| Clathrin-mediated endocytosis is one of the pathways involved in the internalization of extracellular and/or membrane-bound macromolecules. The recycling of clathrin coated vesicle can be divided into the following steps: coat recruitment, invagination, constriction, fission, and uncoating. Each step is regulated by different accessory proteins. These accessory proteins, together with clathrin and lipid, bind each other to from an interaction network which we call the clathrin accessory protein network or C.A.N. for short.; My dissertation work addresses the uncoating step of clathrin-mediated endocytosis. To our knowledge, at least two proteins are required in this step: auxilin, and Hsc70. Auxilin is a brain specific protein with a C-terminal J domain. Previous studies showed that auxilin binds to the vertex of clathrin-coated vesicles via its clathrin binding domain, and then recruits Hsc70 to the vesicles via its J-domain. Hsc70 then disassembles the clathrin-coated vesicles in a step involving ATP hydrolysis.; Hsc70 (70 kDa heat shock cognate protein) is a constitutively expressed member of the 70 kDa heat shock protein (Hsp70) chaperone family. Hsp70s are one of the most important classes of molecular chaperones. Together with its co-chaperones, Hsp70 chaperone proteins are widely involved in protein folding, macromolecular complex assembly/disassembly, protein translocation, and other cellular processes. Hsp70s are two domain proteins containing an ATPase domain and a substrate binding domain that binds to hydrophobic regions of substrate proteins. ATP binding to the ATPase domain causes the substrate binding domain to release substrate, while ADP binding locks substrate tightly into the substrate binding domain. Binding of ATP also causes global conformational changes in the Hsp70 proteins. However, the Hsp70 mechanism remains obscure because only structures of the isolated ATPase and substrate binding domains have been determined. To better understand the mechanism of Hsp70 generally, and of the uncoating step specifically, I successfully determined the structures of bovine Hsc70 and auxilin J-domain by X-ray crystallography and then functionally investigated them with biochemical and biophysical approaches, which markedly advances our understanding of the mechanism of hsp70 chaperones and the molecular details of clathrin coat disassembly. |
