70 KDA HEAT SHOCK PROTEINS AND THE HOMOLOGOUS UNCOATING ATPASE

我们实验室的总体重点是研究70kda的热量

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.