70-kda Heat Shock Proteins And Their Associated Cofactor

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 total internal reflectance microscopy to determine the timing of GAK binding relative to dynamin and clathrin binding during invagination of clathrin-coated pits. Following transient recruitment of dynamin to the clathrin puncta, large amounts of GAK are transiently recruited. GAK and clathrin then disappear from the evanescent field as the pit invaginates from the plasma membrane and finally these proteins disappear from the epifluorescence field, probably as the clathrin is uncoated from the budded vesicles by Hsc70. The recruitment of GAK is dependent on its PTEN-like domain, which we found binds to phospholipids. This suggests that interaction with phospholipids is essential for recruitment of GAK and, in turn, Hsc70, but Hsc70 recruitment alone might not be sufficient to induce irreversible clathrin uncoating. When budding of clathrin-coated pits was inhibited by actin depolymerization, there was repeated flashing of GAK on the clathrin-coated pit but neither scission nor irreversible uncoating occur. Therefore, budding as well as synchronous recruitment of GAK might be required for irreversible clathrin uncoating. In further studies at the animal level during the past year, we continued our studies on the auxilin and GAK knock-out mice. We had previously determined that mice in which GAK is conditionally knocked-out of neuronal cells at embryonic day ten show major developmental defects in the brain and die shortly after birth. We have now found that the mice also die shortly after birth when GAK is conditionally knocked-out of liver or skin cells. Histological analysis of both the liver and the skin show profound developmental defects these in tissues just as occurred in the brain. Furthermore, when the adult animal is treated with tamoxifen to conditionally knock-out the GAK, the mice die within a few days of the treatment. Therefore GAK is required for viability of the adult animal as well as in development.