95-15474 Ungewickell The vectorial transport of selected membrane proteins from the plasma membrane and the trans-Golgi network to the endosomal system involves clathrin coated vesicles. The clathrin coat is connected by hetero-tetrameric adaptor proteins to the underlying membrane and is removed enzymatically before fusion at the targeted membrane. Neuronal clathrin coated vesicles contain in addition to the adaptors the monomeric coat proteins AP180 and auxilin. Results from in vitro reconstituted system imply that uncoating of clathrin requires the cognate form of the heat shock protein hsp70 (hsp70c) and a 100 KDa cofactor. This cofactor has been identified as auxilin. Auxilin was previously characterized as a clathrin assembly protein. The function of auxilin in the uncoating is reminiscent of DnaJ proteins that function together with hsp70 to constitute a chaperone system involved in prokaryotic and eukaryotic cells in numerous functions: protein folding, protein transport across membranes, and activation of DNA binding proteins. DnaJ-like proteins are presumed to target hsp70 to specific substrates. The objective of this proposal is to gain insights into the molecular mechanism of the uncoating reaction and to prove that auxilin or auxilin-like proteins function together with hsp70c to form a chaperone machine that specifically dissociate clathrin coat and regulate clathrin dynamics in vivo. This will be acheived by investigating the domain structure of auxilin to determine which parts interact with assembled clathrin coat and with hsp70c as well as how these interactions are regulated. Truncated forms of auxilin will be expressed in bacteria, purified, and assayed in vitro to determine their ability to bind to clathrin, to recruit hsp70c to the clathrin coat, and to elicit uncoating of the clathrin coat. Chemical cross linking reagents will be used to identify novel uncoating intermediate that presumably contains auxilin, hsp70c, and clathrin. Mutated genes that encode auxilin that are unable to recruit hsp70c to clathrin will be introduced by transfection into neuronal and non-neuronal cells to examine their effects on clathrin mediated membrane traffic. Auxilin homologues will be search for in non-neuronal cells using the in vitro functional assay with fractionated homogenates of various cells. cDNAs encoding possible homologues will be cloned and sequenced. Studies of the chaperone-mediated uncoating of clathrin coated vesicles are relevant not only to understanding clathrin dynamics, but also to understanding the molecular mechanism of a specific eukaryotic chaperone machine. %%% The vectorial transport of selected membrane proteins from the plasma membrane and the trans-Golgi network to the endosomal system involves clathrin coated vesicles. The clathrin coat is connected by hetero-tetrameric adaptor proteins to the underlying membrane and is removed enzymatically before fusion at the targeted membrane. Neuronal clathrin coated vesicles contain in addition to the adaptors the monomeric coat proteins AP180 and auxilin. Results from in vitro reconstituted system imply that uncoating of clathrin requires the cognate form of the heat shock protein hsp70 (hsp70c) and auxilin. Auxilin was previously characterized as a clathrin assembly protein. The objective of this proposal is to gain insights into the molecular mechanism of the uncoating reaction and to prove that auxilin or auxilin-like proteins function together with hsp70c to form a chaperone machine that specifically dissociate clathrin coat and regulate clathrin dynamics in vivo. This will be acheived by investigating the domain structure of auxilin to determine which parts interact with assembled clathrin coat and with hsp70c as well as how these interactions are regulated. Studies of the chaperone-mediated uncoating of clathrin coated vesicles are relevant not only to understanding clathrin dynamics, but also to understanding the molecular mechanism of a specific eukaryotic chaperone machine. ***
