Ngsee J K, Fleming A M, Scheller R H
Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Howard Hughes Medical Institute, Stanford University, California 94305.
Mol Biol Cell. 1993 Jul;4(7):747-56. doi: 10.1091/mbc.4.7.747.
Low molecular weight (LMW) GTP-binding proteins are hypothesized to play a role in the vectorial transport of intracellular vesicles. Mutational studies in yeast and subcellular localization in mammalian cells suggest that a family of LMW GTP-binding proteins, termed rab, target intracellular vesicles to their appropriate acceptor compartment. In this report, we demonstrate that an elasmobranch homologue of rab3A, o-rab3, plays a significant role in the sequestration of regulated secretory vesicles. When transfected into the murine endocrine cell line AtT-20, the wild-type o-rab3 protein is localized exclusively to the tips of the processes, a region of the cell known to accumulate proteins associated with regulated secretory vesicles. Two mutations, Gln81 to Leu (Q81L) and Asn135 Ile (N135I), which alter GTP binding or rate of hydrolysis, blocked the localization of the o-rab3 protein to the tips of cell processes. These mutations also hindered the sequestration of ACTH-containing secretory vesicles to the process tips but did not affect the basal or stimulated release of ACTH. Moreover, the sequestration of the protein VAMP to the process tip was also hindered by the mutation. The results demonstrate a role for the rab3 proteins in localization, sequestration, and storage of secretory vesicles near their release site.
低分子量（LMW）GTP结合蛋白被认为在细胞内囊泡的向量运输中发挥作用。酵母中的突变研究和哺乳动物细胞中的亚细胞定位表明，一类称为rab的LMW GTP结合蛋白可将细胞内囊泡靶向至其合适的受体区室。在本报告中，我们证明rab3A的一种软骨鱼同源物o-rab3在调节性分泌囊泡的隔离中起重要作用。当野生型o-rab3蛋白转染到鼠内分泌细胞系AtT-20中时，它仅定位于突起的尖端，该区域是细胞中已知会积累与调节性分泌囊泡相关蛋白质的区域。两个改变GTP结合或水解速率的突变，即Gln81突变为Leu（Q81L）和Asn135突变为Ile（N135I），阻止了o-rab3蛋白定位于细胞突起的尖端。这些突变还阻碍了含促肾上腺皮质激素（ACTH）的分泌囊泡隔离至突起尖端，但不影响ACTH的基础释放或刺激释放。此外，突变也阻碍了蛋白VAMP隔离至突起尖端。结果表明rab3蛋白在分泌囊泡在其释放位点附近的定位、隔离和储存中发挥作用。
