70-kda Heat Shock Proteins And Their Associated Cofactors

National Heart, Lung, And Blood Institute

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Abstract

Our laboratory is interested in the formation and dissolution of both normal and pathological protein complexes in the cell with an emphasis on the role of molecular chaperones in this process. We have continued our studies on endocytosis, examining what drives the assembly of the clathrin-coated pit. We previously showed that during, clathrin-mediated endocytosis, both clathrin and AP2 on clathrin-coated pits exchanged with free clathrin and AP2 in cells. To better understand the mechanism of clathrin and AP2 exchange, these processes were examined in a cell-free system. We found that Hsc70 was not only required for dissociation of clathrin but, in addition, by binding to the dissociated clathrin, it also facilitated the rebinding of clathrin to the pits. As for the exchange of AP2, we found that it was caused by a cycle of phosphorylation and dephosphorylation of the beta chain of AP2 with the former causing dissociation and the latter rebinding of AP2. Finally, we found that clathrin-coated pits were able to be regenerated by addition of cytosol provided that both PIP2 and a small nucleus of AP2 were present on the plasma membrane. In contrast, irreversible dissolution of the pits occurred upon complete dissociation of AP2 or a decrease in the PIP2 level. [unreadable] [unreadable] Aside from our research on clathrin-mediated endocytosis, we have focused on the propagation of prions, which are infective proteins having an amyloid conformation. In yeast, the molecular chaperone, Hsp104, regulates the inheritance of several yeast prions including PSI+, which is the prion form of the translation termination factor Sup35p. By using live cell imaging, we found that Sup35p-GFP in the non-prion form was diffuse in psi- cells, while Sup35p-GFP in prion-form was aggregated in PSI+ cells. To test whether these foci are the actual seeds or propagons, which are responsible for maintaining the prion phenotype, we examined whether the number of foci in yeast cells correlate with the extent of curing of PSI+ when yeast are cured by inactivation of Hsp104 using a dominant negative Hsp104 mutant. Imaging of the cells followed by plating showed there is a direct correlation between the number of cells with foci and the curing of the PSI+ phenotype. In addition, we re-examined the fluorescence changes that occur upon guanidine addition, which also cure PSI+ yeast by inactivating Hsp104. In agreement with our previous studies, we found in the presence of guanidine, there are no visible foci in yeast that are still PSI+. However, we now find that upon stress treatments, the foci become visible in these cells and show a direct correlation between the percentage of cells having foci and the extent of curing. Therefore, curing cells either using the dominant negative Hsp104 mutant or guanidine treatment to inactivated Hsp104 yield data that are consistent with a model in which inhibiting Hsp104 prevents the seeds or propagons from replicating and cell division is required for dilution of the remaining seeds or propagons to cure yeast of the prion phenotype. [unreadable] [unreadable] We also have been examining the trafficking of prion protein, both the native form and the amyloid form of the protein called scrapie, in mouse neuronal cell lines. Cells were depleted of clathrin using RNA interference to determine whether clathrin-mediated endocytosis was required for the internalization of prion protein. Our results showed that both the constitutive and copper-stimulated internalization of prion occurred via a clathrin-independent, dynamin-dependent pathway. Finally, to understand scrapie propagation, we have been examining the trafficking of the scrapie form of the prion protein through the endocytic pathway to the lysosome