AtCSLD2 is an integral Golgi membrane protein with its N-terminus facing the cytosol.
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.
Zeng
Weiqing
W
DOE-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. zengweiq@msu.edu
Keegstra
Kenneth
K
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
D029681:Arabidopsis Proteins; D008565:Membrane Proteins