it is defined as a series of B-strands alternating with a- helices with two conserved aspartate residues separated by 46 to 120 amino acids (Saxena and Brown, 1997). The “domain A” motif is also the defining signature of the glycosyltransferase family 2 (GT2) and covers a region that is necessary for UDP-binding (Charnock and Davies, 1999). In addition to domain A, processive glycosyltransferases contain a second domain called domain B (Saxena et al., 1995). Processive glycosyltransferases are involved in the synthesis of long-chained polysaccharides (Dhugga, 2001; Saxena and Brown, 2000), and it is believed that the presence of both domain A and domain B is required for the continuous addition of sugars to an elongating polymer (Charnock et al., 2001). The observation that RGP contains domain A but not domain B led Saxena and Brown (1999) to propose that RGP is a non-processive glycosyltransferase. To date, no experimental evidence exists that would attribute a glycosyltransferase activity to RGP. Golgi localization of RGP and Golgi-localized B-glucan synthase (GS-I) activity RGP is localized in the Golgi apparatus. This was demonstrated by an analysis of pea membranes fractionated by sucrose density gradients, which showed that pea RGP glycosylation co-fractionated with Golgi-localized B-glucan synthase (GS-l) activity, and not with fractions containing the endoplasmic reticulum-localized NADPHzcytochrome c reductase activity or the mitochondria-localized cytochrome c oxidase activity (Dhugga et al., 1991). The localization of RGPs to the Golgi apparatus was later confirmed by electron microscopy of pea hypocotyls using pea RGP antibodies (Dhugga et al., 1997). Upon further examination of the localization of pea RGP by electron microscopy, the authors consistently found that RGP was concentrated in only one side in each dictyosome, the side that is the most electron dense or the trans-side (Driouich et al., 1993). This led Dhugga et a1. (1997) to suggest that RGP is localized primarily on the trans cisternae of the Golgi apparatus. Glucan synthase I (GS-I) activity, a Golgi-localized activity that produces [34,4- glucan from UDP-Glc (Hassid, 1972), is believed to be involved in xyloglucan biosynthesis (Ray, 1980). To date, the enzyme(s) responsible for GS-I activity have not been identified. The proteins in the Golgi apparatus involved in the synthesis of polysaccharides have been recalcitrant to purification, largely because their activities are lost during the purification process, a characteristic of membrane-bound proteins. Only recently have the first genes encoding Golgi-localized glycosyltransferases involved in polysaccharide synthesis have been identified (Perrin et al., 1999; Edwards et al., 1999). It was during their attempts at identifying the proteins responsible for GS-I activity that Dhugga et a1. (1991) identified and purified RGP. The purification of RGP in protein fractions enriched for GS-I activity suggests that RGP may be involved in the synthesis of a B-l,4-glucan. RGP glucosylation requires the same divalent cations (Mg2+ or Mn“, but not Ca2+ or Sr“) as the ones required by GS- 1 activity (Dhugga et al., 1991). In addition, both RGP glucosylation and GS-I activity are inhibited by UDP-Xyl and UDP-Gal, but not by UDP-Man or non-uracil-containing nucleotide sugars. These observations suggested that RGP may be a component of GS-I activity in the synthesis of [3-1,4-glucan (Dhugga et al., 1991). Starch synthesis and lipid glycosylation Autoglycosylation is not unique to RGP. Proteins capable of self-glycosylation have been implicated in priming the synthesis of animal glycogen (Lomako et al., 1990; Viskupic et al., 1992) and plant starch (Moreno et a1. 1986; Ardila and Tandecarz, 1992). When the gene encoding the above-mentioned putative starch primer was cloned, it was found to be RGP (Bocca et al., 1999). Since RGP is not localized in plastids (Dhugga et al., 1991 and 1997), and only one sugar at a time is attached to RGP (Singh et al., 1995), it is unlikely that RGP participates in starch synthesis (Dhugga et al., 1991; Bocca et al., 1999). When RGP-containing protein extracts are incubated with UDP-[”C]Glc, a glucosylated lipid is formed in addition to glucosylated RGP (Read et al., 1986; Dhugga et al., 1991). Lipid glucosylation, unlike RGP glucosylation, does not require a divalent cation, is inhibited by UDP-Man, and is not blocked by UDP-Gal (Dhugga et al., 1991). These observations suggested that RGP is not involved in lipid glycosylation (Dhugga et a1,199l) AMYLOGENIN Krisman and Barengo (1975) were the first to identify the protein primer for glycogen synthesis in animals, later named glycogenin (Rodriguez and Whelan, 1985). Glycogenin is a protein that self-glucosylates in the presence of UDP-Glc (Meezan et al., 1988). Evidence for the priming ability of glycogenin includes the observation that one glycogenin protein can be found per glycogen molecule in rabbit muscle (Kennedy et al., 1985). Viskupic et a1. (1992) cloned the first glycogenin gene and confirmed earlier observations that the predicted protein size of glycogenin is 38 kD. Analogous with animal glycogen synthesis by glycogenin, starch synthesis in plants is though to require a protein primer, also known as amylogenin (Moreno et al., 1986; Moreno et al., 1987). Amylogenin has been described as a self-glucosylating protein capable of serving as a primer for protein-bound alpha-1,4-glucan synthesis (Moreno et al., 1987). To date the gene encoding a protein capable of priming alpha-1,4- glucan synthesis has not be cloned. Efforts to isolate the putative protein primer for starch synthesis (Ardila and Tandecarz, 1992) instead have lead to the isolation of the potato RGP (Bocca et al.; 1999). The self-glucosylation of this potato protein, termed UDP-glucosezprotein transglucosylase (UPTG), in the presence of UDP-Glc was hypothesized to be the starting point for the enzymatic elongation of glucan chains by starch synthases or starch pyrophosphorylases (Moreno et al., 1987). Bocca et a1. (1997) later observed that UPTG also self-xylosilated in the presence of UDP-Xyl, a surprising result considering that starch does not contain xylose residues (Martin and Smith, 1995). When antibodies raised against the purified UPTG (Bocca et al., 1997) were used to screen a cDNA expression library from potato stolons, two cDNAs were identified. The cDNA sequences coded for polypeptides of 365 and 366 amino acids (41 kD polypeptides) and shared 89% identity at the protein level (Bocca et al., 1999). When these potato cDNA sequences were used in a GenBank database search, the authors found that their potato genes were 86-93% identical to RGPs from Arabidopsis, pea, corn, rice, and wheat (Bocca et al., 1999). Furthermore, the self-glycosylation of UPTG required an“ and was reversible in the presence of UDP-sugars with the exception of UDP-Man, the same reversible self-glycosylation described for the pea RGP (Dhugga et al., 1997). Both the high sequence similarity and almost identical biochemical properties between UPTG and RGP led Bocca et a1. (1999) to conclude that UPTG was not a protein primer for starch synthesis, or amylogenin, but an RGP homologue. Singh et al. (1995) identified a corn protein that self-glucosylated in the presence of UDP-Glc. Although the authors did not show any evidence that this protein primed alpha-1,4-glucan synthesis, they named it amylogenin. The authors sequenced over 40% of the protein and found that the glucosylation site was a single arginine in the middle of the protein (Singh et al., 1995). Dhugga et a1. (1997) later found that the corn protein sequenced by Singh et al. (1995) was over 80% identical to the pea RGP, and renamed it the corn RGP. The two studies described above aimed at identifying the potato (Ardila and Tandecarz, 1992) and corn (Singh etal., 1995) amylogenin, but instead led to the identification of the potato (Bocca et al., 1999) and corn (Dhugga et al., 1997) RGP. However, in neither case were the proteins analyzed for their priming ability in the synthesis of starch. It was not until the genes were cloned and the activities of the respective proteins have been analyzed that the self-glucosylation of these putative amylogenins will be found to be reversible and to require Mn2+ (Bocca et al., 1999), two characteristics that do not coincide with the activity described for the synthesis of protein-bound alpha-1,4—glucan (Moreno et al., 1987), yet characteristic of RGP reversible self-glucosylation (Dhugga et al., 1997). To date no amylogenin has been shown to be involved in the priming of starch synthesis. A putative amylogenin (AMY), that is not RGP, has been cloned in wheat (de Pater and Kijne, 1997). The wheat AMY (accession Y18625) is only 42% identical to the pea RGP, yet AMYs from wheat, rice (Y18623), and Arabidopsis (BAB09620) are 60-90% identical to each other at the protein level. In addition, AM Ys encode proteins with a predicted size of 38 kD, the same size as the protein primer for glycogen, 10 glycogenin (Meezan et al., 1988). To date no evidence exists for a role of these 38 kD AMYs in starch synthesis, nor any evidence for reversible self-glucosylation. STATEMENT OF PROBLEM AND PRINCIPAL RESULTS Plant cell wall polysaccharide synthesis occurs at the plasma membrane and in the Golgi apparatus (Nebenfiihr and Staehelin, 2001). Only cellulose and callose, both polymers of glucose, are synthesized at the plasma membrane (Delmer, 1999). The remaining polysaccharides that make up the cell wall are synthesized in the Golgi apparatus (Bolwell, 2000). The substrates for polysaccharide synthesis, the nucleotide sugars, are synthesized in the cytoplasm (Bonin et al., 1997; Gibeaut, 2000; Reiter and Vanzin, 2001). The process by which nucleotide sugars are transported from the cytoplasm to the Golgi apparatus is not well understood. Part of this process is thought to require transporter proteins at the Golgi membrane (Gibeaut, 2000), and the first Golgi- localized nucleotide sugar transporter was recently isolated (Baldwin et al., 2001). The localization of RGP to the Golgi apparatus led Dhugga et a1. (1991) to propose a role for RGP in cell wall polysaccharide synthesis. The goal of this dissertation was to investigate the hypothesis proposed by Dhugga et a1. (1997), which stated that RGP might be involved in cell wall polysaccharide synthesis. The way in which RGP is involved in this process is unclear, but it was thought that it may be a protein primer for polysaccharide synthesis or a carrier protein involved in the uptake of nucleotide sugars into the Golgi apparatus. 11 Chapter 2 describes the cloning and characterization of the first RGP from Arabidopsis, AtRGP] . The experiments presented in this chapter have been published as part of a manuscript (Delgado et al., 1998). Zhaohong Wang, under the supervision of Dr. de Rocher, identified and sequenced the AtRGP cDNAs used in this study (Wang, 1995). Antibodies were raised against purified GST-AtRGPl and used to show that AtRGP was localized in the cytoplasm as well as the Golgi apparatus. These antibodies were also used to show that RGP is plant specific. Finally, purified GST-AtRGPl reversibly glycosylated in the same manner as described for the pea RGP (Dhugga et al., 1991). Chapter 3 describes the characterization of the four RGPs present in Arabidopsis. The experiments presented in this chapter have been compiled as a manuscript for publication. Expression analysis showed that AtRGP] and AtRGP2 were expressed in all tissues tested, while AtRGP3 and AtRGP4 were only expressed in siliques. Likewise, AtRGP] and AtRGP2 glucosylated in the presence of UDP-Glc, while AtRGP3 and AtRGP4 did not. The notion that AtRGPl and AtRGP2 share the same function was introduced based on the observation that a double T-DN A insertional mutant lacking both AtRGP] and AtRGP2 may be lethal. Chapter 4 suggests future directions for the elucidation of the function RGPs have in plants. In addition to the results presented in the previous chapters, others results that have no clear explanation were added as an appendix. 12 REFERENCES Ardila FJ, Tandecarz J S (1992) Potato tuber UDPglucose : protein transglucosylase catalyses its own glucosylation. Plant Physiol 99: 1342-1347 Bacic A, Harris PJ, Stone BA (1988) Structure and function of plant cell walls. In The biochemistry of plants: a comprehensive treatise. 14. Stumpf PK and Conn EE, ed., Academic Press, New York, 297-371. Baldwin TC, Handford MG, Yuseff M-I, Orellana A, Dupree P (2001) Identification and characterization of GONSTl , a Golgi-localized GDP-mannose transporter in Arabidopsis (2001) The Plant Cell 13: 2283-2295 Bocca Sn, Rothschild A, Tandecarz J S (1997) Initiation of starch biocynthesis: purification and characterization of UDP-glucosezprotein transglucosylase from potato tubers. Plant Physiol Biochem 35: 203-210 Bocca SN, Rojas-Beltran JA, Gebhardt C, Moreno S, Du Jardin P, Tandecarz J S (1999) Molecular cloning and characterization of the enzyme UDP-glucosezprotein transglucosylase from potato. Plant Physiol Biochem 37: 809-819 Bolwell GP (2000) Biosynthesis of plant cell wall polysaccharides. Trends Glycosci. Glycotech. 65: 143-160 Bonin CP, Potter 1, Vanzin GF, Reiter WD (1997) The M URI gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6—dehydratase, catalysing the first step in the de novo synthesis of GDP-L-fucose. Proc Natl Acad Sci USA 94: 2085-2090 Chamock SJ, Henrissat B, Davis 0] (2001) Three-dimensional structures of UDP-sugar glycosyltransferases illuminate the biosynthesis of plant polysaccharides. Plant Physiol 125: 527-531 Chamock SJ, Davis GJ (1999) Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. Biochemistry 38: 6380-6385 de Pater S, Kijne J (1997) Cloning and characterization of the wheat starch biosynthetic enzyme amylogenin. 5th International Congress of Plant Molecular Biology. Singapore. Abstract No. 746 Delgado 1], Wang Z, de Rocher A, Keegstra K, Raikhel NV (1998) Cloning and characterization of Athpl: a reversibly autoglycosylated Arabidopsis protein implicated in cell wall biosynthesis. Plant Physiol 116: 1339-1349 13 Delmer DP (1999) Cellulose biosynthesis: Exciting times for a difficult field of study. Annu Rev Plant Physiol Plant Mol Biol 50: 245-276 Dhugga KS, Ulvskov P, Gallagher SR, Ray PM (1991) Plant polypeptides reversibly glycosylated by UDP-glucose: possible components of Golgi beta-glucan synthase in pea cells. J Biol Chem 266: 21977-21984 Dhugga KS, Ray PM (1994) Purification of the reversibly glycosylated polypeptides from pea: purified polypeptides exhibit the same properties as the Golgi-bound form. Plant Physiol. Supplement 105, #684 Dhugga KS, Tiwari SC, Ray PM (1997) A reversibly glycosylated polypeptide (RGPl) possibly involved in plant cell wall synthesis: purification, gene cloning and trans Golgi localization. Proc Natl Acad Sci. USA 94: 7679-7684 Dhugga KS (2001) Building the wall: genes and enzyme complexes for polysaccharide synthases. Curr Opin Plant Biol 4: 488-493 Driouich A, Faye L, Staehelin A ( 1993) The plant Golgi apparatus: a factory for complex carbohydrates and glycoproteins. Trends Biochem Sci 18: 210-214 Edwards ME, Dickson CA, Chengappa S, Sidebottom C, Gidley MJ, Reid JSG (1999) Molecular characterisation of a membrane-bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis. Plant J 19: 691-697 Faik A, Desveaux D, Maclachlan G (2000) Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions. Biochem J 347: 857-864 Franz G (1976) The dependence of membrane-bound glucan synthases on glycoprotein which can act as acceptor molecules. Applied Polymer Symposium 28: 611-621 Gibeaut DM (2000) Nucleotide sugars and glycosyltransferases for synthesis of cell wall matrix polysaccharides. Plant Physiol. Biochem. 38: 69-80 Hayashi T (1989) Xyloglucans in the primary cell wall. Annu Rev Plant Physiol and Plant Mol Biol 40: 139-168 Hirschberg CB, Robbins PW, Abeijon C (1998) Transporters of nucleotide sugars, ATP and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 67: 49-69 Krisman CR, Barengo R (1975) A precursor of glycogen biosynthesis: alpha-1,4-glucan protein. FEBS Lett 53: 117-123 Lomako J, Lomako WM, Whelanki WJ (1990) The biogenesis of glycogen: Nature of the carbohydrate in the protein primer. Biochem Int. 21: 251-260 14 Martin C, Smith AM (1995) Starch synthesis. Plant Cell 7: 971-985 Meezan E, Ananth S, Siegal S, Manzella D, Pillion D, Rohen L (1988) Glucose transfer to a glycogen-like glycoprotein from rat kidney. J Cell Biol 107: 191a Moreno S, Cardini CE, Tandecarz J S (1986) Alpha-glucan synthesis on a protein primer, uridine diphosphoglucose : protein transglucosylase 1. Eur J Biochem 157: 539-545 Moreno S, Cardini CE, Tandecarz J S (1987) Alpha-glucan synthesis on a protein primer. Eur J Biochem 162: 609-614 Nebenffihr A, Staehelin A (2001) Mobile factories: Golgi dynamics in plant cells. Trends in Plant Science 6: 160-167 Newman T, de Bruijn FJ, Green P, Keegstra K, Kende H, McIntosh L, Ohlrogge J, Raikhel N, Somerville S, Thomashow M (1994) Genes galore: a summary of methods for accessing results from large- scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol. 106: 1241-1255 Perrin R, DeRocher A, Bar-Peled M, Zeng W, Norambuena L, Orellana A, Raikhel N, and Keegstra K (1999) Xyloglucan Fucosyltransferase, an Enzyme Involved in Plant Cell Wall Biosynthesis. Science 284: 1976-1979 Read SM, Thelen M, Delmer D (1986) Identification of UDP-Glucose-binding proteins in Mung bean membranes. in Proceedings of the 4th Cell Wall Meeting (Vian, B., Reis, D., and Goldberg, R., eds.), pp. 308-311, Universite Pierre et Marie Curie Press, Paris. Reiter WD, Vanzin GF (2001) Molecular genetics of nucleotide sugar interconversion pathways in plants. Plant Mol Biol 47: 95-113 Reiter WD (1998) Arabidopsis thaliana as a model system to study synthesis, structure, and function of the plant cell wall. Plant Physiol Biochem 36: 167-176 Rodriguez IR, Whelan WJ (1985) A novel glycosyl-amino acid linkage : rabbit muscle glycogen is covalently linked to a protein via tyrosine. Biochem Biophys Res Commun 132: 829-836 Saxena IM, Brown Jr. MR, Fevre M, Geremia RA, Henrissat B (1995) Multidomain architecture of B-glycosyl transferases: implications for mechanism of action. J Bacteriol 177: 1419-1424 Saxena IM, Brown Jr. MR (1997) Identification of cellulose synthase(s) in higher plants: sequence analysis of processive B-glycosyltransferases with the common motif ‘D, D, D35Q(R,Q)XRW’. Cellulose 4: 33-49 15 Saxena IM, Brown Jr. MR (1999) Are the reversibly glycosylated polypeptides implicated in plant cell wall biosynthesis non-processive B-glycosyltransferases? Trends Plant Sci 4: 6-7 Saxena IM, Brown RM (2000) Cellulose synthases and related enzymes. Curr Opin Plant Biol 3: 523-531 Singh DG, Lomako J, Lomako WM, Whelan WJ, Meyer HE, Serwe M, Metzger JW (1995) B—Glycosylarginine: a new glucose-protein bond in a self-glucosylating protein from sweet corn. FEBS Lett 376: 61-64 Viskupic E, Cao Y, Zhang W, Cheng C, DePaoli-Roach AA, Roach PJ (1992) Rabbit skeletal muscle glycogenin. Molecular cloning and production of fully functional protein in Escherichia coli. J Biol Chem 267: 25759-25763 Wang Z (1995) Molecular characterization of a conserved Arabidopsis gene involved in cell wall synthesis. Master thesis. Michigan State University. p. 55. 16 CHAPTER II ARABIDOPSIS REVERSIBLY GLYCOSYLATED POLYPEPTIDE-l This chapter is presented exactly as published in: Ivan J. Delgado, Zhaohong Wang, Amy de Rocher, Kenneth Keegstra and Natasha V. Raikhel (1998) Cloning and Characterization of AthpI : a Reversibly Autoglycosylated Arabidopsis Protein Implicated in Cell Wall Biosynthesis. Plant Physiol 116: 1339-1350 17 ABSTRACT A reversibly glycosylated polypeptide (RGP) from pea is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding an Arabidopsis homologue, AtRGP] , and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologues in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGPl protein and RNA concentration are highest in roots and suspension cultured cells. Localization of the protein shows it to be mostly soluble, but also peripherally associated with membranes. We confirmed that AtRGP] , produced in Escherichia coli, could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for RGP in cell wall biosynthesis, although its precise role is still unknown. 18 INTRODUCTION This article is a continuation of the work started by Zhaohong Wang in her Master’s thesis under the supervision of Dr. de Rocher. The primary cell wall of dicotyledonous plants is laid down by young cells prior to the cessation of elongation and secondary wall deposition. Comprising up to 90% of the cell’s dry weight, the extracellular matrix is important for many processes including morphogenesis, growth, disease resistance, recognition, signaling, digestibility, nutrition and decay. The composition of the cell wall has been extensively described (Zablackis et al., 1995) yet, many questions remain unanswered regarding the synthesis and interaction of these components to provide cells with a functional wall (Carpita and Gibeaut, 1993, Carpita et al., 1996) Heteropolysaccharide biosynthesis can be divided into four steps: 1) chain or backbone initiation, 2) elongation, 3) side-chain addition and 4) termination and extracellular deposition (W aldron and Brett, 1985). The similarity between various polysaccharide backbones leads to the prediction that the synthesizing machinery would be conserved between each other. For example, the backbone of xyloglucan polymers, [3- l,4 glucan, can be synthesized independently of, or concurrently with, side chain addition (Campbell et al., 1988; White et al., 1993), and this polymer and the chains that make up cellulose are identical. The later addition of side chains to xyloglucan are catalyzed by specific transferases such as xylosyltransferase (Campbell et al., 1988), galactosyltransferase and fucosyltransferase (Faik et al., 1997), all localized to the Golgi compartment (Staehelin and Moore, 1995). 19 The enzymes involved in wall biosynthesis have been recalcitrant to isolation (Carpita et al., 1996; Albersheim et al., 1997). Only recently have the first genes encoding putative cellulose biosynthetic enzymes, termed celA, been isolated from cotton (Gossypium hirsutum) and rice (Oryza sativa) (Pear et al., 1996). During studies of polysaccharide synthesis in pea Golgi membranes, Dhugga et a1. (1991) identified a 41 kDa protein doublet that they suggested was involved in polysaccharide synthesis. The authors showed that this protein could be glycosylated by radiolabeled UDP-glucose, but this labeling could be reversibly competed with unlabeled UDP-glucose, UDP-xylose, and UDP-galactose, the sugars that make up xyloglucan. The 41 kDa protein was named PsRGPl (Bisum sativum Reversibly Glycosylated Polypeptide-l, Dhugga et al., 1997). Furthermore, the conditions that stimulate or inhibit Golgi localized B-glucan synthase activity (GS-I) are the same conditions that stimulate or inhibit the glycosylation of PsRGPl (Dhugga et al., 1991). In order to address the role of this protein in polysaccharide synthesis, the authors purified the polypeptides and obtained sequence from tryptic peptides (Dhugga and Ray, 1994). Antibodies raised against PsRGPl show that it is soluble and localized to the plasma membrane (Dhugga et al., 1991) and Golgi compartment (Dhugga et al., 1997). In addition to its Golgi localization, the steady state glycosylation of PsRGPl is approximately 10:7:3 (UDP- glucose: -xylose: -galactose), which is similar to the typical sugar composition of xyloglucan (1.0: 0.75: 0.25) (Dhugga et al., 1997). We are interested in studying various aspects of cell wall metabolism, including synthesis of polysaccharides and their delivery to the cell wall. Studies on pea have shown that a 41 kDa protein may be involved in cell wall polysaccharide synthesis, 20 possibly xyloglucan (Dhugga et al., 1997). Here, we report the characterization of an Arabidopsis thaliana Reversibly Glycosylated Polypeptide-1 (AtRGPl), a soluble protein that can also be found associated weakly with membrane fractions, most likely the Golgi. The reversible nature of the glycosylation of this Arabidopsis homolog by the substrates used to make polysaccharides, nucleotide sugars, suggests a possible role for AtRGP] in polysaccharide biosynthesis. 21 MATERIALS AND METHODS Plant and Cell Growth A. thaliana wild type plants (Columbia ecotype) were grown in soil at 22°C, 80% humidity and with 16 hours of light. To obtain large amounts of soil free root tissue, seeds were germinated in liquid culture medium (1 see per 100 mL of 10 g L" sucrose, 4.3 g L" Murashige and Skoog salts [Gibco BRL], 0.5 g L" Mes, 0.1 g L" myo-inositol, 1 mg L'1 thiamine-HCl, 0.5 mg L" nicotinic acid, pH 5.7). Plants grown in liquid culture and maintained under constant agitation (50-60 rpms) and light grew mostly roots. A. thaliana cell suspension cultures (CSC) were obtained from Axelos et al., 1992 and maintained by diluting a week old culture 1:5 with fresh CSC medium (3.2 g L" Gamborgs B5 [Sigma], 20 g L" sucrose, 2.5 uM 2,4 dichlorophenoxyacetic acid, 0.5 g L" Mes, pH 5.7). Cloning, Sequencing and Sequence Analysis Peptide sequences from purified pea proteins (Dhugga and Ray, 1994; Fig 2.1) were used to search the Expressed Sequence Tags database (dBEST) comprised of the GenBank, EMBL, and DDBJ EST databases. Basic BLAST was performed, using the tblastn option which compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames (Altschul et al., 1990, http://www.ncbi.nlm.nih.gov/BLASTI). Twenty-eight ESTs have been obtained so far from the original search as well as subsequent searches (Altschul et al., 1997) using the ESTs themselves to find overlapping ESTs. These ESTs can be grouped based on sequence similarity using the DNAStar software package (MegAlign module, DNAStar 22 Inc., Madison, WI). Based on sequence similarity, eight ESTs (T20512, T46245, T42672, T22507, T22943, H37657, N37306 and T44971) can be assigned to group I (ATRGPl), five to group two (T23020, T46745, AA597661, AA650721, AA650701) (AtRGP2), three (A'I'I‘S4214, R30021 and R90614) can be assigned to a third group (AtRGP3), while the rest (A'I'I‘S3942, T44394, T45672, ATI‘SO381, H76915, N65402, N65528, N65622, AA042694, AA650802, AA651536 and AA597661) were too short to be unambiguously assigned to a specific group. Furthermore, so far similar genomic sequences to AtRGPl have been identified in two Arabidopsis chromosomes. A 129 nucleotide stretch at the end of a bacteria artificial chromosome (BAC) clone (T2482- Sp6) of chromosome I (httpz/lcbil.humgen.upenn.edu/~atgc/SPP.html) is 96% identical to AtRGP], while a 113 nucleotide stretch at the end of a BAC clone (T20M8-Sp6) of chromosome 11 (http://www.tigr.org/tdb/at/atgenome/atgenome.html) is 96% identical to AtRGPl. Full length cDNA clones (T20512 and T46745) were obtained from the PRL2 Arabidopsis cDNA library (Newman et al., 1994) kept by the Arabidopsis Biological Research Center at Ohio State University. Automated sequencing of the cDNAs was performed by the Plant Biochemistry Facility at Michigan State University. BLAST searches of the GenBank database using the full length AtRGP] cDNA also detected 19 rice ESTs (RICC1486A, RICC2546A, RICR0440A, RICR1510A, RICR2980A, RIC81545A, RICS 1750A, RIC81848A, RIC82305A, RICSSO91A, C28170, C73320, C27450, C26584, C72510, C26475, C25974, C71661), four of which (C72510, C26584, RICSSO91A, C27450) can be fused to give a full length clone (Oryza sativa RGP, OsRGPl). Other RGPs identified in this search include RGPs from pea 23 (Pisum sativum RGP or PsRGPl , U31565), maize (Zea mays RGP or ZmRGPl , U89897), and cow pea (Vigna unguiculata RGP or VuRGPl , AF005279). Hydropathy analysis of AtRGPl was performed using the DNAStar package (Protean module, DNAStar Inc., Madison, WI), which uses the method by Kyte and Doolittle (1982). Protein sequence analysis of AtRGP] was performed using the DNAStar software package. In addition, the pSORT program (http://psort.nibb.ac.jp/) was used, which compares a given protein sequence against a database of known sequences that mediate protein sorting to various membranes and organelles. Isolation of RNA and Northern Analysis Total RNA from Arabidopsis flowers, leaves, roots and stems was extracted as described (Puissant and Houdebine, 1990). Total Arabidopsis RNA (30ug/lane) was separated on a gel containing 1% agarose and 6% formaldehyde. Electrophoresis was stopped when the dye front had migrated three-quarters down the gel. Sizes were estimated using the sizes of ribosomal RNAs as markers. Gels were washed for 20 minutes in 10X Standard saline citrate (SSC) and analyzed as described by Sambrook et al., 1992. Briefly, transfer was performed by capillary action onto a nylon membrane (Hybond N, Amersham) overnight. Membranes were washed in 2X SSC for 5 minutes, crosslinked in a UV-Crosslinker and stored at room temperature between filter papers. Hybridization was done overnight at 65°C in Northern hybridization buffer (5X SSC, 10X Denhardts [2 mg mL" Ficoll, 2 mg mL" polyvinylpyrrolidone, 2 mg mL" Bovine serum albumin (BSA)], 0.1 M KPO4, pH 6.8, 100 ug/mL ssDNA [salmon sperm DNA, freshly boiled], 10% Dextran, and 30% deionized forrnamide) using a randomly-primed 24 32P-labeled 1.0 kb EcoRI-Bbvl fragment of the AtRGPl cDN A at 1,000,000 dpm mL". Membranes were washed once with 2X SSC, 0.5% Sodium dodecyl sulfate (SDS), twice with 2X SSC, 0.1 % SDS, and once with 0.2X SSC, 0.1 % SDS (1X SSC is 0.15 M sodium chloride, 15 mM sodium nitrate, pH 7.0) each for 30 minutes at 65°C prior to autoradiographic exposure. Protoplast Preparation Twenty grams of cells from a 5-8 day old cell suspension culture collected on a 80 um filter were incubated with 50 mL of freshly made protoplasting solution (15.4% [w/v] sucrose, 0.32% [w/v] Gamborg’s BS minimal organic, 5 mg mL" Cellulase Onozuka R10 [Yakutt Honsha Co. LTD, Tokyo], 1.2 mg mL" Macerozyme R10 [Yakutt Honsha Co. LTD, Tokyo]) for 2 hours in a rotary shaker (80 rpm). All manipulations were done at room temperature. The cells were then poured into babcock centrifuge bottles and centrifuged for 10 minutes at 1,100 rpms in an [EC HNSII clinical centrifuge swinging bucket rotor. Floated protoplasts were collected, mixed with 20 mL 0.4 M Betaine, 3 mM MES, 10 mM CaClz, pH 5.7 and then pelleted for 5 minutes at 50 g. Protein Extraction Four milliliters of cold lysis buffer (20 mM HEPES-KOH, pH 7.0, 13.5% [w/v] sucrose, 10 mM potassium acetate, 1 mM DDT, 0.5 mM PMSF, 1 mM EDTA) was added per milliliter of packed protoplasts and passed through a 25-5/8-gauge needle at 4°C until no unbroken protoplasts could be detected under the microscope. Cell debris was pelleted by centrifugation at 500g for 5 minutes and this homogenate was called total 25 protoplast protein. Total plant protein from different tissues (flowers, leaves, roots, stems, and roots grown in liquid culture) was extracted by grinding 1-5 g of the given tissue in liquid nitrogen and mixing with 5 mL of lysis buffer at 4°C. For all plant tissue samples, cell debris was pelleted by centrifugation at 500g for 5 minutes. This homogenate was called total tissue protein, where “tissue” specifies the tissue used. Overexpression of a fusion protein in E. Coli and Antibody Preparation The 1.4 kb EcoRI-NotI fragment of AthpI (GenBank accession number AF013627), encoding all but the first nine amino acids from the 5’ end of the cDNA, was cloned into the EcoRI-Notl sites of pGEX-SX-2 (Pharmacia Biotech) to generate an N - terminal in-frame fusion with the 26 kDa domain of Glutathione S-transferase (GST). This fusion was overexpressed in Escherichia coli (strain DH50t) by growing cells in LB media at 37°C to an OD600 of 0.7-0.8, then adding isopropylthio-B-D-galactosidase (1171’ G) to a final concentration of 0.2 mM and finally incubating the culture at 28°C for 4 hours. The soluble GST fusion protein was purified as described (Bar-Peled and Raikhel, 1996). Five hundred microliters (0.4-0.5 mg) of eluted GST-AtRGP] fusion protein was emulsified by sonication with an equal amount of TiterMax adjuvant (Cthx Corp., Norcross, GA) and injected into rabbits (New Zealand White). Rabbits were injected two more times with an equal amount of GST-AtRGP] protein to boost the immune response over a two month period. Bleeds were taken a month after the first injection and every other week thereafter. Bleeds were treated as described (Harlow and Lane, 1988). 26 Timed Studies of RNA and Protein Levels A 0.5 L Arabidopsis cell suspension culture was started by adding 50 mL of a 7- day-old culture to 450 mL of fresh CSC media. Fifteen milliliter aliquots were taken at 24 hour intervals over a 12 day span. Each fraction was centrifuged at 50g for 5 minutes, the supernatant removed and the weight of the packed cells measured. Equal amounts of protein (SOug/lane) were separated by Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli et al., 1970) and AtRGPl presence was determined by immunoblotting. Equal amounts of RNA (30ug/lane) were separated in a 1% agarose, 2% formaldehyde gel and the level of Athpl RNA was determined by Northern analysis. Reaction with UDP-sugars One hundred to 250 ug of total Arabidopsis, pea, tobacco or maize total protein form roots was labeled as described (Dhugga et a1 , 1991). Briefly, 0.1uCi of UDP-D-[U- l"C]-glucose (specific activity 263 uCi umol" [ICN Radiochemicals, Irvine, Ca]), or 0.5uCi of UDP-D-[6—3H]-galactose (specific activity 10.6 mCi timol'l [ICN Radiochemicals, Irvine, Ca]), was added to the protein sample in a volume of 50-150 uL of lysis buffer containing 3 mM MgC12. The reactions were stopped after a 10 minute incubation at room temperature by adding 10-30 uL of SDS-PAGE loading buffer (120 mM Tris pH 6.8, 200 mM DTT, 4% SDS, 0.02% Bromophenol Blue, 20% Glycerol). The protein samples were separated by SDS-PAGE and treated as described below. Since the UDP-Glc and UDP-Gal reactions gave the same results, only the UDP-Glc reactions are presented. 27 Reactions with purified GST-AtRGPl protein were carried out using 2 ug of protein in the absence of M g”. The reactions were mixed by inversion and incubated statically at room temperature for 10 minutes. For displacement reactions, after a 10 minute incubation with 20 pmol (0.2 ptCi) of UDP-[3H]-glucose (specific activity 10.9 Ci mmol" [Sigma, St, Louis, MO)] , UDP, Glc, UDP-Glc, UDP-Xyl, UDP-Gal, or UDP- Man was added to a final concentration of 3 mM and the reactions continued for 10 more minutes. To stop the reactions SDS-PAGE loading buffer (120 mM Tris pH 6.8, 200 mM DTT, 4% SDS, 0.02% Bromophenol Blue, 20% Glycerol) was added. The protein samples were separated by SDS-PAGE and treated as described below. Immunochemical studies Immunoprecipitation Immunoprecipitations were done as described (Harlow and Lane, 1988). Anti- AtRGPl polyclonal antibodies were crosslinked to protein-A Sepharose 6MB (Pharmacia Biotech) and mixed with total protein from roots grown in liquid culture. After washing 3X with Phosphate buffered saline (PBS) and 1X with 10 mM potassium phosphate buffer pH 7.0, proteins bound to the antibodies were eluted with 100 mM Glycine-HCl pH 2.5. Protoplasts labeled with 3sS-methionine were incubated overnight in a rotary shaker at 80 rpm and total protein was prepared as described and immunoprecipitated. 28 SDS-PAGE and Immunoblotting Protein concentration was determined as described (Bradford, 1976) using BSA as standard. Protein molecular weight standards were purchased from BioRad (Low- Range marker and Broad-Range marker, catalog #161-0304 and 161-0317 respectively). All proteins samples were separated on 12.5% SDS-PAGE gels using 1X Running Buffer (250 mM Tris, 1M Glycine, 0.5% SDS). After separation, gels containing UDP-D-[U-"C]-glucose and UDP-D-[3H]- galactose labeled reactions were stained with 0.1% Commassie Blue overnight, destained with 5 volumes of 30% methanol, 10% acetic acid, washed with distilled water 2 times, incubated with Fluoro-Hance (Research Products International Corp., Illinois) for 15 minutes and dried for 2 hours in a BioRad Gel Drier (Model 583). The dried gels were exposed to film for 7-10 days at -80°C prior to film development. For immunoblotting, gels containing various protein samples (30-50ug/lane) were washed with 5 volumes of 1X Transfer Buffer (63 mM Trizma Base, 250 mM glycine, 0.13% SDS, 20% methanol) and transferred in the same buffer to a 0.45 um nitrocellulose membrane (Hybond N, Amersham) using a semi-dry apparatus at 2 mA cm‘2 for 2 hours. Filters were stained with 1X Ponceau-S (2% Ponceau-S, 30% Trichloroacetic acid, 30% Sulfosalicylic acid) and blocked overnight in 10% (w/v) dry milk powder in 1X PBS, 0.1% polyoxyethylenesorbitan monolaurate (Tween-20) (PBST). AtRGPl polyclonal antibodies were used at 1:1000 dilution. Anti-RD28 (plasma membrane, PM, marker) and anti-TIP (tonoplast marker) sera at 1:500 dilution and anti- BiP (ER lumen protein) at 1:1000 dilution were a gift from Maarten Chrispeels (University of California, San Diego). Anti-AtELP (partially Golgi localized protein, 29 Ahmed et al., 1997) and anti-AtPEP12 (post-Golgi compartment protein, Conceicao et al., 1997) polyclonal antibodies were used at 1:500 dilution. ARA4 monoclonal antibody (a Golgi membrane localized and soluble protein, Ueda et al., 1996) was used at 1:500 dilution. After primary antibody incubation for 1-2 hours in PBST, membranes were washed three times with PBST and then reacted with a 1:2500 dilution of alkaline- phosphatase conjugated goat anti-rabbit secondary antibody (Kirkegaard and Perry Laboratories Inc., Gaitherburg) for 1-2 hours except for the ARA4 antibody, which required alkaline-phosphatase conjugated goat anti-mouse secondary antibody. Finally, filters were washed 2X with PBST, 2X with PBS and 1X with Alkaline buffer (100 mM Tris, 100 mM N aCl, 5 mM MgC12, pH 9.5) prior to detection of the immune complexes using the substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 150 ug mL", final concentration) and nitroblue tetrazolium (NBT, 300 ug mL", final concentration) in Alkaline buffer. Localization Differential Centnfugation Total protoplast protein was centrifuged for 20 minutes at 1,000g (4°C). The pellet was washed with lysis buffer, centrifuged again and termed pl. The supernatant (sl) was centrifuged at 5,000g for 30 minutes. The pellet was washed with lysis buffer, centrifuged again and termed p5. The supernatant (55) was treated in the same way for sequential differential centrifugations at 15,000g, 25,000g, 50,000g and 100,000g, with the last two centrifugations carried out for 40 minutes. All pellets (p1, p5, p15, p25, p50, and p100) were resuspended in lysis buffer and the protein quantified. 30 Membrane association Total microsomes were prepared by centrifugation of total protoplast protein at 150,000g for 1 h and washing the pellet with lysis buffer. Pellets were resuspended with lysis buffer (total protein), or lysis buffer containing 0.1%, 0.5% or 1.0% Triton X-100, 0.5 M or 2 M urea, 0.1 M sodium carbonate, 0.1 M, 0.5 M or 1.0 M NaCl, or 0.1 M potassium phosphate pH 7.0, for 1 h on ice, centrifuged again at 150,000g for 1 h. The resulting pellets were washed with lysis buffer, resuspended in loading buffer and separated by SDS-PAGE prior to analysis by immunoblot using anti-AtRGP] antibodies. Sucrose Density Gradient Centrifugation Total protoplast protein was centrifuged for 10 minutes at 1,000g (4°C) and 6 mL of the supernatant (81) was loaded on top of a 16-55% equilibrium density sucrose gradient modified from Gibeaut and Carpita (1994). In order to prepare the gradient, stock solutions of 55%, 40%, 33.5%, 26.5% and 16% (w/v) sucrose buffered in 10 mM HEPES-KOH (pH 7.0), 10 mM KOAc, 2 mM EDTA were prepared. Gradients were prepared by the sequential addition of 2.7 mL of 55% sucrose solution, 8.8 mL of 40% sucrose solution, 7.0 mL of 33.5% sucrose solution, 6.0 mL of 26.5% sucrose solution, and 4.5 mL of 16% sucrose solution to 35 mL polyallomer tubes (Beckman, catalog # 326823). After each one of these solutions were carefully layered on top of each other, they were centrifuged for 18-20 hours at 150,000g in a Beckman SW28 rotor at 4°C. This resulted in an equilibrium density gradient similar to one obtained using a gradient maker. Equal volume fractions were collected, aliquots of the fractions (SO-100 uL) were separated by SDS-PAGE and the separation proteins analyzed by immunoblotting. 31 RESULTS Identification of an Arabidopsis cDNA Encoding an AtRGPl Protein Sequence from three purified tryptic peptides from the pea RGP protein doublet were presented by Dhugga and Ray (1994) and used to search the dBEST database. A total of 28 Arabidopsis ESTs have been obtained so far from the original and subsequent searches. Based on sequence similarity, these ESTs can be assigned into at least three different groups, denoted AthpI (AF013627), AthpZ (AF013628) and Athp3 (AF034255) for Arabidopsis thaliana Reversibly glycosylated polypeptide-1, 2, and 3 respectively. These cDNAs share between 93 and 99% sequence identity at the amino acid level. Because the similarity is spread through the whole sequence only one cDNA, AthpI, was used for further analysis (Fig. 2.1 A). We used the full length cDNA of AthpI to search the dBEST databases and obtained, in addition to the 28 Arabidopsis cDNAs, 19 rice cDNAs, a full length maize cDNA, and a full length cow pea cDNA that shared significant sequence similarity with AthpI . None of these sequences belonged to a gene with an assigned function at the time of the BLAST search. AtRGPl is 84%, 87%, 83%, and 85% identical to the pea, cow pea, maize, and rice proteins, PsRGPl, VuRGP, ZmRGP, and OsRGP respectively. A full length cDNA clone encoding the rice RGP has not been isolated yet, but available ESTs can be combined to make up a full length clone (OsRGPl). Searches of non-plant databases gave mixed results. While similar sequences were found in both the yeast genome database (http://speedy.mips.biochem.mpg.de/mips/yeast/index.htmlx) and the Synechocystis database (http://www.kazusa.or.jp/cyano/cyano.htrnl), none shared significant similarity to AtRGP] (P>0.01), all the sequence alignments were very short 32 Figure 2.1. Predicted protein sequence of AtRGP] (GenBank accession no. AF013627) and comparison with other RGP proteins. A, Amino acid sequence alignment of AtRGP], PsRGPl (U31565), VuRGP (AF005279), ZmRGP (U89897), and OsRGP using the MegAlign module of DNAStar. The black regions represent amino acid differences between AtRGPl and the other RGPs. The overline highlights a putative N - myristoylation site. The shaded box represents the predicted UDP-Glc-binding domain U1 from cotton CelA. The open box is the site of glycosylation as determined in sweet corn. Underlined are the two pea tryptic peptides used to search the EST database. B, Alignment of UDP-Glc-binding domains from various organisms, as determined by Delmer and Amor (1995), with the predicted binding domain of RGP. 33 Figure 2.1 A (top) s—lt-l Ht—l HH Hui-l t—lr-l 0.0.0.0.0. 0.0.0.0.0. 0.0.0.0.CL 0.0.CLQO_ 0.0.0.QCL 22222 32322 22222 22223 33222 446953120 “MES!” “MES” 44015317) 44005300 'IflcNI‘P-D "ICLNL‘J-D {ENDS-D d0.N:>O {lNfi-D magma macaw mEmmm >>>>> }}}}} «mucosa: ._.I._J—I_I_r QCLCLCLCL 131515151: >-->->—>- yaya; HHHHH mango magma “can: 222': mmxmm xxxxx }}}}} HFHHE U-lI-IJUJ LU 22222 did-(ICE LLI-l-l-I-u_|-L 22222 _l_1_1 _J HHHHH }}}}3} DDDDD magma: LLu—LI. 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DDDDD 0.0.0.0.11 c-l‘fii't-I H e-Id't-ILns-I Hd‘t—l H H‘d‘HLfit-l Nr-INLDN \anKDNKQ Hr-le-ls-lt-l fi'l’fid‘U‘ld’ COINWd'W HHHWH HHHHH M Figure 2.1 A (bottom) HH HH HH HH HH °'°"L%% %%%%% %%%%% %%%%% %%%%% #wszm umszm uwszw umszm umszm dam>o daw>o dawbo o dawbo gamma mmmmm mmmm 00000 gamma 444 uuuuu 2222: mm Damon mmMmm EWHH; uuuuu dqddd >%}*> £2222 2232M uUuUu __,|_1_l_.|_l mun/1mm E¥¥¥¥ 00000 2212: 00000 mmmmmliygga h>§> >y>rr HHHHH P 222:: >r*>* H qqddd manna uuu manna 04444 M 05000 uUDDD 2222 HI-IHHH |_.|—l—}—1— qr; _.l_|_J_l_l 3332234 _l_1 mmama }}>>> Ekhhh aa;:= mama“ momma dd {4 hhhhh 0:900 4 444 m m: >>>>h mmumm mo um|ooooo £2222 ddddd 44440 mmmmm {dddd 44444 2:22: mmmmm 00900 22222 DDDDD gamma ddmdi £2222 LJULJUU HHHHH _l._l-‘._l._l 00000 HHHHH HHHHH xxxxx uuuuu >33}; omamo Hfirz> £5232 22222 mmmmm mmmmm manna HHfiflE ooooo *rrrr ummmm uuuuu yggga manna 44404 gaggg mmmmu QQDDD hlkhh uuuuu HHHHH HHH H 151515015 {-I-ct-ct-ct 151315015 It—l—E ¥¥x¥¥ aaaaa xxxxx momma manna £2222 rrrrr 44444 HHHHE DDDDD mmmmm 22222 hhhhh DDDDD xxxxx mmmmm fiafiaz >¥¥¥¥ xxxxu 2223; bfi-y-h} Eflafiflfl .—l.—I.—l—.I_l }}}}3 «(did macaw zzzzz H0000 cacao HHHHH >>333 xxflxw a Hmd Heama Héama HiHMH @0506 imfiaé mnmfiw NHNWN NHNHN NNNNN NNNNN mmm~m fl Figure 2.1 B x—l‘sl' 0000 NH ll OOHNOOOO I\ mmgommt—lr-INNLD N I l l I IDDt—Ikmmmm It—Irlmml s—ls—ls—lr-lv-l<[<[ IIKOI CLQQQQr—Ir—Im H— m UUUUUCDGJ\ NOUOH--2>- I_I-IIIEI_ >wllllll - - —mmE2+—2E ¥¥¥¥¥>>>>LL—l—I —El _Ioowoo>w _IMOI _lmamm —E_ (10-20 amino acids), and none of the predicted open reading frames encoded a 41 kDa protein. These findings are further supported by the immunoscreening of various species described below. Hydropathy profile analysis (Kyte and Doolittle, 1982) predicts that AtRGP] is a hydrophilic protein (data not shown). Visual and computer aided (pSORT, http://psort.nibb.ac.jp/) analyses of the protein sequence predict that the protein lacks a signal sequence that might direct AtRGP] to a membrane compartment within the cell. The same computer program predicts the existence of an N -myristoylation site in AtRGPl (Fig. 2.1 A). N-myristoylation has been shown to aid in the association of otherwise soluble proteins to the cytosolic surface of membranes. The sequence of AtRGPl was compared with a reported predicted binding domains for UDP-glucose. The determination of these UDP-glucose binding domains is based on the observation that there is a substantial sequence conservation between bacterial catalytic subunit and other enzymes that catalyze the polymerization of B- glycosyl residues from either UDP-glucose or UDP-N-acetylglucosamine (Saxena et al., 1994), as well as UDP-glucose binding proteins (Delmer and Amor, 1995; Pear et al., 1996). AtRGPl is 40% identical and 53% similar to the predicted UDP-glucose binding domain, U1, of cotton CelA. (Delmer and Amor, 1995, Pear et al., 1996; Saxena and Brown Jr., 1997). Furthermore, AtRGP] contained the critical Asp residue (Fig. 2.1 B). In contrast, sequence identity between the deduced amino acid sequence of the cotton CelA full length clone and AtRGP] is very low, around 15%. Singh et al. (1995) have demonstrated that the glucose, from UDP-glucose, was attached to an arginine residue of a maize protein with high sequence similarity to 37 AtRGPl. The tryptic peptide containing the glucosylated arginine is 93% identical to a region of AtRGPl (Fig. 2.1 A, open box). Dhugga et a1. (1997) noted this same similarity to PsRGPl and postulated that Arg-158 is the location of sugar addition in the pea protein. Similarly, we postulate that Arg-158 is the location of glucose addition in the Arabidopsis protein. Distribution of RGP] Transcript and Protein The isolation of RGP proteins in both monocots and dicots suggests a general function for this protein. To confirm the presence of proteins similar to RGP, as evidenced by database searches, we used AtRGPl antibodies in immunoblots against —' protein from various organisms. Immunoreactive polypeptides of 41 kDa were detected in Arabidopsis, pea, tobacco and maize (Fig. 2.2, lanes 1-4). Although pea RGP] has been referred to as a polypeptide of about 40 kDa (Dhugga et al.,1991; Dhugga et al., 1997), it is clear from the cloned cDNA that PsRGPl has a predicted size of 41 kDa. Since all RGPs so far identified encode proteins of approximately the same size, we will refer to them as 41 kDa proteins. A 64 kDa protein was also detected by AtRGP] antibodies in immunoblots of Arabidopsis, pea, tobacco, maize and Synechocystis (Fig. 2.2, lanes 1—5). The relationship of this protein to the 41 kDa protein is not yet clear. Although four hypothetical proteins were found in the Synechocystis database (5110524, 5110501, 5110602 and 8111080) that shared limited sequence similarity within separate regions in AtRGPl , none were of the size detected by immunobloting. AtRGP] antibodies did not detect any proteins in either yeast or mammalian extracts (Fig. 2.2, lanes 6 and 7) as expected from database searches. 38 Figure 2.2 1 2 3 4 5 6 7 ~53 litt‘itt" M“ y it ~ Figure 2.2. Homologs of AtRGP] from other species. Total protein (50 fig) from Arabidopsis (lane 1), pea (lane 2), tobacco (lane 3), maize (lane 4), cyanobacteria (lane 5), yeast (lane 6), and HeLa cells (lane 7) was analyzed by immunoblot using anti- AtRGPl antibodies. All plant-derived protein extracts were obtained from root tissue. Molecular mass is indicated in kilodaltons. 39 The AthpI transcript level in different tissues of Arabidopsis was determined by Northern analysis (Fig. 2.3 A). One band of ~1.4 kb was detected in all tissues tested. Although AthpI RNA levels were relatively uniform, the highest level was detected in suspension cultured cells. In whole plants, roots had the highest level and stems had the lowest level of AthpI RNA (Fig. 2.3 A). Immunoblotting using anti-AtRGPl antibodies was used to determine protein levels in the same tissues. Levels of AtRGPl protein mirrored RNA levels, again being highest in suspension cultured cells and roots, and F lowest in leaves (Fig. 2.3 B lanes 8, 5, and 4, respectively). Consistent with these results, immunoblots of similar tissues from the various plant species tested (data not shown) showed that the highest level of RGPl protein was found in roots (Fig. 2.2, lanes 2-4). Although RNA levels are similar in leaves and stems, more AtRGP] protein can be found in stems than in leaves. AtRGP] was only detected as a doublet in tissues where it was highly expressed (Fig. 2.3 B, lane 7; Fig. 2.2, lane 1), but it was clearly not the same kind of doublet as that seen for pea (Fig. 2.2 lane 2). In addition, a 64 kDa protein was detected by immunoblot analysis of total protein in nearly all tissues tested (Fig. 2.3 B, lanes 4-8), as well as in all the other plants species tested (Fig. 2.2). The nature of this protein is not understood and it does not seem to be well recognized by AtRGP] antibodies since immunoprecipitation studies recognize AtRGPl almost exclusively (Fig. 2.3 B, lanes 1 and 2). Since AtRGP] RNA and the corresponding protein were highest in suspension cells, its behavior was studied by starting cell suspension cultures and then following them through a time course. As seen in Figure 2.3 C, cells were grown for a period of 12 days and aliquots taken at 24 hour intervals to determine the amount of AtRGP] RNA 40 Figure 2.3. AtRGPl RNA and protein are highest in suspension-cultured cells and roots from whole plants. A, RNA levels in different tissues. Total RNA (30 jig) from A. thaliana flowers (lane 1), leaves (lane 2), roots (lane 3), stems (lane 4), and suspension- cultured cells (lane 5) was separated in a 1% agarose and 6% formaldehyde gel, transferred onto a nylon membrane, and hybridized with a random-primed, 32P-labeled, 1.0—kb EcoRI-Bva fragment of the AtRGP] cDNA. The single band obtained is of the expected size. Molecular mass is indicated in kilodaltons. B, Protein levels in different tissues. Immunoprecipitations using total protein from [3SS]Met-labeled protoplasts (lane 1) and unlabeled protoplasts (lane 2) were performed. The former was analyzed by SDS- PAGE and autoradiography and the latter was analyzed by immunoblot. Total protein (50 a g) from A. thaliana flowers (lane 3), leaves (lane 4), roots (lane 5), stems (lane 6), root liquid culture (lane 7), and cell-suspension cultures (lane 8) was analyzed by immunoblotting using anti-AtRGP] antibodies. Molecular mass is indicated in kilodaltons. C, AtRGP] RNA and protein levels during the growth cycle of suspension cells. A cell-suspension culture was started by diluting a l-week-old culture 10 times with fresh medium. Samples representing 1/40 of the original volume were collected at 1- d intervals, and protein and RNA were extracted from each sample. Top, Total AtRGPl protein from each sample was analyzed by immunoblot; bottom, total AtRGPl RNA was determined as described in Figure 4A. 41 Figure 2.3 A 1 2 3 4 5 1.8) 1.5) .. 1.3) 1.1) I“ 97.4) 66.2) _w 45) w C-n—n—I-I-‘l" 31) C 123456739101112 AtRGPl) ” w. w-ymr» u,.._‘..‘ . - 123456789101112 AtRGP] RNA > . *~ 42 and corresponding protein. Although there was a clear increase in RNA accumulation during the first 5 days, peaking at day 4 and then almost undetectable by day 8, the corresponding protein level was constant through out the whole experiment. AtRGPl is Soluble and Membrane Associated One strategy for understanding the function of AtRGPl is to localize this protein. h Hydropathy plots predict that AtRGPl is a soluble protein (not shown) although analysis of the sequence shows that it contains a putative N-myristoylation site that may be involved in its association with membranes (Fig. 2.1 A). To determine if AtRGPl is i soluble or membrane associated, fractionation studies were carried out. Protoplasts derived from CSC were lyzed and subjected to sequential differential centrifugation. The resulting pellets were analyzed by immunoblot using AtRGPl antibodies (Fig. 2.4 A). Several intrinsic membrane (AtPEP12, RD28, and AtELP), membrane associated and soluble (ARA4), and soluble (ER lumen, BiP) proteins were analyzed in the same fractions and used as a control. AtRGPl was enriched in fractions composed of soluble proteins ($100), but was also detected in membrane containing fractions (pS-plOO, Fig. 2.4 A). Our results indicate that AtRGP] is both soluble ($100) and membrane associated. The presence of AtRGP] in both soluble and membrane bound fractions is interesting and may provide hints regarding its role in the cell. Since AtRGP] is predicted to be a soluble protein, we were specifically interested in studying the nature of its association with membranes. Equal amounts of total microsomes were prepared (p150) and pellets resuspended under various conditions. After centrifugation of treated membranes at 150,000g, pellets were resuspended in lysis buffer to analyze protein left 43 Figure 2.4. AtRGPl is soluble and membrane associated. A, Total protoplast protein from suspension-cultured cell protoplasts was centrifuged at 1,000, 5,000, 10,000, 15,000, 25,000, 50,000, and 100,000g. The resultant pellets were resuspended in lysis buffer and make up fractions p1, p5, p10, p15, p25, p50, and p100, respectively. 8100 denotes the supernatant after the 100,000g centrifugation and represents total soluble proteins. Equal volumes of protein were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblot. Various soluble proteins (BiP), integral membrane proteins (AtPEP12p, RD28, and AtELP), and peripheral membrane proteins (ARA4) were compared with the membrane association of AtRGPl. The fraction of total protein (T) present in each pellet is shown. B, AtRGPl is a peripheral membrane protein. Total microsomes were prepared by centrifuging total protein at 150,000g for l h and washing the pellet with lysis buffer. Pellets were resuspended with various buffers for 1 h on ice and centrifuged again at 150,000g for l h, and the pellets were analyzed by immunoblot using anti-AtRGPl antibodies. Microsomes were resuspended with lysis buffer (lane 1, total protein) or lysis buffer containing 0.1, 0, or 1.0% Triton X-100 (lanes 2, 3, and 4, respectively), 0.5 or 2 m urea (lanes 5 and 6, respectively), 0.1 m sodium carbonate (lane 7), 0.1, 0.5, or 1.0 m NaCl (lanes 8, 9, and 10, respectively), or 0.1 m potassium phosphate buffer, pH 7.0, alone (lane 11). 44 Figure 2.4 A & 9‘ 4? $9 32‘? 3’“ &® 9°“ AtRGP] > M.-. . _...... ARA4 > .-...... BIP > AtPEPlz > ‘ - w H .. RD28 > AtELP > % of T protein 100 29 15 14 9 9 6 l7 B T | Triton, % lurea, M [CO NaCl, M |KPO4 0.1 0.5 1.0 0.5 2.0 0.1 0.1 0.5 1.0 0.1 1 2 3 4 5 6 7 8 9 10 11 45 associated with membranes by immunoblot. Total protein (Fig. 2.4 B, lane 1) and microsomes resuspended in lysis buffer containing Triton X-100, urea, sodium carbonate, NaCl or potassium phosphate buffer alone were pelleted and resuspended in lysis buffer for immunoblot analysis (Fig. 2.4 B). Nonionic detergents, high levels of urea (2 M) and alkaline treatment elute AtRGPl from the membrane (Fig. 2.4 B, lanes 2-4, 6 and 7) while low levels of urea (0.5 M), salt treatment and hypotonic wash with a neutral buffer left some AtRGPl associated with the membrane (Fig. 2.4 B, lanes 5, 8-11). This T suggests AtRGPl is a peripheral membrane protein. As a further attempt to determine the localization of AtRGPl , a post-nuclear supernatant (5]) fraction of CSC derived protoplast lysates was fractionated on sucrose gradients and the individual fractions analyzed by immunoblot (Fig. 2.5). AtRGPl peaked in fraction 10, corresponding to approximately 30% sucrose. BiP, a soluble protein localized to the ER lumen, peaked in fraction 9 corresponding to approximately 28% sucrose (Fig. 2.5 A). Due to the extreme difficulty in separating ER from Golgi membranes, a separate gradient was performed in the presence of Mg”. Shifts towards denser fractions in the presence of Mg+2 is characteristic of ER proteins (Lord, 1987; Bar- Peled and Raikhel, 1997), and as expected, BiP shifted to denser fractions. The fact that AtRGPl did not shift towards denser fractions in the presence of Mg+2 as BiP did (data not shown), suggests that AtRGPl is not an ER protein. The plasma membrane marker, RD28, was detected at the bottom of the gradient, in fractions that lacked AtRGPl (Fig. 2.5 B). This results suggest that AtRGP] is not localized the plasma membrane. AtPEP12, a recently identified protein believed to reside in a post-Golgi, pre-vacuolar compartment, is present in membranes located at the bottom 46 Figure 2.5. Membrane localization of AtRGP]. Equilibrium-density gradients were used to fractionate A. thaliana total membrane preparations from suspension-cultured cell protoplasts. One-twentieth (100 pL) of equal-volume fractions was separated by SDS- PAGE and transferred to nitrocellulose membranes. A, Fractionation of AtRGPl was determined by immunoblot analysis and compared with that of the membrane marker BiP (ER). Because of the presence of AtRGPl and BiP in the same fractions, similar gradients were analyzed in the presence of Mg+2 and showed that BiP shifted to a denser part of the gradient, but AtRGP] did not (data not shown). B, AtRGPl fractionation was also compared with that of other known membrane markers, AtELP, AtPEP12p, and R28. 47 Figure 2.5 slllvt. .Lla llrtll.llt I; 323an E23& 3.826 \ I 3 mg S 5» mV ow hm cm mm Vm an R mm mm om vm mm m H om M: S a E vacuosmg Anna—M A NHmHmu< A LAM—«AV A “maxi. Am:— A umwfiu< m < 48 of the gradient. Finally, the distribution of AtRGP] partially coincides with that of AtELP (Fig. 2.5 B). Results from our lab have shown that AtELP fractionates mainly with two fractions, one of which coincides with that of Golgi localized fucosyl transferase activity (Ahmed et al., 1997), and preliminary results by electron microscopy partially localize AtELP to the Golgi (Ahmed SU, and Raikhel NV, unpublished data). Our results support the Golgi localization of RGP (Dhugga et al., 1997). Glycosylation of AtRGPl The pea RGPl was originally identified because it was glycosylated by UDP-D- [U-“C]-glucose (Dhugga and Ray, 1991) in a manner that was reversed by using UDP- glucose, UDP-xylose and UDP-galactose. Similar experiments revealed a single labeled protein when total Arabidopsis soluble protein was analyzed (Fig. 2.6 A, lane 1). Reactions using total pea membrane protein yielded the expected PsRGPl doublet (Fig. 2.6 A, lane 2). Only pea seemed to have a clear doublet since tobacco and maize only showed a single protein labeled with UDP-glucose (Fig. 2.6, lanes 3 and 4, respectively), while total protein from Cyanobacteria or purified GST protein showed no labeling (Fig. 2.6, lanes 5 and 6, respectively). In addition to the glycosylation of AtRGP] , a reaction between radiolabeled UDP-Glc and a 30 kDa protein was also detected. Labeling of a 30 kDa protein was detected in maize, which contains a large proportion of RGP in total protein extracts from roots, as well as fraction 10 of the Arabidopsis sucrose gradient (Fig. 2.6 A, lanes 4 and 8, respectively). The presence of this 30 kDa protein seems to coincide with fractions containing high concentrations of RGP, since fraction 10 is where AtRGPl is found at 49 the highest concentration (Fig. 2.5 A and B). The nature of this 30 kDa polypeptide is not known. Glycosylation of AtRGPl using UDP-D-[U-“C]-glucose implies that it corresponds to the A. thaliana homolog of PsRGPl. In pea the association between PsRGPl and the glucose from UDP-glucose was shown to be through a glycosydic bond resistant to boiling in 5% SDS (Dhugga and Ray, 1991). AtRGP] seems to behave in a similar way since all the samples shown in Figure 6 A and B were boiled in loading buffer containing 3% SDS prior to analysis. To test further the nature of this association in Arabidopsis, a purified GST fusion of AtRGPl was prepared. The 68 kDa GST fusion, GST-AtRGPlp, was incubated with UDP-D-['4C]-glucose or UDP-D-[3H]-glucose in the l absence of metal ions and shown to be glycosylated by UDP-glucose (Fig. 2.6 A, lane 7; and Fig. 2.6 B, lane 1, respectively). The reaction with UDP D-[3H]-glucose was tested against UDP, Glc, UDP-Glc, UDP-Xyl, UDP-Gal, or UDP-Man. UDP, UDP-Glc, UDP- Xyl and UDP-Gal were able to displace the label from GST-AtRGP] (Fig. 2.6 B, lanes 2, 4, 5 and 6 respectively), while Glc and UDP-Man did not (Fig. 2.6 B, lanes 3 and 7 respectively). These results demonstrate that AtRGPl is autoglycosylated as shown for PsRGPl (Dhugga et al., 1997) independently of secondary factors. 50 Figure 2.6. AtRGP] is reversibly autoglycosylated. A, Total protein (100-250 F g) from Arabidopsis (lane 1), pea (lane 2), tobacco (lane 3), maize (lane 4), and cyanobacteria (lane 5); 5 pg of purified GST (lane 6) and GST-AtRGPlp (lane 7) and 50 pg of the Arabidopsis Sue-gradient fraction 10 (lane 8) were incubated with 0.1 yCi of UDP- [14C]Glc before analysis by SDS-PAGE and autoradiography. Molecular mass is indicated in kilodaltons. B, Displacement of bound radiolabel from GST-AtRGP]. Two micrograms of GST-AtRGP] was incubated wrth UDP-[3H]Glc for 10 min. Various substrates were then added to a final concentration of 3 mm and the incubations were continued for 10 min more. Lane 1, No substrate added; lane 2, UDP; lane 3, Glc; lane 4, UDP-Glc, lane 5, UDP-Xyl; lane 6, UDP-Gal; and lane 7, UDP-Man. After the second incubation, reactions were stopped and analyzed by SDS-PAGE and autoradiography. 51 Figure 2.6 A 1 2 97.4 > 66.2 > 45 > 31 > B 1 GST-AtRGPl > an 52 DISCUSSION We have isolated and characterized a cDN A clone that encodes the Arabidopsis homologue of the PsRGPl doublet. The PsRGPl doublet has been localized to the Golgi compartment and shown to be reversibly glycosylated by UDP-glucose, this later fact giving it its name, PsRGPl (Pisum sativum Reversibly _G_lycosylated Eolypeptide-l). The existence of RGP in both dicots (Dhugga et al., 1991) and monocots (Singh et al., 1995), but not in other non-plant systems suggests a plant specific function. Database searches using AtRGP] yielded 28 Arabidopsis, 19 rice, a full length maize cDNA, and a full length cow pea cDNA. Computer assisted sequence comparisons among the Arabidopsis ESTs lead to their classification into at least three distinct groups. Furthermore, portions of AtRGP have been sequenced in both chromosomes I and II of Arabidopsis, suggesting that AtRGP] is part of a small multi-gene family. Our results differ from those in pea (Dhugga et al., 1997), where it has been suggested that PsRGPI is a single copy gene. Elucidating the functional role of AtRGPl-related Arabidopsis proteins, such as AtRGP2, will be pursued because they share a high degree of identity, yet include subtle differences such as a reduction of sequence identity at the N -terminus which leads to the absence of a putative N -myristoylation site in AtRGP2 (not shown). The existence of at least two different AtRGP cDNAs could explain the presence of a doublet in Arabidopsis since AtRGP2 is seven amino acids longer than AtRGPl, roughly equal to the small difference in size between the two peptides detected by immunoblot analysis (Fig. 3B lane 7). The larger difference between the two pea polypeptides could be a species specific case since no other plant examined thus far showed this kind of doublet. 53 Immunoblot studies using anti-AtRGP] antibodies detected not only AtRGP] , but also a 64 kDa protein. The nature of this 64 kDa protein is not understood. It could be a modified version of AtRGPl , a related protein or a completely unrelated protein cross- reacting with antibodies raised against AtRGPl. The use of known bacterial UDP-sugar binding motifs to determine the corresponding motifs in plants (Pear et al., 1996) is a strong indication that these motifs are conserved in different polysaccharide synthesizing enzymes. Sequence comparisons with previously defined UDP-glucose binding sites (Delmer and Amor, 1995; Pear et al., 1996; Saxena and Brown Jr., 1997) showed that AtRGPl contains a similar motif which 5 ‘5 may be involved in its binding to UDP-sugars. This motif shares 40% sequence identity with the cotton CelA UDP-glucose binding domain, U1. Singh et al. (1995) isolated a sweet corn protein based on its glycosylation with UDP-glucose, which they named amylogenin. The protein was digested and the sequence from eight tryptic peptides accounted for about 40% of the protein. Dhugga et al. (1997) suggested that this protein be named ZmRGP] since the tryptic peptide sequences of this protein are nearly identical to PsRGPl and AtRGP]. Interestingly a single amino acid, Arg-158, of this protein was found to be labeled with UDP-D-[14C]-glucose, in accord with the single glycosylation of PsRGPl (Dhugga et al., 1991). The U1 motif (Pear et al., 1996), a UDP-glucose binding domain predicted through its sequence conservation with the catalytic subunits of enzymes involved in the polymerization of B-glucosyl residues as well as UDP-glucose binding proteins in other systems, may function in UDP-glucose binding in AtRGPl, prior to the glycosylation of this protein at Arg-158. 54 RGP has been localized to the Golgi both in pea (Dhugga et al., 1997) and possibly in maize (Epel et al., 1996). All the membrane markers used in this study, with the exception of ARA4, have been extensively characterized in our laboratory (Ahmed et al., 1997; Bar-Peled et al., 1997; Conceicao et al., 1997). We were able to show that AtRGPl is found in membrane containing fractions other than the ER or the plasma membrane, presumably the Golgi. The accumulation of ARA4 (Ueda et al., 1996) in the soluble fraction (Fig. 4, 5100), is typical of small GTP-binding proteins, which exist in T both cytosolic and membrane associated forms. Sequence analysis of AtRGPl predict it to be a soluble protein. Membrane association experiments show that most of AtRGP] is indeed soluble, but a small fraction can be found peripherally associated with membranes. Such a distribution within the cell suggests that the reaction of AtRGPl with UDP-sugars takes place in the cytoplasm. Our working hypothesis is that AtRGPl may be in some way involved in polysaccharide synthesis, possibly as a protein primer or as some form of intermediate in polysaccharide synthesis. There is no evidence for a primer function other than an analogy to protein primed starch and glycogen synthesis. AtRGPl ’s reversible glycosylation, its prominent residence in the cytoplasm where nucleotide sugars are found, and its transient association with membranes suggests that it functions as a carrier of UDP-sugars from the cytoplasm to membranes such as the Golgi. Our understanding of the enzymes involved in cell wall biosynthesis is scant at best, and so far, little progress has been made in isolating these enzymes, a requirement for studying their function. With this in mind, the use of cell suspension cultures for the study of cell wall biosynthesis may prove to be highly beneficial. Our preliminary 55 observations show that at the transcriptional level, AtRGP] RNA can accumulate several fold within four days after subculture and completely disappear after day 8. The fact that AtRGP] protein persisted through out this time course is not surprising since suspension cell systems are known to be very active. For example, processes such as the secretion of cell wall polysaccharides continue unhindered since there is less spatial limit to cell wall deposition due to cell-to-cell contact. Further investigation is necessary to determine the function of AtRGPl. Our studies support the notion that AtRGPl is capable of autoglycosylation using UDP- glucose and UDP-galactose, most likely from the cytoplasmic pool of sugar nucleotides, and carry these sugars to membranes, most likely the Golgi. Its function when associated L with membranes is not clear, but may involve the priming of polysaccharide synthesis. These observations, along with the ones made for the PsRGPl doublet (Dhugga et al., 1991), suggest that AtRGP] may play a role in cell wall biosynthesis. 56 REFERENCES Ahmed SU, Bar-Peled Maor, Raikhel NV (1997) Cloning and subcellular localization of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells Plant Physiol 114: 325-336 Albersheim P, Darvill A, Roberts K, Staehelin AL, Vamer JE (1997) Do the structures of cell wall polysaccharides define their mode of synthesis?. Plant Physiol 113:1-3 Atkinson EM, Long SR (1992) Homology of Rhizobium meliloti NodC to polysaccharide polymerizing enzymes. Mol Plant Microbe Interact 5: 439-442 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic Local Alignment Search Tool. J Mol Biol 215: 403-410 Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402 Baba K, Sone Y, Misaki A, Hayashi T (1994) Localization of xyloglucan in the macromolecular complex composed of xyloglucan and cellulose in pea stems. Plant Cell Physiol 35: 439-444 Bacic A, Harris PJ, Stone BA (1988) Structure and function of plant cell walls. In Jack Preiss, ed, The Biochemistry of Plants, Vol 14. Academic Press, San Diego, pp 297-371 Bar-Peled M, Raikhel NV (1996) A method for isolation and purification of specific antibodies to a protein fused to GST. Anal Biochem 241: 140-142 Bar-Peled M, Raikhel NV (1997) Characterization of AtSEC12 and AtSARl proteins likely involved in ER and Golgi traffic. Plant Physiol 114: 315-324 Bolwell GP (1987) Synthesis of cell wall components: aspects of control. Phytochemistry 27: 1235-1253 Bradford MM (1976) A rapid and sensitive method for the quantification of nanogram quantities of proteins utilizing the principle of dye-binding. Anal Biochem 72: 248-254 Brown MR Jr, Saxena IM, Kudlicka K (1996) Cellulose biosynthesis in higher plants. Trends Plant Sci 1: 149-156 Brummell DA, Camirand A, Maclachlan GA (1990) Differential distribution of xyloglucan glycosyl transferases in pea golgi dictyosomes and secretory vesicles. J Cell Sci 96: 705-710 57 Campbell RE, Brett CT, Hillman JR (1988) A xylosyltransferase involved in the synthesis of a protein-associated xyloglucan in suspension-cultured dwarf-french-bean (Phaseolus vulgaris) cells and its interaction with a glycosyltransferase. Biochem J 253: 795-800 Carpita NC, Gibeaut DM (1993) Structural models of the primary cell walls of flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J 3: 1-30 Carpita NC, McCann MC, Griffing LR (1996) The plant extracellular matrix: news from the cell’s frontier. Plant Cell 8: 1451-1463 Conceicao AS, Marty-Mazars D, Bassham DC, Sanderfoot A, Marty F, Raikhel NV (1997) The syntaxin homologue AtPEP12p resides on a late post-Golgi comparment in plants. Plant Cell 9: 571-581 Daniels MJ, Mirkow TE, Chrispeels MJ (1994) The plasma membrane of Arabidopsis thaliana contains a mercury-insensitive aquaporin that is a homolog of the tonoplast water a channel protein TIP. Plant Physiol 106: 1325-1333 1*“ Delmer DP, Amor Y (1995) Cellulose biosynthesis. Plant Cell 7: 987-1000 Dhugga KS, Ulvskov P, Gallagher SR, Ray PM (1991) Plant polypeptides reversibly glycosylated by UDP-glucose: possible components of Golgi B-glucan synthase in pea cells. J Biol Chem 266: 21977-21984 Dhugga KS, Ray PM (1994) Purification of the reversibly glycosylated polypeptides from pea: purified polypeptides exhibit the same properties as the Golgi-bound form. Plant Physiol Supplement 105, #684 Dhugga KS, Tiwari SC, Ray PM (1997) A reversibly glycosylated polypeptide (RGPl) possibly involved in plant cell wall synthesis: purification, gene cloning and trans Golgi localization. Proc Natl Acad Sci 94: 7679-7684 Driouich A, Faye L, Staehelin LA (1993) The plant Golgi apparatus: a factory for complex polysaccharides and glycoproteins. Trends Biochem Sci 18: 210-214 Epel BL, Van Lent JWM, Cohen L, Kotlizky G, Katz A, Yahalom A (1996) A 41 kDa protein isolated form maize mesocotyl cell walls immunolocalizes to plasmodesmata. Protoplasma 191: 70-78 Faik A, Chileshe C, Sterling J, Maclachan G (1997) Xyloglucan galactosyl- and fucosyltransferase activities from pea epicotyl microsomes. Plant Physiol 114: 245-254 58 Freshour G, Clay RP, Fuller MS, Albersheim P, Darvill AG, Hahn MG (1996) Developmental and tissue-specific structural alterations of the cell-wall polysaccharides of Arabidopsis thaliana roots. Plant Physiol 110: 1413-1429 Fry SC (1995) Polysaccharide modifying enzymes in the plant cell wall. Ann Rev Plant Physiol Plant Mol Biol 46: 497-520 Gibeaut DM, Carpita NC (1994) Biosynthesis of plant cell wall polysaccharides. FASEB J 8: 904-915 Harlow E, Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Hayashi T, Matsuda K (1981) Biosynthesis of xyloglucan in suspension-cultured soybean cells: occurrence and some properties of xyloglucan 4-[3-D-glucosyltransferase and 6-a- D--xylosyltransferase. J Biol Chem 256: 1 l 1 l7-11 122 Hayashi T (1989) Xyloglucans in the primary cell wall. Annu Rev Plant Physiol and Plant Mol Biol 40: 139-168 Hirschberg CB (1987) Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 56: 63-87 Hoson T (1989) Structure and function of plant cell walls: immunological approaches. Int Rev Cytol 130: 233-268 Kleene R, Berger EG (1993) The molecular and cell biology of glucosyltransferases. Biochim Biophys Acta 1154: 283-325 Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105-132 Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 Levy S, Staehelin LA (1992) Synthesis, assembly and function of plant cell wall macromolecules. Curr Opin Cell Biol 4: 856-862 Lin FC, Brown RM, Drake RR, Haley BE (1990) Identification of the uridine 5’- diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc. J Biol Chem 265: 4782-4784 Lord JM (1987) Isolation of endoplasmic reticulum: general principles, enzymatic markers, and endoplasmic reticulum-bound polysomes. Methods Enzymol 148: 576-584 59 Lynch MA, Staehelin LA (1992) Domain-specific and cell type-specific localization of two types of cell wall matrix polysaccharides in the clover root tip. J Cell Biol 118: 467- 479 Lynch MA, Staehelin LA (1995) Immunocytochemical localization of cell wall polysaccharides in the root tip of Avena sativa. Protoplasma 188: 115-127 Matthysse AG, White S, Lightfoot R (1995) Genes required for cellulose synthesis in Agrobacterium tumefaciens. J Bacteriol 177: 1069-1075 Mayer R, Ross P, Weinhouse H, Amikam D, Volman G, Ohana P, Calhoon RD, Wong HC, Emerick AW, Benziman M (1991) Polypeptide composition of bacterial cyclic diguanylic acid dependent cellulose synthase and the ocurrence of immunologically crossreacting proteins in higher plants. Proc Natl Acad Sci USA 88: 5472-5476 McCann MC, Wells B, Roberts K (1992) Complexity in the spatial localization and length distribution of plant cell wall matrix polysaccharides. J Microsc 166: 123-136 McNeil M, Darvill A, Fry SC, Albersheim P (1984) Structure and function of the primary cell wall of plants. Annu Rev Biochem 53: 625-663 Moore PJ, Swords KMM, Lynch MA, Staehelin LA (1991) Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants. J Cell Biol 112: 589-602 Moreno S, Cardini CE, Tandecarz J S (1986) a-glucan synthesis on a protein primer, uridine diphosphoglucose: protein transglycosylase 1: separation from starch synthetase and phosphorylase and a study of its properties. Eur J Biochem 157: 539-545 Munoz P, N orambuena L, Orellana A (1996) Evidence for a UDP-glucose transporter in golgi apparatus-derived vesicles from pea and its possible role in polysaccharide biosynthesis. Plant Physiol 112: 1585-1594 Newman T, de Brujn FJ, Green P, Keegstra K, Kende H, McIntosh L, Ohlrogge J, Raikhel NV, Somerville S, Tomashow M, Retzel M, Somerville C (1994) Genes galore: a summary of methods for accessing results from a large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol 106: 1241-1255 Okazawa K, Sato Y, Nakagawa T, Asada K, Kato I, Tomita E, Nishitani K (1993) Molecular cloning and cDNA sequencing of endoxyloglucan transferase, a novel class of glycosyltransferase that mediates molecular grafting between matrix polysaccharides in plant cell walls. J Biol Chem 268: 25364-25368 Palcic MM (1994) Glycosyltransferases in glycobiology. Meths Enzymol 230: 300-339 60 Paulson JC, Colley KJ (1989) Glycosyltranferases: structure, localization, and control of cell type-specific glycosylation. J Biol Chem 264: 17615-17618 Pear JR, Kawagoe Y, Schreckengost WE, Delmer DP, Stalker DM (1996) Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase. Proc Natl Acad Sci USA 93: 12637-12642 Puissant C, Houdebine L-M (1990) An improvement of the single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. BioTechniques 8: 148-149 Quentrneier H, Ingold E, Seitz HU (1987) Purification of an autocatalytic protein- glycosylating enzyme form cell suspension of Daucus carota L.. Planta 171: 483-488 Ray PM (1980) Cooperative action of fi-glucan synthase and UDP-xylose xylosyl transferase of Golgi membranes in the synthesis of xyloglucan-like polysaccharide. Biochim Biophys Acta 629: 431-444 Read SM, Thelen M, Delmer DP (1986) Identification of UDP-glucose-binding proteins in mung bean membrane preparations. in B Vian, D Reis, R Goldberg, eds, Cell Wall ‘86: Proceedings of the 4th Cell Wall Meeting, Universite Pierre et Marie Curie-Ecole Norrnale Superieure, Paris, pp 308-311 Reiter W-D, Chapple CCS, Somerville CR (1993) Altered growth and cell walls in a fucose-deficient mutant of Arabidosis. Science 261: 1032-1035 Roberts K (1990) Structures of the plant cell surface. Curr Opin Cell Biol 2: 920-928 Robertson D, McCorrnack BA, Bolwell GP (1995) Cell wall polysaccharide biosynthesis and related metabolism in elicitor-stressed cells of French bean (Phaseolus vulgaris L.). Biochem J 306: 745-750 Saxena IM, Brown RM Jr, Fevre M, Geremia RA, Henrissat B (1995) Multidomain architecture of B-glycosyl transferases: implications for a mechanism of action J Bacteriol 177: 1419-1424 Saxena IM, Brown Jr. RM (1997) Identification of cellulose synthase(s) in higher plants: sequence analysis of processive B-glycosyltransferases with the common motif “D,D,D35Q(R,Q)XRW”. Cellulose 4: 33-49 Showalter AM (1993) Structure and function of plant cell wall proteins. Plant Cell 5: 9- 23 Silberstein S, Gilmore R (1996) Biochemistry, molecular biology, and genetics of the oligosaccharyltransferase. FASEB J 10: 849-858 61 Singh DG, Lomako J, Lomako WM, Whelan WJ, Meyer HE, Serwe M, Metzger JW (1995) B-Glycosylarginine: a new glucose-protein bond in a self-glucosylating protein from sweet corn. FEBS Lett 376: 61-64 Smythe C, Cohen P (1991) The discovery of glycogenin and the priming mechanism of glycogen biogenesis. Eur J Biochem 200: 625-631 Staehelin LA, Moore I (1995) The plant golgi apparatus: structure, functional organization and trafficking mechanisms. Annu Rev Plant Physiol Plant Mol Biol 46: 261-288 Talbott LD, Ray PM (1992) Molecular size and separability features of pea cell wall polysaccharides. Implications for models of primary wall structure. Plant Physiol 98: 357-368 Tenhaken R, Thulke O (1996) Cloning of an enzyme that synthesizes a key nucleotide- sugar precursor of hemicellulose biosynthesis from soybean: udp-glucose dehydrogenase. Plant Physiol 112: 1127-1134 Ueda T, Anai T, Tsukaya H, Hirata A, and Uchimiya H (1996) Charaterization and subcellular localization of a small GTP—binding protein (ARA-4) from Arabidopsis: conditional expression under the control of the promoter of the gene for heat-shock protein HSP81-1. Mol Gen Genet 250: 533-539 Waldron KW, Brett CT (1985) ) Interaction of enzymes involved in cell wall heteropolysaccharide biosynthesis. In Biochemstry of plant cell walls. CT Brett, JR Hillman, eds, SEB Seminar Series, Cambridge University Press, Cambridge. pp. 79-97 White AR, Xin Y, Pezeshik V (1993) Xyloglucan glucosyltransferase in Golgi membranes from Pisum sativum (pea). Biochemistry Journal 294: 231-238 Zablackis E, Huang J, Muller B, Darvill AG, Albersheim P (1995) Characterization of the cell wall polysaccharides of Arabidopsis thaliana leaves. Plant Physiol 107: 1129- l 138 Zablackis E, York WC, Pauly M, Hantus S, Reiter WD, Chapple CS, Albersheim P, Darvill A (1996) Substitution of L-fucose by L-galactose in cell walls of Arabidopsis murl. Science 272: 1808-1810 62 CHAPTER III CHARACTERIZATION OF THE FOUR AtRGPS OF ARABIDOPSIS This chapter is the foundation 0g 5 manuscript that will be submitted for publication in the near future: Ivan J. Delgado, Kenneth Keegstra and Natasha V. Raikhel (2001) Characterization of the four AtRGPs of Arabidopsis 63 ABSTRACT The plant-specific reversibly glycosylated polypeptides (RGPs) are localized in the cytoplasm and the Golgi apparatus. RGPs self-glycosylate using UDP-sugars, the same substrates utilized by the Golgi apparatus for the synthesis of cell wall polysaccharides. These and other observations have led to the hypothesis that RGPs are involved in plant cell wall polysaccharide synthesis. In an effort to evaluate this hypothesis, we present an expression analysis of the four RGPs found in Arabidopsis, as well as a biochemical analysis of the their respective proteins. In addition, although homozygous T-DNA insertion mutants in AtRGP] or AtRGP2 revealed no phenotypic defects, a double mutant in both genes could not be recovered. This observation suggests that AtRGP] and AtRGP2 are functionally redundant. INTRODUCTION The 41 kD protein, as the reversibly glycosylated polypeptide (RGP) was known prior to its purification, has been implicated in cell wall biosynthesis (Dhugga et al., 1991). This conclusion is mainly based on the fact that it reacts with UDP-sugars (Dhugga et al 1997, Delgado et al., 1998; Faik et al., 2000), the substrates for polysaccharide synthesis, and was localized in the Golgi apparatus (Dhugga et al., 1997), the site of cell wall polysaccharide synthesis (Nebenfiihr and Staehelin, 2001). Cloning of the first RGP (Dhugga et al., 1994) led to the identification of RGPs from various species (Saxena and Brown, 1999; Bocca et al., 1999). Analysis of RGP sequences in the genomes of plants and other organisms, as well as immunoblot analysis using RGP antibodies of protein samples from plant and non-plant species showed that RGPs are plant specific (Delgado et al., 1998). In addition to the localization of RGP in the Golgi apparatus (Dhugga et al., 1997), RGP has also been detected in the cytoplasm (Delgado et al., 1998). The glycosylation of RGPs takes place in the presence of any UDP-sugar, with the possible exception of UDP-mannose (Dhugga et al., 1991; Dhugga et al., 1997; Delgado et al., 1998; Bocca et al., 1999; Faik et al., 2000). The localization of RGPs and their reaction with UDP-sugars introduced the idea that they may be involved in the transport of UDP-sugars from the cytoplasm to the Golgi apparatus (Delgado et al., 1998). RGP is defined by two separate activities. First, in the presence of UDP-[”C]Glc, RGP self-glucosylates. Second, when an excess of unlabeled UDP-Glc or UDP is added to a reaction between UDP-[”C]Glc and RGP, no RGP self-glucosylation can be detected (Dhugga et al., 1991). The reversible self-glycosylation of RGP is the biochemical 65 definition of this protein, and by this definition RGP has been characterized in mung bean (Franz, 1976; Read et al., 1986), pea (Dhugga et al., 1997), Arabidopsis (Delgado et al., 1998), potato (Bocca et al., 1999), and nasturtium (Faik et al., 2000). Another definition for RGP, one that does not require a biochemical analysis, takes advantage of the fact that RGPs are highly conserved proteins in plants (Delgado et al., 1998). Namely, all RGPs identified to date are 41 kD proteins that are at least 68% identical to each other (Delgado et al., 1998). Although multiple RGPs have been identified in Arabidopsis (Delgado et al., 1998) and potato (Bocca et al., 1999) based on their sequence identity, to date no biochemical characterization of the multiple RGPs found in a single plant species has been performed. Self-glucosylation in the presence of UDP-Glc has also been described for the protein primer involved in glycogen synthesis in animals, later named glycogenin (Rodriguez and Whelan, 1985; Meezan et al., 1988). Viskupic et a1. (1992) cloned the first glycogenin gene, and confirmed earlier observations that the predicted protein size of glycogenin is 38-kD. Analogous to animal glycogen synthesis by glycogenin, starch synthesis in plants is thought to require a protein primer, also known as amylogenin (Moreno et al., 1986; Moreno et al., 1987). Amylogenin has been described as a 38-kD self-glucosylating protein capable of serving as a primer for protein-bound alpha-1,4- glucan synthesis (Moreno et al., 1987). To date the gene encoding a protein capable of priming alpha-1,4-glucan synthesis in plants has not be cloned. In an effort to isolate the protein primer for starch synthesis, also known as amylogenin, Singh et a1. (1995) isolated a 41 kD sweet corn protein that self-glucosylated in the presence of UDP-Glc. When the glucosylated corn protein was purified, digested 66 with trypsin and the tryptic peptides sequenced, a single glucose was found covalently attached to an arginine via a B-linkage. Dhugga et a1. (1997) found that the sequence of the tryptic peptides of the corn protein, which amounted to over 40% of the total estimated corn protein sequence (Singh et al., 1995), showed over 84% sequence identity with the pea RGP. This led Dhugga et a1. (1997) to rename the corn “amylogenin” as the corn RGP. Likewise, the predicted protein from the full-length clone of the corn RGP] (accession U89897) is almost 100% identical to the sequenced tryptic peptides obtained by Singh et a1 (1995). These observations indicate that the corn self-glucosylating protein is an RGP homologue. The equivalent arginine shown to be glucosylated in the corn RGP (Singh et al., 1995) is also present in the pea RGP (Dhugga et al., 1997) and every other RGP so far identified (Delgado et al., 1998). Therefore, it has been presumed that this arginine is the site of self-glucosylation of the RGPs (Dhugga et al., 1997). Efforts to isolate the potato amylogenin (Ardila and Tandecarz, 1992) instead have lead to the isolation of the potato RGP (Bocca et al.; 1999). The self-glucosylation of this potato protein, termed UDP-glucosezprotein transglucosylase (UPTG), in the presence of UDP-Glc was hypothesized to be the starting point for the enzymatic elongation of glucan chains by starch synthases or starch pyrophosphorylases (Moreno et al., 1987). Bocca et a1. (1997) later observed that UPTG also self-xylosilated in the presence of UDP-Xyl, a surprising result considering that starch does not contain xylose residues (Martin and Smith, 1995). When antibodies raised against the purified UPTG (Bocca et al., 1997) were used to screen a cDNA expression library from potato stolons, two cDNAs were identified. The cDNA sequences coded for polypeptides of 365 and 366 amino acids (41 kD polypeptides) and their predicted proteins shared 89% sequence 67 identity (Bocca et al., 1999). When these potato cDNA sequences were used in a GenBank database search, the authors found that their potato genes were 86-93% identical to RGPs from Arabidopsis, pea, corn, rice, and wheat (Bocca et al., 1999). Furthermore, the self-glycosylation of UPTG required Mn2+ and was reversible in the presence of UDP-sugars with the exception of UDP-Man, the same reversible self- glycosylation described for the pea RGP (Dhugga et al., 1997). Both the high sequence t similarity and almost identical biochemical properties between UPTG and RGP led Bocca et a1. (1999) to conclude that UPTG was not a protein primer for starch synthesis, or amylogenin, but an RGP homologue. I i. The two studies described above aimed at identifying the potato (Ardila and Tandecarz, 1992) and corn (Singh et al., 1995) amylogenin, but instead led to the identification of the potato (Bocca et al., 1999) and corn (Dhugga et al., 1997) RGP. One of the reasons for this result was the fact that in neither case were the proteins analyzed for their priming ability in the synthesis of starch. It was not until the genes were cloned and the activities of the respective proteins analyzed that the self-glucosylation of these putative amylogenins was found to be reversible and to require an“ (Bocca et al., 1999), two characteristics that do not coincide with the activity described for the synthesis of protein-bound alpha-1,4-glucan (Moreno et al., 1987), yet characteristic of RGP reversible self-glucosylation (Dhugga etal., 1997). To date no amylogenin has been shown to be involved in the priming of starch synthesis. A putative amylogenin (AMY), that is not RGP, has been cloned in wheat (accession Y18625). The wheat AMY is only 42% identical to the pea RGP, yet AMYs from wheat, rice (Y18623), and Arabidopsis (BAB09620) are 60-90% identical to each 68 other at the protein level. In addition, AM Ys encode proteins with a predicted size of 38 kD, the same size as the protein primer for glycogen, glycogenin (Meezan et al., 1988). To date no evidence exists for a role of these 38 kD AMYs in starch synthesis. The low sequence identity between AMYs and RGPs, and the fact that AMYs are only 38 kD proteins, suggests that AMYs and RGPs have different functions in plants. As a result, AMYs were deemed different enough to RGPs not to be included in this analysis. In this report we describe an expression analysis of the four Arabidopsis RGPs, as well as a biochemical analysis of the their respective proteins. In addition, a reverse genetic approach was used to elucidate the function of two RGPs, AtRGP] and AtRGP2. Although no obvious phenotype was identified in both homozygous mutants, a double AtRGPI/AIRGPZ mutant could not be recovered, suggesting that such a genotype is lethal to a plant. 69 MATERIALS AND METHODS Plants and growth conditions Seeds were surface sterilized, sown on Murashige and Skoog plates, and germinated for one to two weeks. Seedlings about two weeks after germination were transferred to soil and grown at 25°C under 16 hrs of light as described (Delgado et al., 1998). Growth stage-based phenotypic analysis of T-DNA insertion mutants was performed at Paradigm Genetics as described (Boyes et al., 2001) Sequencing and Sequence Analysis BLAST analysis was performed to identify all known RGPs (http://www.ncbi.nlm.nih.gov/blast/). Sequences were aligned and fused to obtain full- length cDNAs using the DNAStar software package (MegAlign module, DNAStar Inc., Madison, WI). The identified genes were compared to those annotated by the Institute for Genomic Research, TIGR, as gene indices (Gls) (http://www.tigr.org/tdb/tgi.shtml). The chromosomal locations of the different genes were obtained using MapViewer (httpzllwww.arabidopsis.org/servlets/mapper). The intron-exon structures were obtained by comparing full-length cDNAs with genomic sequences. Automated sequencing of the cDNAs, as well as the T-DNA inserts, was performed at the Genomic Technologies Support Facility at Michigan State University. T-DNA insertion mutant isolation T-DNA inserts into all four Athps were isolated from the collection maintained by the Arabidopsis Knockout Facility (AKF) using the procedures described in their 70 website (http://www.biotech.wisc.edu/Arabidopsis/default.htm). The primers used for each gene were as follows, with the first one being the forward primer and the second the reverse primer. AtRGP] : 5’-AGAACGGTCCAATTAAACTGTACCACGA-3’ and : 5’- CT CGGTCATAAATAGAACT ACACCATTCAC-3 ’; AtRGP2: 5 ’ -ATCCGATCT CAT CT CT CT CA'I'I'I‘CGAAAC-B ’ and 5 ’ -CATACTGCTCTCAAGC'I'ITGCCACTGGCT- 3’; AtRGP3: 5’-CGCAA'ITGTATTC’ITCCGTGAAACCCACG-3’ and 5’-CGAAG TGCACTCTI'TAGGAAGAGTCAC-3 ’; and AtRGP4: 5 ’-CGGGCTACAACCTCGAA GCTATCGAAGCG-3’ and 5’-AA'ITTCCATATAACATITAGCTGCAGTGT-3’. All the procedures, from the PCRs, to the Southern blots and the sequencing were carried out as specified by the AKF. Isolation of RNA and RT-PCR Total RNA from Arabidopsis stems, siliques, roots, and leaves was extracted as described (Delgado et al., 1998). Total Arabidopsis RNA (1 ug/reaction) was utilized for each RT-PCR reaction. The cDNA synthesis reactions were performed using SuperScript II reverse transcriptase as described by the manufacturer (Life Technologies, catalog number 18064-014). Namely, the RNA and primers were denatured at 70°C for 10 mins, chilled on ice, the transcriptase added and the reaction incubated for 50 mins at 42°C. The gene-specific primers were as follows: AtRGP] forward 5’-TGGTTGAGCCGGCGAAC ACCG'I'I‘GGAATI‘C-B’, and reverse 5’-CTCGGTCATAAATAGAACTACACCATI‘C AC-3’ primers. AtRGP2 forward 5’-GGAGGTGAATAAGTCITCATCTGACAC-3’ and reverse 5’- CCAAATGCAAAAACCATAACCGGACAA-3’ primers. AtRGP3 forward 5’- CCAAGAAGTCATCCTTGTTATCGTACA-3’ and reverse 5’- GCTTATGAGG'I'I'I 71 TAAGAGATGGCG'IT-3’ primers. AtRGP4 forward 5 ’- CAAATTAATGAAGGAGTA TGAGAACCA-B’ and reverse 5’- G'ITCGTCAAGCAACCCAATCCGATATA-3’ primers. All these primers were made to either untranslated region of the respective genes. Once the cDN As were synthesized and the reactions were terminated by heating at 70°C for 15 mins, an aliquot of each cDNA was used as template for the PCR reactions. The PCR conditions were as follows: reactions were heated to 95°C (hot start) and the polymerase added. 35 cycles of 94°C for 15 seconds, followed by 65°C for 30 seconds, followed by 2 mins at 72°C were performed. A final step at 72°C for 4 mins finished the PCR reaction. A fraction of each reaction was loaded onto ethidium-bromide stained 1% agarose gels and separated at 100 volts on 1X TBE buffer. The resolved bands were visualized in a UV-box. Protein Extraction Two-weeks old Arabidopsis seedlings were harvested and lyzed in 50 mM M Tris, pH 7.5, 0.25 M sucrose, lmM EDTA using a glass dounce homogenizer. The lysate was centrifuged for 2 mins at 2,000 x g and the supernatant used for further reactions. Overexpression of Fusion Proteins in Escherichia coli A 1.3-kb EcoRI-DraI fragment of AtRGP] was cut from its parental vector pZLl (Newman et al., 1994), and cloned into the EcoRI-EcoRV sites of pBluescript KS 11'. This construct was named AtRGPlpBL. From AtRGPlpBL a 1.4-kb EcoRI-NotI fragment encoding all but the first nine amino acids from the 5’ end of the AtRGP] 72 cDNA was cloned into the EcoRI-NotI sites of pGEX-SX-Z (Pharmacia) to generate an N-terrninal in-frame fusion with the 26-kD domain of GST. To generate GST-AtRGP] mutants, site-directed mutagenesis reactions were performed. Two primers were synthesized for each residue to be mutated. The PCR reactions used the construct AtRGPlpBL as template. The forward primer was phosphorylated and contained the nucleotide substitutions to be introduced, while the reverse primer was synthesized so that it would anneal starting with the first nucleotide upstream of the site where the forward primer annealed. PCR reactions were then carried out that synthesized copies of the whole AtRGPlpBL construct and introduced the desired nucleotide substitutions. The fragments synthesized by the PCR reactions were purified and ligated. EcoRI-NotI fragments from these mutated AtRGPlpBL constructs were purified and ligated into the EcoRI-Notl sites of pGEX-SX-Z to generate an N- terminal in-frame fusion with the 26-kD domain of GST. The GST-fusions obtained were then transformed into E. coli and used for protein expression as described below. The primer combinations used were as follows. For GST-AtRGPl-R158K, the forward primer was 5’-P-GCI‘GACTI‘CGTCA_AA;_GGATACCCTI'TC-3’ and the reverse primer was 5’-ACCTI‘CACGGTATGGGTCGTACAAGGT-3’, where P stands for phosphate and the underline highlights the substituted nucleotides. For GST-AtRGPl- R158H, the forward primer was 5’-P-GCTGACTTCGTCQAIGGATACCCTITC-3’ and the reverse primers was 5’-ACC'I'I‘CACGGTATGGGTCGTACAAGGT-3’. The other AtRGPs were also cloned as GST-fusions. For GST-AtRGP2, restriction sites were introduced by PCR. The forward primer was 5’- GAGCTCGAT ATCGQAATI‘CCGACTATC -3’ and the reverse primer was the Sp6 primer 5’- 73 A'I'ITAGGTGACACTATAG-3’. The EcoRI introduced is underlined in the forward primer. The PCR fragment was purified and digested with EcoRI and NotI and cloned into the EcoRI-Notl sites of pGEX-SX-Z. Unlike AtRGPl and AtRGP2, which were in pZLl vectors, GST-AtRGP3 and GST-AtRGP4 were in pBluescript 11 SK' vectors (Asamizu et al., 2000). For GST- AtRGP3, restriction sites were introduced by PCR. The forward primer was 5’- GCTAGACA'I'I‘GGAATTCCTACGATTCG-3’, and the reverse primer was 5’- GAAAAACAGAGGCGGCCGCACATGTGA-3’. An EcoRI site was introduced in the forward primer and a NotI site was introduced in the reverse primer (underlines, respectively). For GST-AtRGP4, the forward primer was 5’-GGCACCTI'I‘GAGAATI‘C ATCTGGATATT-3’, and the reverse primer was 5’-C'I'I‘GACATC'I'IT GCGGCCGCT C TGC'I'I‘C’IT-3’. An EcoRI site was introduced in the forward primer and a NotI site was introduced in the reverse primer (underlines, respectively). PCR reactions using the above mentioned primers generated the expected fragments, which were digested with EcoRI and NotI restriction enzymes, purified and ligated into the EcoRI-NotI sites of pGEX-SX-Z. The above mentioned GST-fusions were overexpressed in E. coli (strain DH5) by growing cells in Luria-Bertani medium at 37°C to an A600 of 0.7 to 0.8, then adding isopropylthio-d-galactosidase to a final concentration of 0.2 uM, and incubating the culture at 28°C for 4 hrs. The soluble GST-fusion proteins were purified as follows: the cells were lyzed by passing them through a French press at 1,100 psi twice, mixing the resulting lysate with a final concentration of 1% Triton X-100 for 30 minutes at 4°C and centrifuging the mixture at 100,000g for 30 minutes. The supernatant obtained was 74 mixed with Glutathione Sepharose 4B beads (catalog number 52-2303-00, Pharrnacia Biotech) for 30 mins at room temperature in a 15 mL tube, the tube centrifuged at 500g for 5 minutes, and the beads washed a total of three times with 10 volumes of PBS. The GST-fusion proteins were eluted using lOmM reduced glutathione in 50mM Tris-HCl, pH 8.0 and the elution dialized. Protein concentration was determined as described previously (Bradford, 1976) using BSA as the standard and checked for purity by running them on SDS-PAGE gels and commassie staining. Reactions with UDP-Glc Five micrograms of GST-fusion protein was incubated with 0.1-0.2 uCi of UDP- [3H]-Glc (specific activity 11.5 Ci/mmol), 0.5 uCi of UDP-[U-‘4C]Xyl (specific activity 238 mCi/mmol [ICN]), 0.5 uCi of UDP-[U-‘4C]Gal (specific activity 270 mCi/mmol [ICN]), or [U-“C]Glc-1-P (specific activity 218 mCi/mmol [ICN]) in a volume of 50-150 uL of lysis buffer in the presence of absence of 1 mM MgC12. The reactions were stopped after a 10-min incubation at room temperature by adding 10-30 uL of SDS-PAGE loading buffer (120 mM Tris pH 6.8, 200 mM DTT, 4% SDS, 0.02% Bromophenol Blue, 20% Glycerol). The protein samples were separated by SDS-PAGE and treated as described below. For TLC experiments, the reactions were incubated for 2 hrs and terminated by spotting them onto TLC plates. SDS-PAGE and Immunoblotting Protein concentration was determined as described before (Delgado et al., 1998) using BSA as standard. Protein molecular weight standards were purchased from BioRad 75 (Low-Range marker and Broad-Range marker, catalog #161-0304 and 161-0317, respectively). Protein samples were separated on 12.5% SDS-PAGE gels using 1X Running Buffer (250 mM Tris, 1M Glycine, 0.5% SDS). After separation, gels containing UDP-[3H]-Glc were fixed with 5 volumes of 30% methanol, 10% acetic acid, washed with distilled water 2 times, incubated with Fluoro-Hance (Research Products International Corp., Illinois) for 15 mins and dried for 2 hrs in a BioRad Gel Drier (Model 583). Film was exposed to the dried gels for 7-10 days at -80°C prior to film development. Immunoblots were performed as previously described (Delgado et al., 1998), using AtRGP] antibodies at a 1:1000 dilution. This Layer Chromatography Polyethylene imide-cellulose (PEI-cellulose) plates (20 cms x 20 cms) were prepared by incubating them with water for 2 min and letting them air-dry for one hour. 10 to 20 uL of a typical RGP glycosylation reaction was spotted onto the plates and air- dried for 20 minutes. The plates were chromatographed in 1 N acetic acid: 3 M LiCl2 (90: 10, v/v). The plates were then cut into sections that were 1.5 cm in height and 2 cm in width, except the first and last sections, which were 3 cm and 2 cm in height, respectively. Radioactivity in each section was determined by scintillation counting. 76 RESULTS A multi-gene family of distinct Arabidopsis proteins Analysis of the Arabidopsis genome revealed four AtRGPs. AtRGP] (accession AF013627) and AtRGP3 (accession AF034255) are found on chromosome III, while AtRGP2 (accession AF013628) and AtRGP4 (accession AF329280) are found on chromosome V (Fig. 3.1 A). AtRGP2 is localized in a segment of chromosome V known to be a duplication of the segment of chromosome HI where AtRGP] is present (Blane et al., 2000). The localization of AtRGP] and AtRGP2 in these segments suggests that AtRGP2 is a paralogue of AtRGP] (Doyle and Gaut, 2000). When the genomic sequences of AtRGP] and AtRGP2 were compared, they were found to be 62% identical. Furthermore, AtRGP] and AtRGP2 share the same intron phase in two of their three intron-insertion sites (not shown). These observations suggest that AtRGP2 is a recent duplication of AtRGP] . AtRGP] and AtRGP2 consist of four exons, and share an almost identical intron- exon structure (Fig. 3.1 B). AtRGP3 also consists of four exons, but the introns in AtRGP3 are all equal in size. AtRGP4 has the most divergent intron-exon structure with only three exons (Fig. 3.1 B). The predicted proteins for each AtRGP are almost identical in size, with 357, 360, 362, and 364 amino acids for AtRGP], 2, 3, and 4, respectively (Fig. 3.2). Therefore, the size of RGPs is approximately 41 kDa. AtRGPl and AtRGP2 are 95% identical, while AtRGP3 and AtRGP4 are only 72% identical to each other (Fig. 3.2). AtRGP3 is at least 82% identical to AtRGPl and AtRGP2, while AtRGP4 is no more than 75% identical to AtRGPl and AtRGP2. All known RGPs are at least 68% identical to each other (not shown). 77 Figure 3.1 A 4.4" _."il.flih- 't-o '7‘ . '."""I we: -".‘ 4. L \ _ F14P3 T1601] ('9 I —u Figure 3.1 A. Chromosomal location of the different AtRGP genes. The numbers represent the location of each AtRGP gene and each open box indicates the approximate location of the BACs in the chromosome. The BAC number for each gene is shown under each open box. The scale is in centimorgans (cM). For chromosome 111, one cM is equivalent to 233 kb, while in chromosome V, one cM is equivalent to 187 kb. 78 Figure 3.1 B 3nUTR| 3nUTR 3LUTR 3aUTR| Figure 3.1 B. Gene structure of the four AtRGP genes. The black boxes denote exons and the lines connecting them are introns. The scale is broken down into sections of 450 base pairs (450 bp). 79 RGPs are classified as non-processive glycosyltransferases because they contain domain A (Fig. 3.2) but not domain B of processive glycosyltransferases (Saxena and Brown, 1999; Chamock et al., 2001). The domain A is also the defining signature of the glycosyltransferase 2 (GT2) family (httpz/lafmb.cnrs-mrs.fr/~pedro/CAZY/gtf_2.html). The N-terminus of all AtRGPs contains the domain A (Fig. 3.2), and it covers a region that is necessary for UDP-binding, as defined by the crystal structure of the UDP-bound GT2 Spore Coat Polysaccharide Biosynthesis Protein, SpsA, from Bacillus subtilis (Charnock and Davies, 1999). All but one residue in SpsA known to be involved in UDP-binding is conserved in all AtRGPs (circles, Fig. 3.2). The domain A of RGPs is defined as a series of B-strands alternating with a- helices with two conserved aspartate residues separated by 46 to 120 amino acids (Saxena and Brown, 1997; Chamock et al., 2001). In plant glycosyltransferases there is a “P-CR” insertion between strand [3-3 and B-4 that can range from 70 to 190 residues (Turner and Somerville, 1997; Arioli et al., 1998; Chamock et al., 2001). A “P-CR” insertion is also present in all AtRGPs, but it is only 11 residues long (bolded box, Fig. 3.2). Expression analysis of the AtRGPs Primers specific for each AtRGP were used to perform non-quantitative RT-PCR reactions for each gene using as templates RNA samples from various Arabidopsis tissues. The results of these reactions can be seen in Figure 3.3. AtRGP] and AtRGP2 were expressed in all tissues tested, whereas AtRGP3 and AtRGP4 were only detected in siliques (Fig. 3.3). These observations are in agreement with similar conclusions drawn 80 Figure 3.2. Sequence comparison of the AtRGP proteins predicted from their cDNAs. The light gray boxes depict residues that are identical in all AtRGPs. The N-terminal UDP-hiding domain, also known as domain A, includes the first 115 residues of every AtRGP. The residues critical for UDP-binding, as defined by the three-dimensional structure of the UDP-sugar dependent glycosyltransferase SpsA from Bacillus subtillis, are highlighted by filled circles. The Arginine residue shown to be the site of glucosylation in the corn RGP is indicated by the downward pointing arrow. 81 Figure 3.2 domain A xxH U-H-u-u. ((4 WV) UULJU l-l— ma-Qoooo in HO O - DDDD >><> D -.oooo 001.919 a - HHHH “JUNE-I o' .. u...— cacao: > mqrr 44E; H HHH Efimmmfi H >—>—>—>- muugu. .I_I.— -l xxx O'DQO'O'O' I: - xxx 59'>'>'>‘ >- xxx (mama - a: mmw *uma‘m - :: >3») >>>> xxx manna. )->-u. Q d ‘D LDIVIPTIRNHHWENIRPFFEQHHJHVQDGD x V'I a o o a o > H H A x z x * x a m o > A I mm > an o “G IL- wr u wmo I: o a 3 WW U )- _I—l d: o. ILIL VI D you - o r —1—1 x —l 22 VI IL .— 3“ ~< VI 2 HH H a PP u M an - v1 mu. HH < mam x >> x PEP HH a m on - a a r I . dd .4 U ID lthxo “JIM D H _1 ( 1(ax ooa- a A a hang xxx 2 Z I IIIUD d-l—l H X D llflm A In a H o Inwo aaa¢ z : khh >¥> llhx H» dim 000 can max Itux :a-m 2 mm <<< coo lumx zsz >- 4.1 can HHH III). >>>< A 4 »>> can a gnaw m 2 can 000 a H IE * > mac new 2 o :2 a < HHH can 4 > v> x 22: man u PPMD mama 444 as: o z M< MMEW my. 444 1.. («>1 anam 444 mom <4 anau coco one man u mm m on (:2: xxxx 2:: >>> 2 HH >>- i... 213- P > >>>> >>>H (<4 m PH > mmHH NN m ooNN mm m “Had mass 2: : aa§§ fifiafi aa axfia Nme va Hva ENme Hva gNm¢ H~m+ gggg Sgga ages agg essg useg gags “Nd-'43 ##4-‘0 6400M 40%-'49“ U-HHU H990 uauu <(<-( ((dd (dd-d (<44 (did-I <(<< <<<< 82 from analysis of the EST databases as defined by the TIGR website (www.tigr.org), where all ESTs for each individual gene are grouped into gene indices or TGI numbers (TC99078, TC99076, TC86775, and TC87643, for AtRGP] , 2, 3, and 4, respectively). AtRGP] and AtRGP2 are well represented with 79 and 95 ESTs, respectively. Furthermore, these ESTs were obtained from all tissue types, from roots to flowers. ESTs for AtRGP3 and AtRGP4 were very limited in number and obtained only from F developing seeds and green siliques (Fig. 3.3). Four AtRGPs and two distinct activities As shown in Figure 3.3, four AtRGPs are expressed in Arabidopsis. To study the activity of the proteins encoded by these genes, N-terminal Glutathione S-transferase (GST)- fusions to each AtRGP were made. These GST-fusions were expressed in E. coli cells and the proteins purified over a glutathione sepharose column. When each GST-fusion was incubated with UDP-[3H]Glc, UDP-[”C]Xyl or UDP-[”C]Gal, only GST- AtRGPl and GST-AtRGP2 showed any glycosylation (Fig. 3.4 A). The strong glycosylation of GST-AtRGPl (Fig. 3.4 A) allowed a more detailed analysis of the requirements for glycosylation in AtRGP]. In an effort to identify the primer for starch synthesis in corn, Singh et a1. (1995) purified the corn RGP (Dhugga et al., 1997). The purified protein was glucosylated with UDP-[”C]Glc, digested by trypsin, and the tryptic peptides sequenced. These experiments showed that a single glucose is attached to an Arg residue via a B-linkage (Singh et al., 1995). To determine if the glucosylation of the Arabidopsis AtRGPl takes place at the same Arg residue as in the sweet corn RGP, Arg-158 in AtRGPl was substituted with Lys (K) or His (H). All 83 Figure 3.3 3 $8 ===§.§. @4421: H mm ; axm mba‘r"? 1271 a .— H fl 5 H m N N M V!- 8 8 8 8 es 8 8 es <1 < <: < Figure 3.3. Expression profiles of the various AtRGP genes. RT-PCR was performed to generate the expression patterns. RNA samples were obtained from stems (St), siliques (Si), roots (Rt) and leaves (Lv). Gene-specific primers were used for each gene and ethidium brornide-stained gels of the respective reactions are shown. To the right of the indi vidual expression patterns are the number of ESTs found for each gene and the tissues from which these ESTs were obtained. 84 three versions of AtRGP] were fused to GST (GST-AtRGP], GST-AtRGPl-R158K, and GST-AtRGPl-R158H, respectively), expressed in E. coli, and purified over a glutathione sepharose column. When the different GST-AtRGPs were incubated with UDP-[3H]Glc and separated by SDS-PAGE, only GST-AtRGP] was glucosylated in the presence of UDP-[3H]Glc (Fig. 3.4 B). The lack of glucosylation of GST-AtRGPl-R158K and GST- AtRGPl-R158H was likely not due to mis-folding of the GST-fusion protein as both substitutions were made to conserved amino acids. It is thought that the lack of glucosylation in the mutant AtRGPls was a result of the absence of the appropriate sugar acceptor, arginine, but it is also possible that these mutant AtRGPls were unable to bind UDP-Glc. *- The lack of glycosylation of GST-AtRGP3 and GST-AtRGP4 may be due to a number of reasons, including mis-folding of these GST-fusions. To determine if GST- AtRGP3 and GST-AtRGP4 are active, all four GST-fusions were incubated with UDP- [3H]Glc, and the reaction products analyzed by thin-layer chromatography. The radioactivity on each TLC plate was determined by scintillation counting. As can be seen in Figure 3.5 A, most of the UDP-[3H]Glc added to GST-AtRGPl and GST-AtRGP2 was still present at the end of the reaction, while almost none of the UDP-[3H]Glc was left at the end of the reactions containing GST-AtRGP3 and GST-AtRGP4. The majority of the radioactivity at the end of the reactions with GST-AtRGP3 and GST-AtRGP4 was present as [3H]Glc. The reaction products obtained in the reactions described above were a result of the activity of the different GST-AtRGPs because the same reactions in the absence of MnCl2 did not change UDP-[3H]Glc (Fig. 3.5 B). RGPs require Mn2+ for glycosylation 85 (Dhugga et al., 1991). Furthermore, the third peak observed in the reactions between the different GST-AtRGPs and UDP-[3H]Glc may be [3H]Glc-l-P (Fig. 3.5 A). In order to analyze the identity of this peak ['4C]Glc-1-P was incubated with the different GST- AtRGPs and the reactions stopped by spotting them onto TLC plates and the plates analyzed as described above. As can be seen in Figure 3.5 C, none of the GST-AtRGPs reacted with [”C]Glc-l-P. Since ["C]Glc-1-P migrated as a peak at 13.5 cms from the origin of the TLC plate (Fig. 3.5 C), the peak identified between UDP-Glc and Glc (Fig 3.5 A) is likely Glc-l-P. An AtRGPl/AtRGPZ double T-DNA insertion mutant may be lethal To study the role AtRGPs may have in plant cell wall synthesis, T-DN A insertions in all four AtRGPs were screened using the Arabidopsis Knockout Facility (http://www.biotech.wisc.edu/Arabidopsis/default.htm). Despite extensive searches, no individual T-DNA insertion mutant was identified for AtRGP3 or AtRGP4. However, three individual T-DNA insertion mutants were identified for both AtRGP] and AtRGP2, and the ones with insertions in an exon were studied further (Fig. 3.6 A). The insertion site was determined by sequencing both ends of the T-DN A. Gene-specific primers were then used to perform RT-PCR reactions using RNA from leaves of AtRGP] and AtRGP2 T-DNA insertion mutants to show that they lacked AtRGP] and AtRGP2 RNA expression, respectively (Fig 3.6 B). To confirm that the AtRGP] and AtRGP2 T-DNA insertion mutants were homozygous, the F2 progeny of each mutant were screened for the presence of the T- DNA in both copies of each gene (Fig. 3.7 A and B). Once homozygous T-DNA 86 Figure 3.4. AtRGPl and AtRGP2 auto-glycosylate. A. Purified GST-AtRGP fusion proteins were incubated with UDP-[3H]Glc, UDP-['4C]Xyl, or UDP-[”C]Gal and the reaction products analyzed by SDS-PAGE. Typical results are shown of film exposed to dried gels for at least 7 days. Purified GST-AtRGP] (1), GST-AtRGP2 (2), GST- AtRGP3 (3), and GST-AtRGP4 (4) were separated by SDS-PAGE and the gel commassie stained to show the amount of each GST-AtRGP protein used per reaction. B. Arginine- 158 is required for AtRGP] glucosylation. Arginine 158 was mutagenized to lysine or histidine in AtRGPl , and the respective GST-fusions purified. GST-AtRGP] with wild type Arginine at position 158 (R), Lysine (K) or Histidine (H), was incubated with UDP- [3H] glucose and the reaction products analyzed by SDS-PAGE. Typical results are shown of film exposed to dried gels for at least 7 days. A commassie stained protein gel shows the amount of each GST-AtRGP protein used per reaction. 87 Figure 3.4 A UDP-Glc UDP-Xyl ° W " .— UDP-Gal GST-AtRGP ‘w u... ”- w B GST-AtRGPl-Glc “ GST-AtRGP ~ w a 88 Figure 3.5. AtRGP3 and AtRGP4 cleave UDP-Glc to glucose. A. Purified GST-AtRGP fusion proteins were incubated with UDP-[3H]Glc in the presence of lmM MnClz, and the reactions products analyzed by thin layer chromatography (TLC). The radioactivity present in each section of the TLC plate was determined by scintillation counting. Diamonds stand for the reaction with GST-AtRGPl , squares for GST-AtRGP2, circles for GST-AtRGP3, and triangles for GST-AtRGP4. B. The same reactions as in A. in the absence of lmM MnClz. C. The same reactions as in A. with [”C]Glc-l-P instead of UDP-[3H]Glc. Best-fit curves were drawn by Microsoft Excel. 89 Figure 3.5 A 8 UDP-Glc Glc-l-P 7 6 DMP 5 (x1000) 4 3 2 1 0 3 4.5 6 7.5 9 10.5 12 13.5 15 16.5 18 20 (cms) B 14' 12' 10' DMP 8' (x1000) 6’ 4. 2. 0. 3 4.5 6 7.5 9 10.5 12 13.5 15 16.5 18 20 (cms) C 35 30 25 DMP 20 (x1000) 15 10 5 0 3 4.5 6 7.5 9 10.5 12 13.5 15 16.5 18 20 (cms) insertion mutants had been identified for both AtRGP] and AtRGP2 (Fig. 3.7. atrgpI-I and atrng-I, respectively), these plants were studied morphologically from germination to maturity as described in Boyes et al. (2001). No gross phenotypic alteration was observed in either AtRGP] or AtRGP2 homozygous T-DNA mutants (not shown). Despite a lack of detectable levels of AtRGP] in atrgpI -I and AtRGP2 in atrng- 1, AtRGPl protein may still be present in atrgpI -1 and AtRGP2 in atrng-I . In order to r. determine if AtRGPl was completely absent in atrgpI-I, and AtRGP2 in atrng-I, a two-dimensional (2-D) western blot analysis was carried out using AtRGPl antibodies. AtRGP] and AtRGP2 can be differentiated by 2-D SDS-PAGE, and 2-D western blot analysis using AtRGPl antibodies detected no AtRGP] in protein samples from atrgpI-I i seedlings, and no AtRGP2 in equivalent protein samples from atrng-I seedlings (not shown). A lack of phenotypic alterations in atrgpl -I and atrng-I suggest that AtRGPl and AtRGP2 may perform similar functions. To study atrgpI-I and atrng-I further, these mutants were crossed to each other to obtain a T-DNA insertion mutant lacking both AtRGP] and AtRGP2. The progeny from such a cross was shown to produce double heterozygous plants, having a single T-DNA insert in both AtRGP] and AtRGP2 (Fig. 3.7). Finally, a PCR screen of 48 individual progenies from a double heterozygous plant revealed no double T-DNA homozygous mutant plant (Fig. 3.7 C). Of the 48 plants screened, three should have been double homozygous T-DN A mutants. Likewise, although nine of each atrgp] -I and atrng-I mutant should have been rescued, only 7 atrgpI-I (Fig. 3.7. lanes 11, 16, 20, 27, 35, 46, and 48) and 5 atrng-I (Fig. 3.7. lanes 8, 14, 18, 29, and 42) mutants were identified. A similar PCR 91 screen of a second set of 48 individual progenies from the above-mentioned double heterozygous plants again did not reveal a double homozygous T-DNA mutant (not shown). The probability of not getting a single double homozygous T-DNA insertion mutant in 96 progenies of a double heterozygous AtRGPI/AtRGPZ T-DNA insertion mutant is between 1.0 and 0.1%. 92 Figure 3.6 A atrgpl -1 V AtRGP! I “U“ n E E El 3"UTR| atrng-l V AtRGP2 | “In a E WUTRI B atrgp l — 1 atrng- l 1 2 l 2 | _I- - AtRGPl— V U -AtRGP2- Figure 3.6. T-DNA insertion mutants in AtRGP] and AtRGP2. A. The location of the T- DNA inserts is indicated by downward pointing arrows (atrgpI -1 and atrng-I for AtRGP] and AtRGP2, respectively). B. RT-PCR was performed to identify individual AtRGP T-DN A mutants unable to express the AtRGP] or AtRGP2 message (lane 1 in atrgpI -1 and atrng-I, respectively). RNA samples were obtained from leaves. Gene- specific primers were used to identify the AtRGP] and AtRGP2 message in both T-DNA mutants. Ethidium bromide-stained gels of the respective reactions are shown. 93 Figure 3.7. In seach of a double atrgpI-I/atrng-I mutant. A. To identify homozygous T-DNA insertion mutants in AtRGP] and AtRGP2, PCR reactions using genomic DNA extracted from leaves of six individual atrgpI-I and atrng-I mutants were performed. AtRGPI-specific primers did not amplify AtRGP] in atrgpl -1 mutants, while AtRGP2- specific primers did not amplify AtRGP2 in atrng-I mutants. To generate a double atrgpI-I /atrgp2-1 mutant, homozygous atrgpI-I and atrng-I plants were crossed to each other and the progeny analyzed for the presence of AtRGP] and AtRGP2. As expected, all six progeny analyzed from this cross (rI-I x r2-I) were heterozygous for both AtRGP] and AtRGP2. B. To test for the presence of both T-DNAs in the progeny obtained from the cross between the atrgpI-I and atrng-I homozygous T-DNA mutants, PCR reactions were carried out using gene-specific primers and primers within the T-DNA. The presence of the T-DNAs from both T-DNA mutants (atrgpI-I and atrng-I) was confirmed in individual plants from the F1 population (rI-I x r2-1, lanes 1-5). Genomic DNA from a wild type plant was used a negative control of amplification (WT). C. The progeny from double heterozygous atrgpI-I/atrng-I plants, or the F2 population from the original cross between atrgpI -1 and atrng-I homozygous plants, was screened for the presence of a double homozygous atrgpI -I/atrgp2-I mutant. Although homozygous atrgpI-I (lanes ll, 16, 20, 27, 35, 46, and 48) and atrng-I (lanes 8, 14, 18, 29, and 42) plants were identified, no double homozygous atrgpl -1/atrgp2-I mutant plants were rescued. 94 Figure 3.7 Iii), 3.3.3336va :evamnmgummanna—nemanafixmn vaNNN—nenan 3 H.— u— m— : 2 NH 3 3 1 mm in length (10.3-12.5 days) F. Stage 1.04, four rosette leaves >1 mm in length (14.4-16.5 days) G. Stage 1.10, ten rosette leaves >1 mm in length (21.6 days) H. Stage 5.10, first flower buds visible (indicated by arrow in inset) (26.0 days) 1. Stage 6.00, first flower open (31.8 days) J. Stage 6.50, midflowering (43.5 days) K. Stage 6.90, flowering complete (49.4 days) L. Stage 9.70, senescent and ready for harvest A. to F. were determined in the early analysis platform. G. to L. were determined in the soil-based platform. 125 Figure A.3.l 126 Figure A.3.2. Growth stage progression and detection of phenotypic differences between wild type and atrgp mutants. A. Growth stage progression as determined in the plate- based platform (stages 0.1-1.04) and the soil-based platform (stages 104-6.90) for wild type (Col-0), and AtRGP] (atrgpI) and AtRGP2 (atrgp2) mutants. Boxes represent the time elapsed between the occurrence of successive growth stages. Days are given relative to the date of sowing, including a 3-day stratification at 4°C to synchronize seed germination. B. Detection of phenotypic difference between wild type and AtRGP] mutant plants. C. Detection of phenotypic difference between wild type and AtRGP2 mutant plants. The differences shown were deemed statistically significant (p<0.05) based on t-tests. The t-test score represents the number of standard deviations (std) of difference between the control mean and the mutant mean. A negative t-test score represents standard deviations below the control mean, while positive t-test scores represent standard deviations above the control mean. “n” represents the sample size or number of plants measured for either the control (ctrl) or the mutant (mut). The degrees of freedom (df) are included for reference purposes. 127 Figure A.3.2 A component component mut mut mut t test rosette 128 REFERENCES Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Gorlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. The Plant Cell 13: 1499-1510 Delgado IJ, Wang Z, de Rocher A, Keegstra K, Raikhel NV (1998) Cloning and characterization of Athpl: a reversibly autoglycosylated Arabidopsis protein implicated in cell wall biosynthesis. Plant Physiol 116: 1339-1349 Harlow E, Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Mufioz P, Norambuena L, Orellana A (1996) Evidence for a UDP-glucose transporter in Golgi aparatus-derived vesicles from pea and its possible role in polysaccharide biosynthesis. Plant Physiol 112: 1585-1594 Orellana A, Neckelmann G, Norambuena L (1997) Topography and function of Golgi uridine-S-diphosphatase from pea stems. Plant Physiol 114: 99-107 129 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII llllllllllllllllllllllllllllllllllllllHl