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.
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.
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