Grant: Discovery Grants Program - Individual

The folding of many proteins requires chaperones. Chaperones also untangle misfolded proteins or target them for degradation. Chaperones are particularly critical in the response to stress; thus global warming is making mechanisms to modulate their activity genetically or chemically increasingly valuable. Our studies focus on the TTT chaperone complex. The components of this complex, Tel2, Tti1 and Tti2, are essential in eukaryotes. Using genetic selections in S. cerevisiae we identified mutations in TTI2 that enhance the folding of a member of the PIKK (Phosphatidylinositol 3-kinase-related kinase) protein family. This effect results from partial loss of Tti2 function, leading us to hypothesize that Tti2 targets slowly folding proteins for degradation. Similar mutations are not found in TEL2 or TTI1, suggesting that Tti2's function may be unique. The goal of our research program is to determine the function of the components of the TTT-complex. An initial emphasis will be placed on Tti2, because of its potentially unique activities and since we have many of the key tools for its analysis. Our studies use yeast because the combination of experimental approaches yeast allow is ideal for solving fundamental biological problems. Specific objectives for this five-year period are: 1. Identify the protein interaction networks of the components of the TTT-complex. The functions of Tel2, Tti1 and Tti2 will be more clearly defined when their interacting partners are determined. We will determine the interaction partners of the three proteins using biochemical approaches. The significance of novel interactions will be examined by mapping the interaction surface through mutagenesis, then introducing the mutagenized alleles into cells. 2. Characterize the structure/function relationships of Tti2. Using unigenic evolution we have identified clusters of invariant residues in Tti2. These residues will be mutated and their phenotypic consequences determined by plasmid shuffling in a tti2 knockout strain we constructed. The effects of the mutations on Tti2 expression, localization, folding of the PIKK proteins, and protein-protein interactions will be examined. Alleles that reduce cell growth will be valuable tools for suppressor genetic studies that will guide experiments to determine the function of Tti2. 3. Identify the ""client"" proteins for Tti2. The importance of the TTT proteins in the folding of members of the PIKK family has been demonstrated. Whether other clients exist is unknown. We will search for proteins whose expression depends on Tti2 and Tel2 using conditional alleles and protein identification through stable isotope labeling with amino acids in cell culture. A second approach to identify both targets and interacting proteins will be through the BioID strategy. This method was designed for mammalian cells, so we will start by identifying targets of mammalian Tti2 in cell culture.