70 KDA HEAT SHOCK PROTEINS AND THEIR ASSOCIATED COFACTORS

我们的实验室正在研究70-kDa类热休克蛋白（Hsp70）作为分子伴侣，即参与ATP依赖的折叠和解折叠的蛋白质，蛋白质复合物的形成和溶解，以及蛋白质的跨膜转运。在许多这些过程中，DnaJ类蛋白质的成员作为Hsc70蛋白质的必要辅因子，将蛋白质底物呈递给Hsc70。其中Hsc70作用需要DnaJ同系物的过程之一是网格蛋白包被的囊泡的去包被。在研究脱壳机制中，我们发现神经特异性100 kDa网格蛋白组装蛋白，生长素，是脱壳过程发生所需的，我们还表明，生长素是DnaJ同系物。我们现在已经发现，GAK，一个最近从人类和大鼠克隆的150 kDa的蛋白质，具有一个C-末端结构域，显示出与生长素的高度同源性。在体外，GAK，如生长素，支持通过Hsc70的网格蛋白篮的去涂层。此外，GAK存在于从大鼠肝脏分离的网格蛋白包被的囊泡中，其可以不被Hsc70包被。因此，GAK显然是DnaJ同源物，其支持Hsc 70对非神经元网格蛋白包被的囊泡的去包被。我们还发现C.线虫产生与生长素同源的107kDa蛋白质。重组C.线虫生长素在体外与Hsc70和网格蛋白的相互作用中能够替代哺乳动物生长素。利用RNA介导的基因干扰（RNAi）抑制C.在线虫中，与野生型相比，Ce-生长素（RNAi）蠕虫中的卵母细胞具有减少的来自假体腔的卵黄原蛋白-GFP的摄取，并且F1 Ce-生长素（RNAi）幼虫具有许多含有大量网格蛋白重链-GFP的突出斑点。此外，所有的F1 Ce-生长素（RNAi）蠕虫逮捕的发展在第一幼虫阶段，L1。因此C.线虫生长素是网格蛋白介导的胞吞作用所必需的，而线虫生长素是网格蛋白介导的胞吞作用所必需的。线虫生长素基因是生存所必需的。最后，我们一直在研究在其活性位点突变的热休克蛋白70进行野生型热休克蛋白70的各种功能的能力。DnaJ同源物诱导野生型Hsc70快速水解ATP，并且认为这是DnaJ同源物将底物呈递给Hsp70的能力中的关键步骤。然而，我们现在发现，生长素可以诱导不能水解ATP的Hsp70突变体与网格蛋白篮结合，这表明将底物呈递给Hsp70的关键步骤是DnaJ同系物瞬时打开Hsp70底物结合位点而不是激活ATP水解步骤的能力。- 70-kDa类热休克蛋白作为分子伴侣，参与蛋白质的ATP依赖性。

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 DnaJ class of proteins act as necessary cofactors with the Hsc70 proteins, presenting protein substrates to Hsc70. One of the processes where a DnaJ homolog is required for Hsc70 action is the uncoating of clathrin- coated vesicles. In studying the mechanism of uncoating, we discovered that the nerve-specific 100 kDa clathrin assembly protein, auxilin, is required for the uncoating process to occur, and we also showed that auxilin is a DnaJ homolog. We have now found that GAK, a 150 kDa protein recently cloned from both humans and rats, has a C-terminal domain that shows a high homology to auxilin. In vitro, GAK, like auxilin, supports the uncoating of clathrin baskets by Hsc70. In addition, GAK is present in clathrin-coated vesicles isolated from rat liver that can be uncoated by Hsc70. Therefore, GAK is apparently the DnaJ homolog that supports uncoating of non-neuronal clathrin-coated vesicles by Hsc70. We have also found that C. elegans produces a 107 kDa protein that is homologous to auxilin. Recombinant C. elegans auxilin is able to substitute for mammalian auxilin in its interaction with Hsc70 and clathrin in vitro. Furthermore, when RNA-mediated gene interference (RNAi) is used to inhibit auxilin expression in C. elegans, oocytes in Ce-auxilin (RNAi) worms have reduced uptake of vitellogenin-GFP from the pseudocoelom and F1 Ce-auxilin (RNAi) larvae have many prominent puncta containing large amounts of clathrin heavy chain-GFP compared to wild type. Moreover, all of the F1 Ce-auxilin (RNAi) worms arrested development at the first larval stage, L1. Therefore C. elegans auxilin is required for clathrin-mediated endocytosis in vivo and the C. elegans auxilin gene is essential for viability. Finally we have been studying the ability of Hsp70s mutated at their active sites to carry out various functions of wild type Hsp70. DnaJ homologs induce wild type Hsc70 to rapidly hydrolyze ATP and it is thought that this is a key step in the ability of DnaJ homologs to present substrates to Hsp70. However, we now find that auxilin can induce Hsp70 mutants that cannot hydrolyze ATP to bind to clathrin baskets showing that the key step in the presentation of substrates to Hsp70 is the ability of the DnaJ homolog to transiently open up the Hsp70 substrate binding site rather than to activate the ATP hydrolysis step. - 70-kDa class of heat shock proteins act as molecular chaperones, involved in the ATP-dependent of proteins.