70 KD Heat Shock and their associated cofactors

我们的实验室研究70 kda的热休克蛋白（Hsp70s）的作用机制，这些蛋白被称为分子伴侣蛋白，因为它们参与蛋白质的折叠和展开以及蛋白质复合物的形成和解离。在这些研究中，我们集中探索了Hsp70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。在它们的许多活动中，Hsp70需要被称为j结构域蛋白的辅助因子来诱导蛋白质底物与Hsp70结合，并且我们之前发现，脱壳还需要j结构域蛋白，即较小的100 kda的网格蛋白组装蛋白（AP），辅助蛋白。Auxilin是一种神经特异性蛋白，我们后来发现Auxilin的非神经元同源物是150 kda蛋白GAK，它与Auxilin非常相似，但也含有n端激酶结构域。我们发现秀丽隐杆线虫有一个促生长素的单一基因，当促生长素的表达被rna介导的干扰抑制时，促生长素介导的胞吞作用明显受到抑制，从而导致线虫在幼虫发育过程中停滞。我们还发现酵母有一个单一的辅助蛋白基因，当这个基因被删除时，产生的单倍体酵母突变体显示出与囊泡相关的网格蛋白增加，而细胞质中自由网格蛋白相应减少。从这些数据中，我们得出结论，Hsp70和auxilin是网格蛋白介导的内吞作用的一个基本步骤所必需的。我们随后证明，在体内，这一基本步骤不仅涉及在网格蛋白包被的囊泡脱出后的网格蛋白解离，还涉及在网格蛋白包被的窝内陷过程中发生的网格蛋白交换。结果表明，网蛋白及其接头蛋白AP2在胞浆中与游离的网蛋白和AP2交换。同样，我们发现反式高尔基网络上的网格蛋白和AP1与胞浆中的自由网格蛋白和AP1交换。从这些数据我们得出结论，在质膜和反式高尔基网络上，网格蛋白包覆的凹坑都是动态结构。我们还得出结论，网格蛋白交换是网格蛋白结构重排所必需的，这种重排发生在网格蛋白包覆的凹坑中。然后，我们使用渗透性细胞来证明Hsc70和辅助蛋白是这种网格蛋白交换所必需的，这表明它们诱导网格蛋白从包覆网格蛋白的凹坑中分离。然而，除非存在胞浆，否则网格蛋白不会重新结合到渗透细胞的凹坑上。同样，我们发现细胞质溶胶对于AP2的解离和重结合也是必需的。我们目前正在研究细胞质中哪些因子参与了网格蛋白的再结合和AP2的解离和再结合。特别是，关于后一个问题，我们正在研究PIP2和磷酸化和去磷酸化在AP2交换中的作用。

Our laboratory studies the mechanism of action of the 70-kDa class of heat shock proteins (Hsp70s), which have been termed molecular chaperones because they are involved in the folding and unfolding of proteins and in the formation and dissociation of protein complexes. In these studies we have concentrated on exploring the role of Hsp70 in clathrin-mediated endocytosis, in particular its ability to dissociate clathrin from clathrin-coated vesicles. In many of their activities the Hsp70s require cofactors known as J-domain proteins that induce protein substrates to bind to Hsp70, and we previously discovered that uncoating also requires a J-domain protein, the minor 100-kDa clathrin assembly protein (AP), auxilin. Auxilin is a nerve specific protein and we later discovered that the non-neuronal homolog of auxilin is the 150-kDa protein GAK that is very similar to auxilin but also contains an N-terminal kinase domain. We then showed that C. elegans has a single gene for auxilin and when auxilin expression is inhibited by RNA-mediated interference, there is a marked inhibition of clathrin-mediated endocytosis which in turn causes the worms to arrest during larval development. We also showed that yeast has a single gene for auxilin and that when this gene is deleted the resulting haploid yeast mutants showed an increase of clathrin associated with vesicles and a corresponding decrease in free clathrin in the cytosol. From these data, we concluded that Hsp70 and auxilin are required for a fundamental step in clathrin-mediated endocytosis. We then showed that in vivo this fundamental step not only involves dissociation of clathrin after clathrin-coated vesicles bud off but also clathrin exchange occurring during invagination of the clathrin-coated pit. Our results showed that clathrin and the adaptor protein, AP2, exchange with free clathrin and AP2 in the cytosol. Likewise we found that clathrin and AP1 on the trans-Golgi network exchanged with free clathrin and AP1 in the cytosol. From these data we concluded that clathrin-coated pits at both the plasma membrane and the trans-Golgi network are dynamic structures. We also concluded that clathrin exchange is required for the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate. We then used permeabilized cells to demonstrate that Hsc70 and auxilin are required for this clathrin exchange showing that they induce clathrin dissociation from clathrin-coated pits. However, clathrin did not rebind to the pits in the permeabilized cells unless cytosol was present. Likewise we found that cytosol was also required for both the dissociation and rebinding of AP2. We are currently investigating what factors in the cytosol are involved in the rebinding of clathrin and in the dissociation and rebinding of AP2. In particular, in regard to the latter question we are investigating the role of PIP2 and phosphorylation and dephosphorylation in the exchange of AP2. During the past year we have investigated two other aspects of Hsc70 and auxilin activity. First, we used optical reconstruction techniques to determine how Hsc70 dissociates individual clathrin triskelions from clathrin baskets. The clathrin baskets used in these structural studies were polymerized using C58J, a hybrid assembly protein that we designed in which the J-domain of auxilin is fused to a 58-kDa portion of the clathrin assembly protein, AP180. At pH 6 C58J-clathrin baskets bind three Hsc70s per clathrin triskelion but uncoating does not occur unless the pH is raised to 7. Our structural studies suggest that three Hsc70s bind to the vertex of each clathrin triskelion in these baskets and these Hsc70s disentangle the clathrin triskelion from the legs of adjacent clathrins. The Hsc70s then remain bound to the vertex of the clathrin triskelion after it dissociates. Second, we investigated the effect of GAK RNAi on clathrin-mediiated endocytosis in tissue culture cells. Interestingly, these studies suggested that, not only are Hsc70 and GAK required for uncoating of clathrin-coated vesicles, but they are also necessary to chaperone the dissociated clathrin so that it is able to return to the membrane and form new clathrin-coated pits. We found that, in the absence of GAK, the number of clathrin-coated pits markedly decreased and large clathrin aggregates appeared in the cytosol. There was no membrane associated with these aggregates suggesting that they were not simply clathrin-coated vesicles that had not been uncoated. Therefore these data suggest that the three Hsc70s bound to each dissociated clathrin triskelion may be required to chaperone the clathrin in the cytosol and may also be involved in clathrin rebinding to new clathrin-coated pits. During the past year our studies on clathrin associated with the TGN have also provided results that are relevant to a crucial question regarding the mechanism by which the trans-Golgi-network (TGN) partitions between the two daughter cells during mitosis. Our results suggest that clathrin and several TGN-associated membrane proteins do not simply fragment during mitosis but actually divide on the mitotic spindle during metaphase. Thus our results suggest that, in contrast to other portions of the Golgi, the TGN does not form de novo in daughter cells from elements dispersed throughout the cytosol during mitosis but rather is partly formed from vesicles that divide on the mitotic apparatus. Furthermore, our results suggest that, following mitosis, the TGN-derived vesicles coalesce into clathrin-coated TGN compartments at the spindle poles before the rest of the Golgi becomes functional. In addition to these studies on permeablized cells we have continued our studies on both the auxilin and GAK knock-out mice. Interestingly, the homozygous auxilin knock-out mice seem to be born with an eye defect suggesting that brain maturation is abnormal. They also seem to have a defect in coat color, a particularly interesting result since it is now known that in addition to being present in the nervous system auxilin is also found in melanosomes. In regard to the GAK knock-out mice, we have developed a fibroblast line from the flox-flox GAK mouse and when we infect these cells with adeno-cre virus we should be able to observe what happens to cells that are completely deprived of GAK. We will also be able to determine if GAK missing its N-terminal kinase domain but containing the part of the molecule homologous to auxilin can rescue these cells.