RGD Reference Report - Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase. - Rat Genome Database

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Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase.

Authors: Marks, D L  Wu, K  Paul, P  Kamisaka, Y  Watanabe, R  Pagano, R E 
Citation: Marks DL, etal., J Biol Chem. 1999 Jan 1;274(1):451-6.
RGD ID: 14390055
Pubmed: PMID:9867864   (View Abstract at PubMed)

Glucosylceramide synthase (GCS) catalyzes the transfer of glucose from UDP-glucose to ceramide to form glucosylceramide, the precursor of most higher order glycosphingolipids. Recently, we characterized GCS activity in highly enriched fractions from rat liver Golgi membranes (Paul, P., Kamisaka, Y., Marks, D. L., and Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293), and human GCS was cloned by others (Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K. I.-P. J., and Hirabayashi, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638-4643). However, the polypeptide responsible for GCS activity has never been identified or characterized. In this study, we made polyclonal antibodies against peptides based on the predicted amino acid sequence of human GCS and used these antibodies to characterize the GCS polypeptide in rat liver Golgi membranes. Western blotting of rat liver Golgi membranes, human cells, and recombinant rat GCS expressed in bacteria showed that GCS migrates as an approximately 38-kDa protein on SDS-polyacrylamide gels. Trypsinization and immunoprecipitation studies with Golgi membranes showed that both the C terminus and a hydrophilic loop near the N terminus of GCS are accessible from the cytosolic face of the Golgi membrane. Treatment of Golgi membranes with N-hydroxysuccinimide ester-based cross-linking reagents yielded an approximately 50-kDa polypeptide recognized by anti-GCS antibodies; however, treatment of approximately 10,000-fold purified Golgi GCS with the same reagents did not yield cross-linked GCS forms. These results suggest that GCS forms a dimer or oligomer with another protein in the Golgi membrane. The migration of solubilized Golgi GCS in glycerol gradients was also consistent with a predominantly oligomeric organization of GCS.



Gene Ontology Annotations    Click to see Annotation Detail View

Cellular Component

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
UgcgRatGolgi membrane located_inIDA PMID:9867864UniProt 
UgcgRatmembrane located_inIDA PMID:9867864UniProt 

Objects Annotated

Genes (Rattus norvegicus)
Ugcg  (UDP-glucose ceramide glucosyltransferase)


Additional Information