Doms R W, Keller D S, Helenius A, Balch W E
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Cell Biol. 1987 Nov;105(5):1957-69. doi: 10.1083/jcb.105.5.1957.
We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.
我们利用在轻度酸性蔗糖梯度中进行密度梯度离心的方法，对水泡性口炎病毒（VSV）G蛋白获得其最终寡聚体结构的过程进行了表征。成熟的野生型VSV G蛋白是一种非共价结合的三聚体。三聚体由新合成的G单体组装而成，其半衰期为6 - 8分钟。为了确定三聚化的位点，并将三聚体形成与内质网（ER）和高尔基体复合体之间运输步骤相关联，我们研究了温度敏感型突变VSV株ts045的组装动力学。在非允许温度（39℃）下，ts045 G蛋白不会从内质网转运。发现在非允许温度下抑制从内质网输出的表型缺陷是ts045 G蛋白在聚集体中的积累。转移到允许温度（32℃）后，ts045 G蛋白聚集体迅速解离（半衰期小于1分钟）为单体G蛋白，随后以与野生型G蛋白相同的动力学三聚化。只有三聚体被转运到高尔基体复合体。动力学研究以及三聚化在阻断内质网到高尔基体运输的条件下（在15℃和4℃）发生的发现表明，三聚体在内质网中形成。细胞ATP的消耗抑制了ts045 G蛋白聚集中间体的解离以及稳定三聚体的形成。结果表明，G蛋白的寡聚化分几步进行，对细胞ATP敏感，并且是从内质网运输所必需的。
