Schwaninger R, Beckers C J, Balch W E
Department of Cell, Biology Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1991 Jul 15;266(20):13055-63.
The vectorial transport of vesicular stomatitis virus (VSV) G protein between the ER and the cis and medial Golgi compartments has been reconstituted using semi-intact (perforated) cells. The transport of VSV-G protein between successive compartments is measured by the sequential processing of the two N-linked oligosaccharide chains present on VSV-G protein to the endoglycosidase (endo) H-resistant structures which have unique electrophoretic mobilities during sodium dodecyl sulfate-gel electrophoresis. The appearance of a form of VSV-G which contains only one endo H-resistant oligosaccharide chain (GH1) is kinetically and biochemically indistinguishable from the appearance of the Man5, endo D-sensitive form (GD), the latter being a processing reaction diagnostic of transport from the ER to the cis Golgi compartment. These results provide evidence that the cis Golgi compartment may contain in addition to alpha-1,2-mannosidase I, both N-acetylglueosamine transferase I and alpha-1,2-mannosidase II. VSV-G protein is subsequently processed to the form which contains two endo H-resistant oligosaccharides (GH2) after a second wave of vesicular transport. Processing of GH1 to GH2 in vitro occurs only after a lag period following the appearance of GH1; processing is sensitive to N-ethylmaleimide, guanosine-5'-O-(3-thiotriphosphate), and a synthetic peptide homologous to the rab1 protein effector domain, and processing is inhibited in the absence of free Ca2+ (in the presence of EGTA), reagents which potently inhibit ER to cis Golgi transport. These results suggest that VSV-G protein proceeds through at least two rounds of vesicular transport from the ER to the medial Golgi compartment for processing to the GH2 form, providing a model system to study the regulation of the vectorial membrane fission and fusion events involved in vesicular trafficking and organelle dynamics in the early stages of the secretory pathway.
利用半完整（穿孔）细胞重建了水泡性口炎病毒（VSV）G蛋白在内质网与顺式和中间高尔基体区室之间的向量运输。通过将VSV - G蛋白上存在的两条N - 连接寡糖链顺序加工成在十二烷基硫酸钠 - 凝胶电泳期间具有独特电泳迁移率的内切糖苷酶（endo）H抗性结构，来测量VSV - G蛋白在连续区室之间的运输。仅包含一条内切糖苷酶H抗性寡糖链（GH1）的VSV - G形式的出现，在动力学和生物化学上与Man5、内切糖苷酶D敏感形式（GD）的出现没有区别，后者是从内质网到顺式高尔基体区室运输的诊断性加工反应。这些结果提供了证据，表明顺式高尔基体区室除了含有α - 1,2 - 甘露糖苷酶I之外，还可能含有N - 乙酰葡糖胺转移酶I和α - 1,2 - 甘露糖苷酶II。随后，在第二轮小泡运输后，VSV - G蛋白被加工成含有两条内切糖苷酶H抗性寡糖（GH2）的形式。GH1在体外加工成GH2仅在GH1出现后的延迟期之后发生；加工对内马来酰亚胺、鸟苷 - 5'-O - （3 - 硫代三磷酸）以及与rab1蛋白效应结构域同源的合成肽敏感，并且在没有游离Ca2 + （在EGTA存在下）时加工受到抑制，这些试剂强烈抑制内质网到顺式高尔基体的运输。这些结果表明，VSV - G蛋白从内质网到中间高尔基体区室至少经过两轮小泡运输才能加工成GH2形式，提供了一个模型系统来研究分泌途径早期阶段小泡运输和细胞器动态中涉及的向量膜分裂和融合事件的调控。
