Hemicellulosic polysaccharides are produced in the Golgi apparatus. The enzymes responsible for synthesizing the glycan "backbones" of the hemicellulosic polysaccharides xyloglucan and galactomannan have been identified. CSLA proteins synthesize the (gluco)mannan backbone of galacto(gluco)mannan, while CSLC proteins are the best candidates for to synthesize the glucan backbone of xyloglucan (Liepman et al. 2007, Cocuron et al. 2007). CSLA and CSLC proteins have multiple transmembrane domains, and the topology of these proteins is likely to impact how they function. For example, a CSL protein with a cytosolic active site may couple synthesis with translocase activity similarly to cellulose synthase. Alternatively, glycan synthesis by a protein with an active site in the Golgi lumen might be dependent on sugar-nucleotide transporters. Here I present fluorescent protein localization by confocal microscopy showing that CSLA and CSLC proteins reside in Golgi membranes. The colocalization of three proteins involved in the biosynthesis of xyloglucan (CSLC4, XXT1, and FUT1) within the secretory pathway was also examined. I probed the topologies of CSLA and CSLC proteins by protease protection methods designed to determine the position of the proteins' termini and their active sites. I show that AtCSLC4, a Golgi-localized xyloglucan glucan synthase, has a topology characterized by a cytosolic active site and an even number (likely 6) of transmembrane domains. Whereas the most abundantly expressed mannan synthase in Arabidopsis, AtCSLA9, is predicted to have 5 TMDs and has an active site facing the Golgi lumen. These topologies have important implications for understanding the biosynthesis of cell wall polysaccharides.
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