Regulation of the Heat Shock Response in Yeast

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The heat shock response is characterized by the induction of transcription of genes encoding protein chaperones. Many stresses induce this response, foremost among them oxygen stresses. Most stresses that activate the heat shock response cause the unfolding and aggregation of proteins (as well as producing the oxygen radical, superoxide anion). Hence, the current paradigm suggests that regulation of the response in eukaryotes occurs through competition for chaperones between unfolded protein and the Heat Shock Transcription Factor, HSF. However, recent data indicate that HSF responds to superoxide anion in vitro, and suggest that superoxide may be critical for the heat shock response in whole cells of yeast, Drosophila, and humans. Thus, the heat shock response may be more properly characterized as a response to superoxide, with protein unfolding being a consequence of the stress but not the direct trigger of HSF activation. The purpose of this project is to distinguish whether unfolded protein or superoxide is the functional, in vivo, trigger of the response. The experiments to achieve this goal include comparing kinetics of production of superoxide and unfolded protein, examining complexes between chaperones and unfolded protein as well as chaperones and HSF, examining oxidative damage to protein, and determining the effects of stresses in yeast cells that are grown anaerobically. The project will also begin the pursuit of cellular components that counteract the effect of superoxide via biochemical activity that is dependent on reduced glutathione. To complement these biochemical approaches, genetic analyses will be performed to seek genes that, when overexpressed, interact with the heat shock system. In addition to providing genetic confirmation of the biochemistry, these experiments will provide an inroad into future analyses.a