Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase
Oligomerization and topology of the Golgi membrane protein glucosylceramide synthase
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DOI：
10.1074/jbc.274.1.451
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发表时间：
1999-01-01
影响因子：
4.8
通讯作者：
Pagano, RE
中科院分区：
文献类型：
--
作者：
Marks, DL;Wu, KJ;Pagano, RE
葡萄糖神经酰胺合酶 (GCS) 催化葡萄糖从 UDP-葡萄糖转移到神经酰胺，形成葡萄糖神经酰胺，这是大多数高级糖鞘脂的前体。最近，我们表征了大鼠肝高尔基体膜高度富集级分中的 GCS 活性（Paul, P.、Kamisaka, Y.、Marks, D. L. 和 Pagano, R. E. (1996) J. Biol. Chem. 271, 2287-2293），并且其他人克隆了人类 GCS（Ichikawa, S.、Sakiyama, H.、Suzuki, G.、Hidari, K. I.-P, J. 和 Hirabayashi, Y, (1996) 美国科学院院刊，A. 93, 4638-4643）。然而，负责GCS活性的多肽从未被鉴定或表征。在本研究中，我们根据预测的人GCS氨基酸序列制备了针对肽的多克隆抗体，并使用这些抗体来表征大鼠肝凝胶膜中的GCS多肽。对大鼠肝高尔基体膜、人类细胞和细菌中表达的重组大鼠 GCS 进行的蛋白质印迹表明，GCS 在 SDS 聚丙烯酰胺凝胶上以类似于 38 kDa 的蛋白质的形式迁移。高尔基膜的胰蛋白酶消化和免疫沉淀研究表明，GCS 的 C 末端和 N 末端附近的亲水环均可从高尔基膜的胞质面接近。用基于 N-羟基琥珀酰亚胺酯的交联剂处理高尔基体膜，产生与抗 GCS 抗体识别的类似 50-kDa 多肽；然而，用相同的试剂处理类似于 10,000 倍纯化的高尔基体 GCS 并没有产生交联的 GCS 形式。这些结果表明GCS与高尔基体膜中的另一种蛋白质形成二聚体或寡聚体。溶解的高尔基体GCS在甘油梯度中的迁移也与GCS的主要寡聚组织一致。
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 Gels membranes. Western blotting of rat liver Golgi membranes, human cells, and recombinant rat GCS expressed in bacteria showed that GCS migrates as an similar to 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 similar to 50-kDa polypeptide recognized by anti-GCS antibodies; however, treatment of similar to 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.