The long term goal of this proposal is to understand the biochemistry and cell biology of protein folding in eukaryotic cells. The general strategy will be to concentrate on the folding of two model proteins, actin and luciferase. The proposed research will focus on folding events as they occur at the ribosome during synthesis of a polypeptide and will examine the role of molecular chaperones in the folding process. The conceptual framework to understand the folding of proteins during translation originates from the applicant's earlier work, which indicates that the newly translated polypeptides are guided to their final conformation through a sequential and highly coupled chaperone pathway. This pathway combines two distinct principles of chaperone action, namely the Hsp70/Hsp40 proteins that act by stabilizing extended polypeptides, and the chaperonin TRiC, which by virtue of its ring-like structure creates an environment that is favorable for polypeptide chain folding. The objective of the proposal is to investigate critical aspects of the folding of newly synthesized proteins by: 1) defining the complement of chaperones that interact with nascent chains as the polypeptide length increases, 2) assessing the requirement of individual chaperone proteins in the folding of newly synthesized proteins and determine whether folding occurs in association with the chaperone complex, 3) investigating the role of the translational apparatus and possible novel components in the recruitment of chaperones to the nascent chains, and 4) examining the interaction of chaperones with newly translated polypeptides in intact mammalian cells.
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