title: AtCSLD2 is an integral Golgi membrane protein with its N-terminus facing the cytosol.
abstract: Cellulose synthase-like proteins in the D family share high levels of sequence identity with the cellulose synthase proteins and also contain the processive beta-glycosyltransferase motifs conserved among all members of the cellulose synthase superfamily. Consequently, it has been hypothesized that members of the D family function as either cellulose synthases or glycan synthases involved in the formation of matrix polysaccharides. As a prelude to understanding the function of proteins in the D family, we sought to determine where they are located in the cell. A polyclonal antibody against a peptide located at the N-terminus of the Arabidopsis D2 cellulose synthase-like protein was generated and purified. After resolving Golgi vesicles from plasma membranes using endomembrane purification techniques including two-phase partitioning and sucrose density gradient centrifugation, we used antibodies against known proteins and marker enzyme assays to characterize the various membrane preparations. The Arabidopsis cellulose synthase-like D2 protein was found mostly in a fraction that was enriched with Golgi membranes. In addition, versions of the Arabidopsis cellulose synthase-like D2 proteins tagged with a green fluorescent protein was observed to co-localize with a DsRed-tagged Golgi marker protein, the rat alpha-2,6-sialyltransferase. Therefore, we postulate that the majority of Arabidopsis cellulose synthase-like D proteins, under our experimental conditions, are likely located at the Golgi membranes. Furthermore, protease digestion of Golgi-rich vesicles revealed almost complete loss of reaction with the antibodies, even without detergent treatment of the Golgi vesicles. Therefore, the N-terminus of the Arabidopsis cellulose synthase-like D2 protein likely faces the cytosol. Combining this observation with the transmembrane domain predictions, we postulate that the large hydrophilic domain of this protein also faces the cytosol.
authors.affiliation: DOE-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. zengweiq@msu.edu
authors.forename: Weiqing
authors.initials: W
authors.lastname: Zeng
authors.forename: Kenneth
authors.initials: K
authors.lastname: Keegstra
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000378:metabolism*; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins