The long range goal of this project is an understanding of the molecular mechanisms of gene regulation in eukaryotes. Genes involved in maltose fermentation in yeast are used as a model system. Previous studies have shown that transcriptional induction of genes required for maltose utilization depends on an activator protein that binds to sequences upstream of maltose-inducible genes. Molecular analyses carried out in the past project period have subdivided the activator encoded by the MAL63 gene into three functional domains. The DNA-binding domain is located within the amino-terminal 100 amino acids and the transcriptional activation domain is located between residues 100 and 250. Residues 250 through 470 constitute a regulatory domain that somehow responds to maltose. Mutational analysis of this regulatory region suggests that it consists of multiple positive and negative sequence elements, leading to the conclusion that this domain interacts with more than one other protein. The main goal of the next project period is to understand how maltose activates the transcriptional activator. In principle, maltose-dependent regulation of the Mal-activator could occur at any one of several levels - DNA-binding, nuclear localization, post-translational modification and sequestration of the protein into a multi-protein complex. In vivo footprinting of the MAL upstream activator sequence will be carried out under induced and uninduced conditions to determine whether DNA binding is regulated. Indirect immunofluorescence will be used to determine whether nuclear localization of the Mal63 protein varies in response to maltose. Isoelectric focusing of GST-Mal63 fusion protein purified from induced and uninduced cells will be used to test for post-translational modifications, such as phosphorylation or glycosylation. To investigate the possibility that Mal63 is sequestered in an inactive complex under uninducing conditions, cell extracts will be fractioned on gel filtration columns and the fractions analyzed by Western blotting. Several different approaches will be employed to identify Mal63- interacting proteins. The two-hybrid protein system will be used to screen for proteins that interact with various LexA-Mal63 fusion proteins. If Mal63 is found to be part of a high molecular weight complex by gel filtration, then the other proteins present in this complex will be sequenced and this information will be used to clone the corresponding genes. In addition, MAL63 mutants will be screened for second-site and multicopy suppressors. Screening for suppressors will be preceded by extensive in vitro mutagenesis of the MAL63 gene to generate potentially suppressible mutants, such as constitutive or uninducible alleles. Three different approaches will be used to generate mutants: (i) charged cluster-to-alanine scanning mutagenesis of the regulatory domain, (ii) site-directed mutagenesis of an interesting proline-rich region, (iii) construction of hybrids between one wild-type activator and one constitutive mutant and (iv) random mutagenesis of a MAL63 deletion mutant that lacks the carboxy-terminal regulatory domain, but still retains a negative regulatory component. Genes encoding putative Mal63-interacting proteins will be disrupted and the resulting mutants will be characterized. A final set of experiments aims to determine the mechanism of inducer sensing and signalling. Cells carrying the MAL63 gene are unable to ferment a-methylglucoside, whereas cells carrying the closely related MAL43 gene are able to utilize this sugar. It is assumed that these activator proteins differ with respect to their ability to interact either with a-methylglucoside (or a metabolite thereof) or with some other protein that binds a-methylglucoside. Efforts will be made to isolate mutations that broaden inducer specificity; such mutations might affect the transcriptional activator gene itself or a gene product that interacts with the activator. In addition, the MGL1 gene will be cloned and sequenced; this gene is believed to encode an activator that promotes transcription of genes involved specifically in fermentation of alpha-methylglucoside.
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