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的热震 蛋白质及其在正常细胞过程和热休克中的作用。 首先，我们正在调查70 kDa的唯一已定义的功能之一 热休克蛋白--70 kDa脱壳(UC)ATPase的能力 从牛脑中分离出包被蛋白的囊泡中的蛋白 在三磷酸腺苷依赖的反应中。我们之前发现，当UC ATPase 与结合的三磷酸腺苷与包被囊泡混合后，有一次最初的爆发 脱膜后缓慢稳定脱膜。基于这些数据，我们 提出了一个简单的模型，其中UC ATPase只有一个结合位点 ATP和一个与笼状蛋白结合的位点。这与更多的 施密德和罗斯曼提出的复杂模型，其中UC ATPase 两个网状蛋白位点和两个三磷酸腺苷位点。我们现在已经获得了数据 这表明UC ATPase实际上只有一个网状蛋白和 一个用于ATP的站点。我们的数据还表明，ATP和ADP都非常结合 与UC ATPase强烈结合(Kbindingca=1 x 108)，从而使UC ATPase、AS 通常制备的每个分子含有大约一个结合的ADP，这是 很难去除。然而，移除此绑定的ADP并不 变性UC-ATPase。我们还研究了三磷酸腺苷酶的活性。 UC ATPase。在没有底物的情况下，UC ATPase没有显示出初始 三磷酸腺苷水解酶的爆发。然而，在组装蛋白存在的情况下 它们需要在包被的囊泡上聚合网状蛋白，非常快速 观察到ATP水解酶的初始猝发。与之相反的是，clathrin可以 不会引起最初的ATP水解性猝发。这些数据表明， 组装蛋白是UC ATPase非常特殊的底物，可能 在脱膜过程中起到重要作用。除了这些酶之外 研究表明，我们克隆了牛脑UC-ATPase基因，并对其进行了表达 放在酵母里。我们之前证明了三种酵母70 kDa蛋白质，至少 其中一种必须存在才能保证酵母的活性，显示出功能性 与牛脑UC ATPase相似。现在进行初步实验 提示牛脑UC-ATPase可以替代这三种酶 酵母70 kDa蛋白质对维持酵母活力的作用。

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. We previously found that, when the UC ATPase with bound ATP is mixed with coated vesicles, there is an initial burst of uncoating followed by slow steady-state uncoating. Based on these data we proposed a simple model where the UC ATPase has only one binding site for ATP and one binding site for clathrin. This contrasts with a more complicated model proposed by Schmid and Rothman where the UC ATPase has two sites for clathrin and two sites for ATP. We have now obtained data which show that the UC ATPase, indeed, has only one site for clathrin and one site for ATP. Our data also show that both ATP and ADP bind very strongly to the UC ATPase (Kbinding ca= 1 x 108) so that the UC ATPase, as it is normally prepared contains ca one bound ADP per molecule which is very difficult to remove. However removal of this bound ADP does not denature the UC ATPase. We have also studied the ATPase activity of the UC ATPase. In the absence of substrate the UC ATPase shows no initial burst of ATP hydrolysis. However, in the presence of the assembly proteins which are required to polymerize clathrin on coated vesicles, a very rapid initial burst of ATP hydrolysis is observed. In contrast, clathrin does not cause an initial burst of ATP hydrolysis. These data suggest that the assembly proteins are a very specific substrate of the UC ATPase and may be important in the uncoating process. In addition to these enzymatic studies, we have cloned the bovine brain UC ATPase and have expressed it in yeast. We previously showed that three yeast 70 kDa proteins, at least one of which must be present for yeast viability, show a functional similarity to the bovine brain UC ATPase. Preliminary experiments now suggest that the bovine brain UC ATPase can substitute for these three yeast 70 kDa proteins in keeping the yeast viable.