70-kda Heat Shock Proteins And Their Associated Cofactor

我们的实验室一直在研究70 kDa类热休克蛋白（Hsp 70）的作用机制，这被称为分子伴侣，因为它们参与蛋白质的折叠和解折叠以及蛋白质复合物的形成和解离。在这些研究中，我们集中探讨热休克蛋白70的作用，内吞作用，特别是它的能力，uncoat网格蛋白包被的囊泡。在它们的许多活动中，Hsp 70需要称为J-结构域蛋白的辅因子，其诱导蛋白质底物与Hsp 70结合，并且我们先前发现，脱壳还需要J-结构域蛋白，100 kDa网格蛋白组装蛋白（AP），生长素。生长素是一种神经特异性蛋白质，我们后来发现生长素的非神经元同源物是150 kDa的蛋白质GAK。在过去的一年里，我们表明，C。线虫具有生长素的单一基因，当生长素的表达被RNA介导的干扰抑制时，网格蛋白介导的内吞作用受到显著抑制，这又导致蠕虫在幼虫发育期间停滞。我们还表明，酵母有一个单一的生长素基因，当这个基因被删除，产生的单倍体酵母突变体表现出增加网格蛋白包被的囊泡和相应的减少游离网格蛋白在胞质溶胶中。此外，有一个显着减少运输羧肽酶Y和G-蛋白偶联受体Ste 3的液泡，这两种蛋白质的运输通常发生通过网格蛋白介导的内吞作用。从这些数据中，我们得出结论，由HSP 70和生长素同系物的网格蛋白包被的囊泡的脱壳是网格蛋白介导的内吞作用的基本步骤。我们现在正在获得auxilin和GAK基因敲除小鼠，以确定这些基因在哺乳动物中的功能。 在过去的一年中，我们也开始调查网格蛋白的解离和重新结合，即网格蛋白交换，是否是网格蛋白介导的内吞作用的正常部分，独立于囊泡形成后发生的网格蛋白的不可逆解离，以及Hsc 70和生长素是否参与这种交换。通过阻断网格蛋白介导的胞吞作用的条件下，网格蛋白包被的坑的质膜上保持完整，我们表明，在ATP的存在下，网格蛋白包被的坑中的网格蛋白交换与游离网格蛋白在胞质溶胶中。这些数据表明，ATP依赖性交换的自由和结合网格蛋白是网格蛋白包被的坑的一个基本属性，因此可能涉及网格蛋白的结构重排，发生网格蛋白包被的坑内陷。这些数据还表明，网格蛋白包被的凹坑是动态结构，因此提出了网格蛋白包被的凹坑中的网格蛋白组装蛋白AP 2也可能与胞质溶胶中的游离AP 2交换的可能性。因此，在过去的一年中，我们调查了这个问题，并调查是否网格蛋白和AP 1绑定到trans-Golgi网络交换与游离网格蛋白和AP 1在胞质溶胶中，分别。正如我们对网格蛋白所观察到的，当网格蛋白介导的内吞作用在网格蛋白包被的凹坑保持完整的条件下被阻断时，网格蛋白包被的凹坑中的AP 2与游离AP 2交换，并且这种交换以与网格蛋白交换大约相同的速率发生。同样，我们发现，在低温下，运输出的trans-Golgi网络被阻止，网格蛋白和AP 1交换与游离网格蛋白和AP 1在胞质溶胶中，分别。我们以前还发现，当网格蛋白介导的胞吞作用在质膜被高渗蔗糖或K耗竭，已报道的影响网格蛋白包被的坑的结构的条件下，网格蛋白交换被完全阻断。因此，在本研究中，我们研究了高渗蔗糖和钾耗竭如何影响AP 2交换在质膜和网格蛋白和AP 1交换在trans-Golgi网络。有趣的是，我们发现，高渗蔗糖和K耗竭不仅阻止网格蛋白交换在质膜，但也在trans-Golgi网络。然而，在质膜上的AP 2和在trans-Golgi网络上的AP 1在这些条件下继续交换。我们的结论是网格蛋白涂层的坑在质膜和trans-Golgi网络的动态结构，显示快速交换的网格蛋白和AP。此外，我们的结论是，虽然网格蛋白和AP一般交换在大约相同的速率，AP能够交换独立的网格蛋白时，网格蛋白交换被阻断。

Our laboratory has been studying 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 endocytosis, in particular its ability to uncoat 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 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. During the previous year we 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 in clathrin-coated vesicles and a corresponding decrease in free clathrin in the cytosol. In addition, there was a marked decrease in transport of both carboxypeptidase Y and the G-protein-coupled receptor Ste3 to the vacuole; transport of both of these proteins normally occurs through clathrin-mediated endocytosis. From these data, we concluded that uncoating of clathrin-coated vesicles by Hsp70 and an auxilin homolog is a fundamental step in clathrin-mediated endocytosis. We are now well on the way to obtaining both auxilin and GAK knock-out mice to determine the function of these genes in mammals. During the previous year we also began an investigation of whether dissociation and rebinding of clathrin, i.e. clathrin exchange, is a normal part of clathrin-mediated endocytosis independent of the irreversible dissociation of clathrin that occurs after vesiculation takes place, and also whether Hsc70 and auxilin are involved in this exchange. By blocking clathrin-mediated endocytosis under conditions where clathrin-coated pits on the plasma membrane remain intact, we demonstrated that, in the presence of ATP, clathrin in clathrin-coated pits exchanges with free clathrin in the cytosol. These data suggested that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits and therefore may be involved in the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate. These data also suggested that clathrin-coated pits are dynamic structures and therefore raised the possibility that the clathrin assembly protein AP2 in clathrin-coated pits might also exchange with free AP2 in the cytosol. Therefore, during the past year we investigated this question and also investigated whether clathrin and AP1 bound to the trans-Golgi network exchange with free clathrin and AP1 in the cytosol, respectively. As we observed for clathrin, when clathrin-mediated endocytosis was blocked under conditions where clathrin-coated pits remain intact, AP2 in the clathrin-coated pits exchanged with free AP2 and this exchange occurred at about the same rate as clathrin exchange. Similarly, we found that at low temperature where transport out of the trans-Golgi network is blocked, both clathrin and AP1 exchanged with free clathrin and AP1 in the cytosol, respectively. We also previously found that, when clathrin-mediated endocytosis at the plasma membrane was blocked by hypertonic sucrose or K depletion, conditions that have been reported to affect the structure of clathrin-coated pits, clathrin exchange was completely blocked. Therefore, in the present study we investigated how hypertonic sucrose and K depletion affected AP2 exchange at the plasma membrane and clathrin and AP1 exchange at the trans-Golgi network. Interestingly, we found that both hypertonic sucrose and K depletion not only blocked clathrin exchange at the plamsa membrane but also at the trans-Golgi network. However, both AP2 at the plasma membrane and AP1 at the trans-Golgi network continued to exchange under these conditions. We conclude that clathrin-coated pits at both the plasma membrane and the trans-Golgi network are dynamic structures that show rapid exchange of both clathrin and APs. In addition, we conclude that, although clathrin and APs generally exhange at about the same rate, APs are able to exchange independently of clathrin when clathrin exchange is blocked.