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J Biol Chem, Vol. 274, Issue 1, 451-456, January 1, 1999
From the Department of Biochemistry and Molecular Biology, Thoracic
Diseases Research Unit, Mayo Clinic and Foundation,
Rochester, Minnesota 55905
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 ~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 ~50-kDa polypeptide recognized by
anti-GCS antibodies; however, treatment of ~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.
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