70 KDA HEAT SHOCK PROTEINS AND THEIR ASSOCIATED COFACTORS

我们的实验室正在研究70 kDa的热休克蛋白(Hsp70)，它作为分子伴侣，参与蛋白质依赖于ATP的折叠和去折叠，蛋白质复合体的形成和溶解，以及蛋白质的跨膜转移。在许多这样的过程中，J结构域的蛋白质类成员充当与Hsc70蛋白的必要辅助因子，与蛋白质底物结合，然后将它们转移到Hsc70。在Hsc70作用需要J-结构域蛋白的过程中，其中一个过程是Hsc70揭开笼蛋白包裹的小泡。我们已经发现了两个J-结构域蛋白质，Axlin A 100 kDa神经特异性蛋白和GAK，它是Axlin的一个150 kDa的非神经元同源物，支持Hsc70揭开被Cathrin覆盖的小泡。我们还发现线虫产生一个与Axin同源的107 kDa的蛋白质。当RNA介导的干扰(RNAi)被用来抑制线虫体内的生长素表达时，蠕虫卵母细胞对YP170：：GFP(卵黄蛋白)的受体介导的内吞作用明显减少。此外，大多数线虫的生长素(RNAi)在幼虫发育过程中停止，并在许多细胞类型中显示出GFP：：clathrin的不均匀分布。光漂白后的荧光恢复(FRAP)显示，它们还显示出体细胞中存在的笼状蛋白包裹的凹坑中的笼状蛋白交换显着减少。因此，很明显，线虫的生长素是网状蛋白介导的体内内吞作用和线虫发育所必需的。为了更好地了解Hsc70和生长素在网状蛋白介导的内吞作用中的作用，我们目前正在利用FRAP技术研究不同条件下组织培养细胞中的网状蛋白动力学。由于在体内，网状蛋白介导的内吞作用需要生长素，所以我们有兴趣确定其结构域。我们的结果表明，在N端紧张素结构域之后，该分子具有一个与笼蛋白组装蛋白AP-180的笼蛋白结合域同源的笼蛋白结合域。然而，令人惊讶的是，我们发现即使在这个笼状蛋白结合域被删除后，得到的20 kDa的生长素C-末端片段仍然被Hsc70支持，表明它含有第二个与N-末端J-结构域相邻的笼状蛋白结合域。我们还创建了一个由AP-180的笼蛋白结合域和Axin的J-结构域组成的嵌合体，并表明该嵌合体支持Hsc70的去涂层。然而，与粘附素不同的是，它的作用是化学计量的，而不是催化的，因为在Hsc70去掉笼蛋白后，嵌合体与未包被的笼蛋白和Hsc70形成了一种复合体，与粘附素不同，它不能从该复合体中解离。我们目前正在试图破译生长素的特定方面，使其能够在支持Hsc70脱涂层的过程中发挥催化作用，而不是化学计量作用。我们目前还在使用核磁共振技术来确定Axin的20 kDa C-末端片段的结构，特别是笼蛋白结合域与J-结构域的结构关系。理解这种结构关系应该有助于阐明底物从J-结构域蛋白转移到Hsc70的基本机制。

Our laboratory is studying the 70-kDa class of heat shock proteins (Hsp70s) which act as molecular chaperones, that is, are involved in the ATP-dependent folding and unfolding of proteins, the formation and dissolution of protein complexes, and the translocation of proteins across membranes. In many of these processes members of the J-domain class of proteins act as necessary cofactors with the Hsc70 proteins, binding protein substrates and then transfering them to Hsc70. One of the processes where a J-domain protein is required for Hsc70 action is the uncoating of clathrin-coated vesicles by Hsc70, We have discovered two J-domain proteins, auxilin a 100 kDa nerve-specific protein and GAK, a 150 kDa, non-neuronal homolog of auxilin that support uncoating of clathrin-coated vesicles by Hsc70, We have also found that C. elegans produces a single 107 kDa protein that is homologous to auxilin. When RNA-mediated interference (RNAi) is used to inhibit auxilin expression in C. elegans, the worm oocytes show markedly reduced receptor-mediated endocytosis of YP170::GFP (yolk protein). In addition, most of the C. elegans auxilin (RNAi) worms arrest during larval development and show a maldistribution of GFP::clathrin in many cell types. They also show a marked decrease in clathrin exchange in clathrin-coated pits present in coelomocytes as shown by fluorescence recovery after photobleaching (FRAP). It is therefore clear that C. elegans auxilin is required for clathrin-mediated endocytosis in vivo and for C. elegans development. In order to better understand the role of Hsc70 and auxilin in clathrin-mediated endocytosis, we are currently studying clathrin dynamics under various conditions in tissue culture cells using FRAP Since auxilin is required for clathrin mediated endocytosis in vivo, we were interested in determining its domain structure. Our results showed that following the N-terminal tensin domain, the molecule has a clathrin-binding domain that is homologous to the clathrin-binding domain of the clathrin assembly protein AP-180. Surprisingly, however, we found that even after this clathrin-binding domain was deleted, the resulting 20 kDa C-terminal fragment of auxilin still supported uncoating by Hsc70 showing that it contains a second clathrin binding domain that abuts the N-terminal J-domain. We have also created a chimera consisting of the clathrin-binding domain of AP-180 and the J-domain of auxilin and shown that this chimera supports uncoating by Hsc70. However, unlike auxilin it acts stoichiometrically rather than catalytically because following uncoating of clathrin by Hsc70 the chimera forms a complex with the uncoated clathrin and Hsc70 from which, in contrast to auxilin, it cannot dissociate. We are currently attempting to decipher the specific aspects of auxilin that allow it to act catalytically rather than stoichiometrically in supporting uncoating by Hsc70. We are also currently using NMR to determine the structure of the 20 kDa C-terminal fragment of auxilin, in particular, the structural relationship of the clathrin-binding domain to the J-domain. Understanding this structural relationship should shed light on the basic mechanism by which substrates are transfered from J-domain proteins to Hsc70s.