Schweizer A, Clausen H, van Meer G, Hauri H P
Department of Pharmacology, University of Basel, Switzerland.
J Biol Chem. 1994 Feb 11;269(6):4035-41.
The identification of an endoplasmic reticulum-Golgi intermediate compartment (ERGIC), defined by the 53-kDa transmembrane marker protein ERGIC-53, has added to the complexity of the exocytic pathway of higher eukaryotic cells. Recently, a subcellular fractionation procedure was established for the isolation of the ERGIC from Vero cells (Schweizer, A., Matter, K., Ketcham, C. M., and Hauri, H.-P. (1991) J. Cell Biol. 113, 45-54) which provides a means to study more precisely the compartmentalization of the various enzymic functions along the early secretory pathway. Here, we have investigated if O-glycan initiation and sphingomyelin synthesis are associated with the ERGIC by analyzing both the responsible enzyme activities and their corresponding products. Moreover, the synthesis of glucosylceramide, the precursor of most glycosphingolipids, was also analyzed. In the purified ERGIC fraction UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase) was only minimally enriched, sphingomyelin synthase was not enriched, and UDP-glucose:ceramide-glucosyl transferase specific activity was lower than in the homogenate. On Percoll gradients all three enzymes cofractionated with Golgi markers rather than ERGIC-53. Accordingly, sphingomyelin concentrations were extremely low in the ERGIC fraction. Double immunofluorescence localization of core N-acetylgalactosamine, the product of GalNAc transferase, by monoclonal antibodies against GalNAc-Ser/Thr (Tn antigen) revealed only little apparent overlap with ERGIC-53. This was particularly evident in brefeldin A-treated cells which showed entirely different patterns of Tn antigens and ERGIC-53. The results suggest that in the secretory pathway of Vero cells O-glycan initiation and sphingomyelin as well as glucosylceramide synthesis mainly occur beyond the ERGIC in the Golgi apparatus.
由53-kDa跨膜标记蛋白ERGIC-53所定义的内质网-高尔基体中间区室（ERGIC）的鉴定，增加了高等真核细胞胞吐途径的复杂性。最近，建立了一种亚细胞分级分离程序，用于从Vero细胞中分离ERGIC（施韦泽，A.，马特，K.，凯查姆，C.M.，和豪里，H.-P.（1991年）《细胞生物学杂志》113卷，45 - 54页），这为更精确地研究早期分泌途径中各种酶功能的区室化提供了一种方法。在这里，我们通过分析相关酶活性及其相应产物，研究了O-聚糖起始和鞘磷脂合成是否与ERGIC相关。此外，还分析了大多数糖鞘脂的前体葡糖神经酰胺的合成。在纯化的ERGIC级分中，UDP-GalNAc：多肽N-乙酰半乳糖胺基转移酶（GalNAc转移酶）仅略有富集，鞘磷脂合酶未富集，且UDP-葡萄糖：神经酰胺-葡糖基转移酶的比活性低于匀浆。在Percoll梯度上，所有这三种酶都与高尔基体标记物共分级分离，而不是与ERGIC-53共分级分离。因此，ERGIC级分中的鞘磷脂浓度极低。针对GalNAc-Ser/Thr（Tn抗原）的单克隆抗体对GalNAc转移酶产物核心N-乙酰半乳糖胺的双重免疫荧光定位显示，与ERGIC-53的明显重叠很少。这在布雷菲德菌素A处理的细胞中尤为明显，这些细胞显示出Tn抗原和ERGIC-53完全不同的模式。结果表明，在Vero细胞的分泌途径中，O-聚糖起始、鞘磷脂以及葡糖神经酰胺的合成主要发生在ERGIC之后的高尔基体中。
