70 KDA HEAT SHOCK PROTEINS AND THEIR ASSOCIATED COFACTORS

Heart, Lung, And Blood Institute

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Abstract

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.