Lacey M L, Haimo L T
Department of Biology, University of California, Riverside 92521.
J Biol Chem. 1992 Mar 5;267(7):4793-8.
Microtubule-based organelle transport is thought to be mediated by the force-generating proteins cytoplasmic dynein and kinesin. These motor proteins have been characterized based on their ability to associate with and translocate microtubules. We show here that cytoplasmic dynein is also present as a peripheral membrane protein of purified synaptic vesicles. The vesicle-associated cytoplasmic dynein is identified by its photo-induced cleavage in the presence of ATP and vanadate. Purified, soluble cytoplasmic dynein is competent to bind to vesicle membranes stripped of endogenous peripheral membrane proteins by alkaline pH. Dynein binding to membranes is saturable at a concentration of 1.00 +/- 0.15 pmol/micrograms vesicle protein and has a dissociation constant of 22.3 +/- 2.4 nM. The association of cytoplasmic dynein with the membrane cannot be reversed by incubation with ATP. Furthermore, following binding to membranes, dynein retains its ability to bind ATP and to be photo-cleaved in the presence of vanadate. The presence of cytoplasmic dynein on synaptic vesicles and its ability to bind to extracted membranes supports current models of microtubule-based organelle translocation.
基于微管的细胞器运输被认为是由产生力的蛋白质胞质动力蛋白和驱动蛋白介导的。这些运动蛋白已根据它们与微管结合并使其移位的能力进行了表征。我们在此表明，胞质动力蛋白也以纯化突触小泡的外周膜蛋白形式存在。通过在ATP和钒酸盐存在下其光诱导切割来鉴定与小泡相关的胞质动力蛋白。纯化的可溶性胞质动力蛋白能够结合通过碱性pH去除内源性外周膜蛋白的小泡膜。动力蛋白与膜的结合在浓度为1.00±0.15 pmol/微克小泡蛋白时达到饱和，解离常数为22.3±2.4 nM。胞质动力蛋白与膜的结合不能通过与ATP孵育来逆转。此外，在与膜结合后，动力蛋白保留其结合ATP并在钒酸盐存在下被光切割的能力。突触小泡上存在胞质动力蛋白及其与提取膜结合的能力支持了当前基于微管的细胞器转运模型。
