The overall goal of the proposed research is to acquire a set of mutations covering the entire surface of conserved proteins already known to have important cellular functions in yeast. After characterization, these mutations will be used to find interacting genes and proteins using a variety of genetic techniques, including isolation and analysis of pseudo-revertants, unlinked non-complementing mutants, and mutations displaying synthetic lethality. We expect to find different genetic interactions with mutations linking different regions of a protein's surface, and it is for this reason that we plan to start with a """"""""synoptic"""""""" set of mutations made with a recent modification of a new technology, the charged-to-alanine scan. Genetic studies of mutations in the interacting genes will be supplemented with limited biochemical investigations aimed at characterizing the interactions of normal and mutant gene products. We hope in the end to make the finding of interacting genes and proteins by genetic methods as reliable and routine as mutagenesis has become. These technologies will be developed and applied to several specific projects: First, we will construct and characterize a synoptic set of new tubulin mutants of yeast. Second, we will identify genetically genes encoding ligands to the actin and tubulin cytoskeletons starting with a synoptic set of alanine-scanning mutants covering actin (already in hand) and the tubulins. Third, we will carry out homolog-scanning hybrid mutagenesis between pairs of genes encoding small GTP-binding proteins in yeast (e.g. the products of the YPTI and SEC4 genes) in order to try to identify, in complementation tests using these hybrids, the sub-domains of these highly similar proteins that determine their specificity for a particular function in protein secretion. Fourth, we will construct mutations with scorable non-lethal or conditional-lethal phenotypes within specificity-determining subdomains of the aforementioned GTP-binding proteins and use these to identify genetically genes encoding protein ligands to particular GTP-binding proteins.
Showing the most recent 10 out of 75 publications
