The long range goal of this project is to identify and characterize molecular mechanisms regulating gene expression in eukaryotes. We have chosen maltose fermentation in Saccharomyces as an example of a regulated system. We have chosen maltose fermentation in Saccharomyces as an example of a regulated system. The initial steps of maltose fermentation are carried out by maltose permease and maltase. Synthesis of these enzymes is induced by maltose and the Mal-activator mediates this regulation. The Ma1-activator is a DNA-binding transcription activator. We identified the functional domains of the Ma-1 activator, and localized the maltose- responsive regulatory domain to approximately the 220 residues at the C- terminus of this 470 residue protein. Our results indicate that several negative and positive regulatory sequence elements are found in the regulatory domain suggesting that several proteins interact with this region of Ma163p and regulate the transactivation domain of Ma163p in a sequence-specific manner. During the next grant period, we plan to test our model of maltose-dependent regulation of the Mal-activator. We will carry out a biochemical analysis of the Mal-activator protein and identify interacting proteins involved in maltose-regulated transcription activation. We will explore the regulatory role of subcellular localization, dimerization, formation of high molecular weight complexes, and post-translational modification in controlling DNA-binding and transcription activation by the Mal-activator. We will carry out a systematic mutation analysis of the regulatory domain of the Mal-activator to identify and localize the negative and positive regulatory sequence elements in this region. Allele-specific and high-copy suppressors of these mutant alleles will be isolated in an effort to identify proteins which interact with the Mal-activator at these sites. The Two-hybrid method will be used top identify proteins which interact with the Mal- activator. We will use molecular genetic approaches to determine whether maltose binds directly to the Ma1-activator. Mutations which broaden inducer specificity will be isolated. The alpha-methylglucoside-activator gene will be isolated and used to characterize the inducer sensing- signaling pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028216-23
Application #
2684718
Study Section
Genetics Study Section (GEN)
Project Start
1980-07-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
23
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Queens College
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Flushing
State
NY
Country
United States
Zip Code
11367
Ran, Fulai; Gadura, Nidhi; Michels, Corinne A (2010) Hsp90 cochaperone Aha1 is a negative regulator of the Saccharomyces MAL activator and acts early in the chaperone activation pathway. J Biol Chem 285:13850-62
Ran, Fulai; Bali, Mehtap; Michels, Corinne A (2008) Hsp90/Hsp70 chaperone machine regulation of the Saccharomyces MAL-activator as determined in vivo using noninducible and constitutive mutant alleles. Genetics 179:331-43
Gadura, Nidhi; Michels, Corinne A (2006) Sequences in the N-terminal cytoplasmic domain of Saccharomyces cerevisiae maltose permease are required for vacuolar degradation but not glucose-induced internalization. Curr Genet 50:101-14
Gadura, Nidhi; Robinson, Lucy C; Michels, Corinne A (2006) Glc7-Reg1 phosphatase signals to Yck1,2 casein kinase 1 to regulate transport activity and glucose-induced inactivation of Saccharomyces maltose permease. Genetics 172:1427-39
Wang, Xin; Michels, Corinne A (2004) Mutations in SIN4 and RGR1 cause constitutive expression of MAL structural genes in Saccharomyces cerevisiae. Genetics 168:747-57
Bali, Mehtap; Zhang, Bin; Morano, Kevin A et al. (2003) The Hsp90 molecular chaperone complex regulates maltose induction and stability of the Saccharomyces MAL gene transcription activator Mal63p. J Biol Chem 278:47441-8
Danzi, Sara E; Bali, Mehtap; Michels, Corinne A (2003) Clustered-charge to alanine scanning mutagenesis of the Mal63 MAL-activator C-terminal regulatory domain. Curr Genet 44:173-83
Wang, Xin; Bali, Mehtap; Medintz, Igor et al. (2002) Intracellular maltose is sufficient to induce MAL gene expression in Saccharomyces cerevisiae. Eukaryot Cell 1:696-703
Jiang, H; Tatchell, K; Liu, S et al. (2000) Protein phosphatase type-1 regulatory subunits Reg1p and Reg2p act as signal transducers in the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae. Mol Gen Genet 263:411-22
Hu, Z; Yue, Y; Jiang, H et al. (2000) Analysis of the mechanism by which glucose inhibits maltose induction of MAL gene expression in Saccharomyces. Genetics 154:121-32

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