70 KDA HEAT SHOCK PROTEINS AND THE HOMOLOGOUS UNCOATING ATPASE

我们实验室的总体重点是研究70 kDa的热 休克蛋白及其在正常细胞过程和细胞周期中的作用 热休克。首先，我们正在调查唯一被定义为 70 kDa热休克蛋白的功能--70 kDa的能力 去包被牛脑中分离的(UC)ATPase以去除笼状蛋白 在ATP依赖的反应中从被包裹的小泡中提取。在……里面 与早先的报道相反，UC ATPase 催化从包被的囊泡中去除笼蛋白，我们的电流 结果表明UC ATPase可以去除笼状蛋白 化学计量学上，一个酶分子与每个 三条网状突起腿。由此产生的酶-笼蛋白复合体是 在溶液中至少稳定24小时，结合的酶是 不能揭开新添加的涂层小泡的外衣。除了……之外 结合紧密，我们也有证据表明UC ATPase 将ADP绑定得非常紧密，这可能解释了为什么 在许多其他的ATPase中，70 kDa的蛋白质与ATP结合得如此紧密 地缘柱。令人惊讶的是，与紧绑在一起的 从包膜上解离出来的酶到网状蛋白 囊泡中，该酶似乎不与游离膜蛋白结合。 解决方案，表明一种特殊的复合体正在形成 UC ATPase将被包裹的囊泡中的网状蛋白解离。我们也 有证据表明去涂层反应可能受 包被囊泡的磷酸化。除了这些之外， 牛脑UC-ATPase的研究，我们考察了它的能力 从酵母菌中分离出70 kDa蛋白质用于牛脑脱壳 包被网状蛋白的囊泡。我们的结果表明，酵母菌70 kDa 蛋白质的作用远不如牛脑UC-ATPase有效； 需要5到10倍的酵母酶才能完成同样的任务 由脑酶进行的脱涂层的量。自.以来 酵母70 kDa蛋白质由几种同工酶组成，我们是 调查这种低脱涂层活跃度是否是由于 这些同工酶中只有一种的活性，或者酵母 一般来说，蛋白质的活性比大脑UC低得多 ATPase。

The overall focus of our laboratory is the study of the 70-kDa heat shock proteins and their role in both normal cellular processes and heat shock. First, we are investigating one of the only defined functions of a 70-kDa heat shock protein-the ability of the 70-kDa uncoating (UC) ATPase isolated from bovine brain to remove clathrin from clathrin coated vesicles in an ATP dependent reaction. In contrast to earlier reports suggesting that the UC ATPase catalytically removes clathrin from coated vesicles, our current results suggest that the UC ATPase removes clathrin stoichiometrically with one enzyme molecule binding to each of the three clathrin legs. The resulting enzyme-clathrin complex is stable for at least 24 hours in solution, and the bound enzyme is not able to uncoat freshly added coated vesicles. In addition to binding clathrin tightly, we also have evidence that the UC ATPase binds ADP extremely tightly which may explain why, in contrast to the many other ATPases, the 70-kDa proteins bind so tightly to ATP affinity columns. Surprisingly, in contrast to the tight binding of the enzyme to clathrin which it has dissociated from coated vesicles, the enzyme does not appear to bind to free clathrin in solution, suggesting that a special kind of complex is forming when the UC ATPase dissociates clathrin from coated vesicles. We also have evidence that the uncoating reaction may be controlled by phosphorylation of the coated vesicles. In addition to these studies on bovine brain UC ATPase, we have investigated the ability of the 70-kDa proteins isolated from yeast to uncoat bovine brain clathrin coated vesicles. Our results show that, the yeast 70-kDa proteins are much less effective than the bovine brain UC ATPase; 5 to 10-fold more yeast enzyme is required to carry out the same amount of uncoating as carried out by the brain enzyme. Since the yeast 70-kDa proteins are composed of several isoenzymes, we are investigating whether this low uncoating activity is due to full activity of only one of these isoenzymes, or whether the yeast proteins, in general, are much less active than the brain UC ATPase.