Our research is directed towards understanding the regulation of SUC2 gene expression in the eucaryote Saccharomyces cerevisiae (yeast). The SUC2 structural gene for invertase encodes two differently regulated mRNAs (1.8 and 1.9 kb) with different 5 feet ends. A secreted, glycosylated form of invertase is encoded by the 1.9 kb mRNA, which is regulated by glucose repression. An intracellular, nonglycosylated invertase is encoded by the 1.8 kb mRNA, which is synthesized constitutively at a low level. The major aim of this work is to understand the molecular mechanisms responsible for regulation of SUC2 gene expression by glucose repression. One approach will be to identify 5 feet noncoding regions essential for regulated SUC2 expression. We have already found a region required for derepression by deletion analysis. Additional deletions will be constructed in vitro and inserted into the yeast genome to assay the effects on in vivo expression. We also propose to treat cloned SUC2 DNA with a mutagen and select in yeast for mutations causing constitutive invertase expression. The effects of both types of mutations will also be examined in strains carrying unlinked regulatory mutations to gain insight into the mode of action of regulatory molecules. The second major approach will be to identify all the unlinked genes involved in the regulation of invertase synthesis. We have already isolated and characterized mutations in five genes (SNF1 - SNF5) which prevent derepression and one gene (SSN6) which causes constitutive secreted invertase synthesis, and we have formulated a working model for glucose regulation of SUC2. To identify possible additional regulatory genes, we will select suppressors of the snf mutations and mutations causing constitutivity; the regulatory effects of these mutations and their interactions with one another will be examined. Finally, as a step towards determining their regulatory roles, we propose to clone and characterize the SNF2 - SNF5 and SSN6 genes. Multiple copies of each gene will be introduced into wild-type and mutant cells to determine the regulatory consequences, and the structure of each gene and the regulation of its expression will be analyzed. Such studies have already been carried out on the cloned SNF1 gene, and we are now ready to construct a protein fusion with 1acZ. Antibody prepared to the fusion protein will be used to characterize the protein product of the SNF1 gene.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034095-03
Application #
3284575
Study Section
Genetics Study Section (GEN)
Project Start
1984-07-01
Project End
1987-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Ruiz, Amparo; Xu, Xinjing; Carlson, Marian (2013) Ptc1 protein phosphatase 2C contributes to glucose regulation of SNF1/AMP-activated protein kinase (AMPK) in Saccharomyces cerevisiae. J Biol Chem 288:31052-8
Ruiz, Amparo; Liu, Yang; Xu, Xinjing et al. (2012) Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases. Proc Natl Acad Sci U S A 109:8652-7
Momcilovic, Milica; Carlson, Marian (2011) Alterations at dispersed sites cause phosphorylation and activation of SNF1 protein kinase during growth on high glucose. J Biol Chem 286:23544-51
Ruiz, Amparo; Xu, Xinjing; Carlson, Marian (2011) Roles of two protein phosphatases, Reg1-Glc7 and Sit4, and glycogen synthesis in regulation of SNF1 protein kinase. Proc Natl Acad Sci U S A 108:6349-54
Liu, Yang; Xu, Xinjing; Carlson, Marian (2011) Interaction of SNF1 protein kinase with its activating kinase Sak1. Eukaryot Cell 10:313-9
Amodeo, Gabriele A; Momcilovic, Milica; Carlson, Marian et al. (2010) Biochemical and functional studies on the regulation of the Saccharomyces cerevisiae AMPK homolog SNF1. Biochem Biophys Res Commun 397:197-201
Hedbacker, Kristina; Carlson, Marian (2008) SNF1/AMPK pathways in yeast. Front Biosci 13:2408-20
Momcilovic, Milica; Iram, Surtaj H; Liu, Yang et al. (2008) Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase. J Biol Chem 283:19521-9
Rudolph, Michael J; Amodeo, Gabriele A; Iram, Surtaj H et al. (2007) Structure of the Bateman2 domain of yeast Snf4: dimeric association and relevance for AMP binding. Structure 15:65-74
Hong, Seung-Pyo; Carlson, Marian (2007) Regulation of snf1 protein kinase in response to environmental stress. J Biol Chem 282:16838-45

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