The goal of this project is to characterize two maltose sensing/signaling pathways identified in Saccharomyces. The first regulates inducible MAL gene transcription via maltose permease and the MAL-activator. The second stimulates enhanced levels of pseudohyphal differentiation and is distinct from the pathway regulating MAL gene expression.
Specific Aim 1 investigates the role of maltose permease in regulating MAL gene expression. Is it a maltose receptor? Methods are described to isolate constitutive mutations in maltose permease and in other proteins that might function as downstream components of the postulated maltose signaling pathway. Additionally, we will test the ability of a heterologous maltose transporter to induce MAL gene expression.
Specific Aim 2 investigates the intracellular sorting of maltose permease during synthesis. Included is an analysis of glucose-induced changes in this trafficking pattern and the role of Snfl kinase. Snfl kinase is required posttranscriptionally for maltose permease synthesis. Suppressor analysis and biochemical approaches are described to identify this function. The roles of Stdlp and casein kinase I (Yck1,2p) in the glucose-induced proteolysis of maltose permease will be investigated.
Specific Aim 3 explores the role of Hsp90 molecular chaperone in the maltose-induced activation of the MAL- activator. The MAL-activator is an Hsp90 chaperone client protein. We will use our collection of MAL-activator mutants to test a proposed chaperone-mediated model of induction. Mutant MAL-activator turnover, chaperone complex formation, and response to permease overexpression will be assayed. Carbon source regulation of MAL-activator degradation will be examined and the ubiquitin-conjugation pathway components determined, particularly the E3 ubiquitin-protein ligase.
Specific Aim 4 will define the upstream components of the maltose sensing/signaling pathway used to stimulate pseudohyphal differentiation, including the maltose receptor. Gpr1 receptor and Gpa2 Galpha protein are not utilized. Tpk2 kinase (PKA) is required but no maltose- stimulated increase in cAMP synthesis is seen. Mutants capable of filamentation on glucose but not maltose will be isolated using transposon-mutagenesis.
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