Organisms respond to a changing environment in multiple ways. Animals can walk away. For plants and single-celled microbes, leaving a stressful environment is not an option. Nor is it an option for individual cells within a multicellular organism. In each of these cases the cells have to adapt. Perhaps the most common environmental change is a change in the nutrients the cell is bathed in. This influences their most basic property: the ability to generate ATP to maintain metabolic homeostasis. In the simple single celled microbe, the budding yeast Saccharomyces cerevisiae, the simplest experimental paradigm for a nutrient shift is the response to loss of glucose, the preferred energy and carbon source in this as in all organisms. In nature, yeast derive their nutrient supply primarily from fermentable sugars and have thus evolved very efficient pathways to take up and metabolize sugars over a wide range of concentrations. Their glycolytic pathway is so efficient that they can dispense altogether with respiration, making them a so-called "petite-negative" yeast, meaning they can grow without a functioning mitochondrial respiratory chain. However, when sugar is exhausted a robust respiratory metabolism is induced that allows them to utilize a variety of secondary carbon sources, ranging from ethanol and glycerol to complex stored carbohydrates and lipids. To activate these pathways an intricate intracellular signaling cascade is initiated by the protein kinase Snf1 and its accessory proteins. Snf1 is a homolog of the ubiquitous AMP-activated protein kinases (AMPKs) found in all multicellular organisms. AMPK functions as an intracellular energy sensor, re-directing metabolic activity to correspond to the availability and need for metabolites and energy. AMPKs modify enzyme activities directly by phosphorylation and indirectly by setting in motion a complex transcriptional cascade that activates transcription factors that in turn activate downstream target genes. Our primary interest is in understanding at a biochemical and molecular level the mechanisms by which Snf1 activates gene expression in yeast, acting through two of its downstream effectors, the transcription factors Adr1 and Cat8. In the last funding period we discovered an inactive pre-initiation complex that was formed when chromatin had become permissive for binding Adr1 and Cat8, but remained repressive for transcription activation. We propose to isolate and characterize the inactive "poised" RNA pol II complex. In the last year we discovered that Adr1 activity is regulated by a repressor, a 14-3-3 protein called Bmh in yeast. Bmh binds to a phosphorylated Regulatory Domain of Adr1. We propose to characterize the binding site for Bmh and to determine the mechanism whereby Bmh represses Adr1 activity.

Public Health Relevance

Alterations in transcriptional regulation brought about by changes in AMP-activated protein kinase (AMPK) activity are thought to occur in heart disease, metabolic syndrome, diabetes, development, and cancer. Our major goal is to understand how AMPK influences the transcription of a large set of genes that are regulated by nutrient stress in yeast. Understanding how the yeast AMPK, the Snf1 complex, regulates downstream genes could shed light on the mechanisms by which AMPK alters the transcription of human genes in response to nutrient stress. This information in turn, might lead to new insights into treatment and diagnosis of metabolic disorders brought about by pathological conditions related to glucose metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026079-32
Application #
8316230
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Reddy, Michael K
Project Start
1978-12-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
32
Fiscal Year
2012
Total Cost
$473,045
Indirect Cost
$169,811
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Braun, Katherine A; Vaga, Stefania; Dombek, Kenneth M et al. (2014) Phosphoproteomic analysis identifies proteins involved in transcription-coupled mRNA decay as targets of Snf1 signaling. Sci Signal 7:ra64
Parua, Pabitra K; Young, Elton T (2014) Binding and transcriptional regulation by 14-3-3 (Bmh) proteins requires residues outside of the canonical motif. Eukaryot Cell 13:21-30
Braun, Katherine A; Young, Elton T (2014) Coupling mRNA synthesis and decay. Mol Cell Biol 34:4078-87
Braun, Katherine A; Parua, Pabitra K; Dombek, Kenneth M et al. (2013) 14-3-3 (Bmh) proteins regulate combinatorial transcription following RNA polymerase II recruitment by binding at Adr1-dependent promoters in Saccharomyces cerevisiae. Mol Cell Biol 33:712-24
Infante, Juan Jose; Law, G Lynn; Young, Elton T (2012) Analysis of nucleosome positioning using a nucleosome-scanning assay. Methods Mol Biol 833:63-87
Abate, Georgia; Bastonini, Emanuela; Braun, Katherine A et al. (2012) Snf1/AMPK regulates Gcn5 occupancy, H3 acetylation and chromatin remodelling at S. cerevisiae ADY2 promoter. Biochim Biophys Acta 1819:419-27
Humston, Elizabeth M; Dombek, Kenneth M; Tu, Benjamin P et al. (2011) Toward a global analysis of metabolites in regulatory mutants of yeast. Anal Bioanal Chem 401:2387-402
Infante, Juan J; Law, G Lynn; Wang, I-Ting et al. (2011) Activator-independent transcription of Snf1-dependent genes in mutants lacking histone tails. Mol Microbiol 80:407-22
Parua, P K; Ratnakumar, S; Braun, K A et al. (2010) 14-3-3 (Bmh) proteins inhibit transcription activation by Adr1 through direct binding to its regulatory domain. Mol Cell Biol 30:5273-83
Ratnakumar, Sooraj; Young, Elton T (2010) Snf1 dependence of peroxisomal gene expression is mediated by Adr1. J Biol Chem 285:10703-14

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