GLUCOSE SENSING AND SIGNALING IN YEAST

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DESCRIPTION: This proposal focuses on glucose control of gene expression in yeast. Previous work has identified many of the players in glucose- regulated gene expression and ordered these proteins into elaborate genetic pathways. Research proposed for the next project period will examine in detail the functions of individual proteins and attempt to identify as yet unknown regulators of the regulators. The first major aim is an understanding of the mechanism of glucose repression. Mig1 is a DNA-binding protein that prevents transcription in the presence of glucose; Snf1 is a protein kinase that acts upstream of Mig1 and inhibits Mig1 activity. The possibility that Mig1 serves as a substrate for Snf1 will be tested in vivo and/or in vitro. Mig1 acts in conjunction with two other proteins, Ssn6 and Tup1. To determine whether Mig1 recruitment of Ssn6 and Tup1 is regulated by glucose, Mig1 immunoprecipitates will be tested for the presence of epitope- tagged versions of Ssn6 and Tup1. Indirect immunofluorescence will be used to determine whether Mig1 import into the nucleus depends on glucose. In addition, Mig1 mutants will be isolated and characterized. Studies of glucose repression will also include analysis of Gal83 and its homologs, Sip1 and Sip2, which are thought to activate the Snf1 kinase. GAL83, SIP1 and SIP2 will be mutagenized in order to generate dominant mutants defective in glucose repression. To search for other members of the Gal83 family, a gal83 sip1 sip2 triple mutant (which has no obvious phenotype) will be mutagenized and screened for mutants in which gene expression is no longer derepressible by glucose. In addition, multicopy suppressors of a GAL83 mutant will be sought. A final aspect of the proposed studies of glucose repression focuses on Reg1, which is assumed to act upstream of Gal83, Sip1 and Sip2. Reg1 will be immunolocalized and the two-hybrid protein system will be used to screen for interacting proteins. The second major aim is to understand the mechanism of glucose induction. Rgt1, like Mig1, is a DNA-binding protein that acts in collaboration with Ssn6 and Tup1 to inhibit gene expression. In contrast to Mig1, Rgt1 is inhibited in the presence of glucose. The Rgt1 binding sites in sequences upstream of glucose-induced genes will be defined precisely. The possibility that Rgt1 activity is regulated at the level of nuclear import or interaction with Ssn6/Tup1 will be investigated as described for Mig1. Rgt1 mutants will be isolated and characterized in order to define functional domains. Studies of glucose induction will also examine Grr1, which is believed to regulate Rgt1 positively. Results obtained elsewhere suggest that Grr1 might play a role in proteolysis; thus, the effect of grr1 mutations on Rgt1 protein stability will be investigated. The two-hybrid protein system will be used to identify Grr1-interacting proteins. Additional grr1 mutants will be generated and characterized in hopes of understanding how Grr1 receives the glucose signal. In order to identify novel components of the glucose induction pathway; multicopy suppressors of grr1 mutants will be sought and new non-derepressible mutants will be isolated. The third aim of the proposal is to identify the glucose sensor. A key question is whether glucose or a glucose metabolite is the signal or whether glucose transport is coupled to a mechanism (such as protein phosphorylation) that generates the signal. If glucose or a metabolite is the signal, then any glucose transporter should generate the signal. To test this possibility, several different transporters will tested individually for their ability to generate the signal. The Snf3 protein is an obvious candidate for a sensor because it is the only glucose transporter that is produced constitutively. Attempts will be made to generate snf3 mutants that generate a signal even in the absence of glucose and others that never transmit the signal even though they transport glucose. In addition, Snf3-interacting proteins will be identified in the two-hybrid protein system. Finally, various glucose analogs will be tested for their ability to activate glucose-induced gene expression. The fourth and last aim of the proposal is to identify trans- acting factors involved in transcription of the GAL4 gene. The sequence upstream of GAL4 lacks a TATA box, but recent studies have identifed a UES (for upstream essential sequence) believed to play the role of a TATA box. This sequence will be defined precisely by random mutagenesis and proteins that bind the UES will be identified in a one-hybrid protein screen. The UASs from other genes do not function in conjunction with the GAL4 UES. It is postulated that the protein that binds to the GAL4 UAS has a novel transcriptional activation domain that interacts specifically with a protein bound to the GAL4 UES. Attempts will be made to identify such a transcriptional activation domain by screening a library of yeast sequences fused to the lexA DNA binding domain for activation of a hybrid promoter consisting of the lexA DNA binding site and the GAL4 UES. Two proteins that appear to bind the GAL4 UAS have been identified in one-hybrid protein screen using the GAL4 UAS as bait. These proteins, Sth1 and Sth2, are homologs of Snf2, which is part of a large complex believed to promote chromatin remodeling. The possibility that Sth1 and/or Sth2 are UES-specific activators will be investigated using appropriate reporter genes and hybrid proteins.