70-kda Heat Shock Proteins and Associated Cofactors

我们的实验室对细胞中正常和病理蛋白复合物的形成和溶解感兴趣，重点是分子伴侣在这一过程中的作用。特别是，我们正在研究无处不在的分子伴侣Hsc70和诱导特定底物与Hsc70结合的j结构域辅因子蛋白。在我们之前的工作中，我们研究了Hsc70在网格蛋白介导的内吞作用中的作用，特别是它从网格蛋白包被的囊泡中分离网格蛋白的能力。我们首先发现，脱膜不仅需要Hsc70，还需要100 kDa的神经特异性j结构域蛋白auxilin或auxilin的非神经元同源物，150 kDa的蛋白GAK，与auxilin相似，但也含有n端激酶结构域。然后我们发现，在体内，网格蛋白包被的坑是动态结构，并且网格蛋白和网格蛋白包被的坑的其他成分，包括网格蛋白介导的内吞过程中网格蛋白接头蛋白AP2的交换。同样，反式高尔基网络上的网格蛋白和网格蛋白接头蛋白AP1与胞浆中的自由网格蛋白和AP1交换。从我们的数据中我们得出结论，网格蛋白交换是网格蛋白结构重排所必需的，当网格蛋白包覆的凹坑发生内陷时。然后，我们通过渗透细胞证明，Hsc70不仅在网格蛋白包被的囊泡脱落后解离网格蛋白，而且在质膜上网格蛋白包被的凹坑或TGN上网格蛋白包被的芽内陷时发生的网格蛋白交换也需要Hsc70。

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. In particular we are studying the ubiquitous molecular chaperone Hsc70 and the J-domain cofactor proteins that induce specific substrates to bind to Hsc70. In our previous work we have studied the role of Hsc70 in clathrin-mediated endocytosis, in particular its ability to dissociate clathrin from clathrin-coated vesicles. We first discovered that uncoating not only requires Hsc70 but also the 100 kDa nerve-specific J-domain protein auxilin or the non-neuronal homolog of auxilin, the 150 kDa protein GAK that is similar to auxilin but also contains an N-terminal kinase domain. We then showed that in vivo clathrin-coated pits are dynamic structures and both clathrin and other components of clathrin-coated pits including the clathrin adaptor protein AP2 exchange during clathrin-mediated endocytosis. Similarly, clathrin and the clathrin adaptor protein AP1 on the trans-Golgi network exchanges with free clathrin and AP1 in the cytosol. From our data we concluded that clathrin exchange is required for the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate. We then showed using permeabilized cells that Hsc70 not only dissociates clathrin after clathrin-coated vesicles bud off but is also required for the clathrin exchange that occurs during invagination of clathrin-coated pits on the plasma membrane or clathrin-coated buds on the TGN. During the past year we used RNA interference to deplete cells of GAK and these studies along with further studies on permeabilized cells showed that Hsc70 and GAK not only cause exchange by dissociating clathrin but also by chaperoning it and facilitating its rebinding to pits. Surprisingly, these studies also showed that Hsc70 directly recruits binding of clathrin adaptors to both the plasma membrane and the trans-Golgi network independent of its effects on clathrin. In further studies at the animal level during the past year, we continued our studies on our auxilin and GAK knock-out mice. We are now certain that auxilin knock-out mice have decreased live births, smaller initial birth weights, and lower litter sizes. Furthermore, we find that these effects are ameliorated when GAK production is highly up-regulated in the brain, an effect that apparently occurs naturally in some of the mice. Since GAK and auxilin are normally present in nearly equal amounts in the brain, it appears that relatively large amounts of GAK are needed to overcome loss of auxilin from the brain. Similarly, we have found that mice in which GAK is conditionally knocked-out of neuronal cells at day 10 pc show highly abnormal brain development and die shortly after birth despite the presence of auxilin in these cells. Therefore we find that auxilin and GAK are not interchangeable. Rather each plays an important and independent role in nerve cells. During the past year we also began to work on both yeast and mammalian prion proteins labeled with GFP to study their trafficking and aggregation. In particular in our studies on yeast prion we used fluorescence photobleaching to follow aggregation of the yeast prion protein Sup35p fused to GFP. Surprisingly, in contrast to current dogma, we found that, after the molecular chaperone Hsp104 is inactivated, which is known to prevent prion propagation, cell division is not needed to dilute out the remaining Sup35p prion. Rather, loss of aggregated Sup35p occurs even in non-dividing cells, perhaps because inactivated Hsp104 does not continuously facilitate formation of new Sup35p prion. We are currently investigating whether this unexpected phenomenon also occurs with other yeast prions.