Genome-wide Function and Substrate Recognition of the Chaperone Prefoldin

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DESCRIPTION (provided by applicant): Chaperones assist in the proper folding of many proteins and protect against protein aggregation, which is increasingly recognized as a pathogenic mechanism in human diseases such as Alzheimer's. Prefoldin is a protein chaperone that is known to play a role in the folding of newly-translated actin and tubulin. We wish to understand the overall cellular function of prefoldin and determine how it recognizes its substrates. Studies of prefoldin function on actin and tubulin suggest that it binds nascent peptides and sequesters them while delivering them to the TRiC/CCT chaperonin. Prefoldin has six homologous subunits, each of which has a long, coiled-coil projection. The tips of certain subunits bind to actin and tubulin, and defined residues in each of these substrates are necessary for this interaction. Thus, prefoldin binding is specific ' and may involve a combinatorial binding of different subunits for different targets. Our specific aims are: 1) Determine the cellular functions of prefoldin. We will use whole-genome measurements of genetic interactions with chaperone mutations to determine which processes are affected by prefoldin. 2) Identify new prefoldin targets and test for TRiC/CCT dependence. We will find additional proteins that depend on prefoldin by using the characteristic pattern of epistasis between mutations in a chaperone and its target. We will then test candidate substrates for direct physical interaction with the chaperone and determine whether loss of chaperone function affects their folding in vivo. 3) Understand how prefoldin binds its various, unrelated substrates. We will use deletions and point mutants based on the solved structure of prefoldin to determine which regions of prefoldin are required by specific target proteins. We will use genetic tests for prefoldin function as well as biochemical measurements of prefoldin binding. Protein aggregation plays a major role in many human diseases, including Alzheimer's. Protein chaperones bind to newly-produced proteins and help them to fold while preventing them from aggregating. We want to know how the chaperone prefoldin binds specific target proteins and what role this has in normal cell function.