Davidson H W, Balch W E
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1993 Feb 25;268(6):4216-26.
Using the glycoprotein of the tsO45 mutant of vesicular stomatitis virus (VSV-G) as a marker, we have developed a system capable of measuring vesicular transport from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) in vitro. Movement from the ER to the cis Golgi compartment was assessed by the conversion of VSV-G from a totally endoglycosidase D (endo D)-resistant form to a species containing one endo D-resistant and one endo D-sensitive oligosaccharide (GD1). Similarly, delivery to the medial cisternae was measured by the appearance of the completely endo D-sensitive form of VSV-G (GD2) or by the acquisition of complete resistance to endoglycosidase H (endo H) (GHr) and delivery to the TGN by the appearance of an endo H-resistant form of VSV-G which was sensitive to digestion with neuraminidase and subsequently beta-galactosidase (GHt). Movement between each sequential compartment required ATP and soluble proteins (cytosol) and was inhibited by nonhydrolyzable analogues of GTP and by an antibody toward the N-ethylmaleimide-sensitive factor NSF. In contrast, fractionation of the cytosol by ammonium sulfate precipitation indicated that distinct proteins were required for movement between successive compartments. Similarly, inclusion of a mutant form of the small molecular weight GTP-binding protein rab1A inhibited movement between the ER and cis Golgi, and between the cis and medial cisternae, but did not affect transport from the medial Golgi to the TGN. Conversely, the protein kinase inhibitor staurosporine prevented movement between the medial Golgi and the TGN but did not influence transport between the ER and early Golgi compartments. This study provides the first demonstration that vesicular transport between the ER and TGN can be reconstituted in a cytosol-dependent fashion in vitro, allowing a direct analysis of the roles of individual components in multiple transport events.
利用水泡性口炎病毒（VSV）tsO45突变体的糖蛋白作为标记，我们开发了一种能够在体外测量从内质网（ER）到反式高尔基体网络（TGN）的囊泡运输的系统。通过将VSV-G从完全抗内切糖苷酶D（内切D）的形式转化为含有一个抗内切D和一个对内切D敏感的寡糖（GD1）的形式，评估从ER到顺式高尔基体区室的转运。同样，通过VSV-G完全对内切D敏感的形式（GD2）的出现或通过获得对内切糖苷酶H（内切H）的完全抗性（GHr）来测量向中间潴泡的转运，并通过出现对神经氨酸酶和随后的β-半乳糖苷酶（GHt）消化敏感的抗内切H形式的VSV-G来测量向TGN的转运。每个连续区室之间的转运需要ATP和可溶性蛋白质（胞质溶胶）并且被GTP的不可水解类似物和针对N-乙基马来酰亚胺敏感因子NSF的抗体所抑制。相反，通过硫酸铵沉淀对胞质溶胶进行分级分离表明，连续区室之间的转运需要不同的蛋白质。同样，包含小分子量GTP结合蛋白rab1A的突变形式会抑制ER和顺式高尔基体之间以及顺式和中间潴泡之间的转运，但不影响从中间高尔基体到TGN的运输。相反，蛋白激酶抑制剂星形孢菌素阻止了中间高尔基体和TGN之间的转运，但不影响ER和早期高尔基体区室之间的运输。这项研究首次证明，ER和TGN之间的囊泡运输可以在体外以胞质溶胶依赖性方式重建，从而可以直接分析单个成分在多个运输事件中的作用。
