70 KDA HEAT SHOCK PROTEINS AND THE HOMOLOGOUS UNCOATING ATPASE

我们实验室正在研究70 kDa热休克蛋白在 各种细胞过程。具体地说，我们正在研究这些属性 从牛脑中分离出70 kDa脱壳(UC)ATPase 从包被网状蛋白的囊泡中去掉网状蛋白。我们之前发现， 当结合了ATP的UC ATPase与包被的囊泡混合时， 先是化学计量的脱涂层，然后是缓慢稳定的- 状态为脱漆。在此基础上，我们提出了一个简单的模型，其中UC ATPase有一个与ATP的结合部位，一个与笼状蛋白的结合部位，以及一个化学计量结合部位 三磷酸腺苷的水解伴随着脱膜。相比之下，施密德和罗斯曼 提出了一个更复杂的模型，该模型包含两个网状蛋白和两个网状结构。 为三磷酸腺苷。在我们目前的工作中，我们首先确认了UC ATPase 只有一个位置用于笼状蛋白，一个位置用于ATP。第二，我们研究了 网状蛋白篮法测定UC-ATPase的活性 纯化的网状蛋白和组装蛋白；网状蛋白篮子 比包衣囊泡更清洁的检测系统，后者具有相当大的 外源ATPase活性。使用由 组装蛋白，AP180，我们发现了一个化学计量比的初始爆发 揭开涂层。与最初的脱涂层有关的是一种 ATPase活性的初始猝发，每3个ATP分子被水解 未涂覆的笼状三倍体。因此，这些研究支持UC的模型 我们之前提出的ATPase操作。然而，令人惊讶的是，我们 还观察到需要一种热稳定的蛋白质辅因子来 去涂层才能发生。除了这些关于去涂层的研究外，我们还发现 UC ATPase的其他肽底物结合到与 网状蛋白。此外，结合的核苷酸对 网状蛋白和其他多肽底物与UC-ATPase的结合。使用 当与底物结合时，结合的ATP会迅速附着和脱落 与ADP结合后，贴壁率和剥离率均大大降低。 令人惊讶的是，在没有结合核苷酸的情况下，这种酶的作用就像是它 结合了ATP而不是结合了ADP，这表明结合的ADP可能 改变酶的构象，使底物附着和脱离 都被屏蔽了。

Our laboratory is studying the role of the 70-kDa heat shock proteins in various cellular processes. Specifically, we are studying the properties of the 70-kDa uncoating (UC) ATPase isolated from bovine brain which uncoats clathrin from clathrin coated vesicles. We previously found that, when the UC ATPase with bound ATP was mixed with coated vesicles, there was a stoichiometric initial burst of uncoating followed by slow steady- state uncoating. On this basis we proposed a simple model where the UC ATPase has one binding site for ATP, one for clathrin, and stoichiometric ATP hydrolysis accompanies uncoating. In contrast Schmid and Rothman proposed a more complex model with two sites for clathrin and two sites for ATP. In our current work we first confirmed that the UC ATPase has only one site for clathrin and one site for ATP. Second, we studied the ATPase activity of the UC ATPase using clathrin baskets prepared with purified clathrin and assembly proteins; clathrin baskets are a much cleaner assay system than coated vesicles which have considerable extraneous ATPase activity. Using clathrin baskets prepared with the assembly protein, AP180, we found a stoichiometric initial burst of uncoating. Associated with this initial burst of uncoating there was an initial burst of ATPase activity with about 3 ATP molecules hydrolyzed per clathrin triskelion uncoated. Thus these studies support the model of UC ATPase action which we previously proposed. Surprisingly, however, we also observed that a heat-stable protein cofactor is required for uncoating to occur. In addition to these studies on uncoating, we found that other peptide substrates of the UC ATPase bind to the same site as clathrin. Furthermore, bound nucleotide has a very specific effect on the binding of clathrin and other peptide substrates to the UC ATPase. With bound ATP, attachment and detachment of substrate occur rapidly while with bound ADP the rates of attachment and detachment are greatly decreased. Surprisingly, in the absence of bound nucleotide, the enzyme acts as if it had bound ATP rather than bound ADP suggesting that the bound ADP may alter the enzyme conformation so that substrate attachment and detachment are blocked.