70-kDa Heat Shock Proteins And Their Associated Cofactor

我们的实验室一直在研究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，我们集中于探索Hsp70在胞吞作用中的作用，特别是它剥离网格蛋白包被囊泡的能力。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，我们之前发现，脱壳还需要j结构域蛋白，即较小的100 kDa的编织蛋白组装蛋白（AP），辅助蛋白。Auxilin是一种神经特异性蛋白，我们后来发现Auxilin的非神经元同源蛋白是150kda的GAK蛋白。在过去的一年里，我们继续研究辅助素是否存在于低等生物中，如果存在，阻止其表达是否会导致体内网格蛋白介导的内吞作用的主要缺陷。我们之前的研究表明，秀丽隐杆线虫有一个单一的促生长素基因，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。在过去的一年里，我们确定了酿酒葡萄球菌中是否表达了auxilin。我们发现酵母基因组编码了一个668个氨基酸的蛋白，我们将其命名为Aux1，它的c端j结构域与哺乳动物auxilin的c端具有40%的氨基酸一致性。虽然在分子的其他部分（可能是网格蛋白结合的地方）几乎没有相似之处，但我们发现Aux1能够将哺乳动物的网格蛋白聚合成篮子。此外，聚合到这些筐中的Aux1在pH为6时招募Hsp70与筐结合，并在pH为7时支持Hsp70脱包。尽管它不如哺乳动物的辅助素那么活跃。当我们随后删除Aux1基因时，我们发现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，而细胞质中相应的游离网格蛋白减少。此外，在不表达Aux1的酵母中，羧基肽酶Y和g蛋白偶联受体Ste3向液泡的运输都明显减少；这两种蛋白质的运输通常通过网格蛋白介导的内吞作用进行。最后，利用免疫电子显微镜，我们发现与野生型细胞相比，不表达AUX1的酵母中网格蛋白包被的囊泡增加了5倍以上。根据这些数据，我们得出结论，Hsp70和辅助蛋白同源物揭开网格蛋白包被的囊泡是酵母中网格蛋白介导的内吞作用的基本步骤。在过去的一年里，我们也开始研究Hsp70和auxilin在体内的功能，特别是在哺乳动物细胞中。我们首先问的是，是否网格蛋白的解离和再结合，即网格蛋白交换，是网格蛋白介导的内吞作用的正常部分，独立于囊泡发生后发生的网格蛋白的不可逆解离。我们在体外用牛脑网格蛋白包被囊泡和在体内用HeLa细胞研究了这个问题。后一项研究采用Keen及其同事首创的方法，用绿色荧光蛋白（GFP）标记网格蛋白轻链，然后测量光漂白质膜上网格蛋白包覆的凹点后荧光恢复的速率和幅度。我们发现，在体外实验中，即使在Hsp70和ATP存在的情况下，网格蛋白包被囊泡和网格蛋白篮中的网格蛋白也不与自由网格蛋白交换。另一方面,收紧在野生型细胞的研究表明,在photo-bleaching质膜的小区域,有一个直接的荧光强度下降50 - 80%其次是恢复80%的荧光半衰期约15秒37 c .当然至少这个复苏的一部分是由于clathrin-mediated内吞作用本身,同时内陷的漂白clathrin-coated坑和形成新的原色的坑。因此，为了确定网格蛋白交换是否发生在现有的凹坑中，必须对网格蛋白包被的凹坑进行FRAP测量，即使在网格蛋白介导的内吞作用被阻断后，这些凹坑仍保持不变。我们使用了两种不同的方法来阻断网格蛋白介导的内吞作用，同时仍然保持了质膜上网格蛋白包裹的凹坑。首先，我们表达了动力蛋白突变体K44A，其次，我们耗尽了胆固醇膜。令人惊讶的是，我们发现在这两种情况下，光漂白的网格蛋白的替换发生的速度和幅度与内吞作用发生时大致相同。此外，通过跟踪单个网格蛋白包覆坑的命运，我们发现这种替换很少是由于漂白坑的溶解和新坑的改造。相反，这是由于几乎所有胞穴中的网状蛋白与胞浆中的游离网状蛋白快速交换所致。此外，我们发现这种交换是atp依赖的。当ATP从细胞中耗尽时，交换的速率和幅度都显著降低。当质膜上包覆网格蛋白的凹坑被钾耗尽或用高渗蔗糖处理细胞转化为膜表面下的网格蛋白篮时，交换也不会发生。因此，与我们的体外数据一致，笼蛋白交换似乎没有发生在体内的笼蛋白篮子中。综上所述，这些数据表明，自由和结合的网格蛋白依赖于atp的交换是网格蛋白包被凹坑的基本特性，而不是网格蛋白篮，因此可能参与了网格蛋白在包被凹坑中发生的结构重排61。Jiang， R., Gao， B., Prasad， K., Greene, l.e.，和Eisenberg， E.: Hsc70伴随网格蛋白并启动其与囊泡膜相互作用。生物。化学学报，27:843 - 847,2000。

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 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. During the past year we have continued our investigation of whether auxilin occurs in lower organisms and, if so, whether preventing its expression causes major defects in clathrin-mediated endocytosis in vivo. We previous 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. During the past year we determined whether auxilin was expressed in S. cerevisiae. We found that the yeast genome encodes a 668-amino acid protein that we named Aux1, which has a C-terminal J-domain that shares 40% amino acid identity with the C-terminal of mammalian auxilin. Although there is little similarity in the rest of the molecule, where presumably clathrin binds, we found that Aux1 is able to polymerize mammalian clathrin into baskets. Furthermore, the Aux1 polymerized into these baskets recruits Hsp70 to bind to the baskets at pH 6 and supports Hsp70 uncoating of these baskets at pH 7. although it is not nearly as active as mammalian auxilin. When we then deleted the Aux1 gene, we found that the resulting haploid yeast mutants showed an increase of clathrin associated with vesicles and a corresponding decrease in free clathrin in the cytosol. Furthermore, in yeast not expressing Aux1, there is 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. Finally, using immuno-electron microscopy we showed that there was more than a 5-fold increase in clathrin-coated vesicles in yeast not expressing AUX1 compared to wild-type cells. 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 in yeast. During the past year we also began an investigation of the functions that Hsp70 and auxilin carry out in vivo, in particular in mammalian cells. We first asked 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. We investigated this question both in vitro using bovine brain clathrin-coated vesicles and in vivo using HeLa cells. The latter studies were carried out by tagging the clathrin light chain with GFP using the method pioneered by Keen and his associates and then measuring the rate and magnitude of the recovery of fluorescence following photo bleaching of the clathrin-coated pits at the plasma membrane. We found that, in vitro, the clathrin in clathrin-coated vesicles and clathrin baskets does not exchange with free clathrin even in the presence of Hsp70 and ATP when partial uncoating occurs. On the other hand, FRAP studies in wild-type cells showed that, after photo-bleaching a small region of the plasma membrane, there was an immediate 50-80% decrease in fluorescence intensity followed by an 80% recovery of fluorescence with a half-life of about 15 s at 37 C. Of course at least part of this recovery was due to clathrin-mediated endocytosis itself, that is the simultaneous invagination of bleached clathrin-coated pits and formation of new unbleached pits. Therefore, to determine whether clathrin exchange occurs in existing pits, FRAP measurements had to be made on clathrin-coated pits that were maintained even after clathrin-mediated endocytosis was blocked. We used two different methods to block clathrin-mediated endocytosis while still maintaining the clathrin-coated pits on the plasma membrane. First, we expressed the dynamin mutant, K44A and second, we depleted the membrane of cholesterol. Surprisingly, we found that in both cases replacement of the photo bleached clathrin occurred at about the same rate and magnitude as when endocytosis was occurring. Furthermore, by following the fate of individual clathrin-coated pits, we found that very little of this replacement was due to dissolution of bleached pits and reformation of new pits. Rather, it was caused by rapid exchange of almost all of the clathrin in the pits with free clathrin in the cytosol. Furthermore, we found that this exchange was ATP-dependent. When ATP was depleted from the cell both the rate and the magnitude of the exchange markedly decreased. Exchange also did not occur when the clathrin-coated pits at the plasma membrane were transformed into clathrin baskets just below the surface of the membrane either by potassium depletion or by treatment of the cells with hypertonic sucrose. Therefore, in agreement with our in vitro data, clathrin exchange did not appear to occur in clathrin baskets in vivo. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits but not clathrin baskets, and therefore may be involved in the structural rearrangement of clathrin that occurs as clathrin-coated pits invaginate61. Jiang, R., Gao, B., Prasad, K., Greene, L.E., and Eisenberg, E.: Hsc70 chaperones clathrin and primes it to interact with vesicle membranes. J. Biol. Chem. 275:8439-8447, 2000.