This is a competitive renewal from Dr. Marian Carlson to study the glucose regulation of the invertase gene, SUC2, in baker's yeast. This application focuses on the Snf1 protein kinase complex that plays a central role in the signal transduction pathway that senses glucose and alters transcription of a large number of glucose repressed and activated genes. The PI's current model for glucose regulation of SUC2 involves three tiers of complexes. 1). The presence of glucose is sensed through the Reg1-PP1 (protein phosphatase I) complex which dephosphorylates 2) a member of the Snf1 kinase complex. The Snf1 kinase complex includes at least the Snf4, and the Sip1, 2, and Gal83 proteins. When dephosphorylated by the glucose signal it isnon-functional, but when glucose is absent, it phosphorylates 3) the Mig1 repressor, inactivating it. When active, the Mig1 repressor complexes with the up1-Ssn6 general repressor to repress transcription of SUC2. The link between each of these tiers is tenuous, although the evidence suggests that each interacts as described. However, there are a number of missing factors including: the proposed Snf1 kinase kinase, the glucose sensing mechanism, and the transcriptional activators of SUC2 which may well be glucose regulated through Snf1, too. This application proposes to detail the known interactions and identify and characterize the missing factors. A combination of biochemical and genetic approaches will be used. The Snf1 kinase will be purified for both in vitro phosphorylation studies, especially focused on Mig1, and protein- protein interaction studies. The latter will be carried out using fusion proteins which will allow tethering one member of the complex to a column. The genetic studies place heavy reliance on the two-hybrid system to isolate factors that interact with Snf1 and members of the SNf1 complex as well as to detail the interacting regions. This approach will be complemented with mutant and high copy suppression hunts designed to identify factors that function upstream of the Snf1 kinase. For analyses of the downstream targets, phosphorylation of both wildtype Mig1 and mutant versions altered in the putative Snf1 target sites, will be studied in vitro and in vivo. Also a strategy is described using a modified two-hybrid approach to identify the SUC2 activator(s) based upon the assumption that these proteins are either essential or redundant since they were never identified in the original SUC2 uninducible mutant hunts.
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