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Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity
W Görner1, E Durchschlag, M T Martinez-Pastor
1Vienna Biocenter, Institut für Biochemie und Molekulare Zellbiologie der Universität Wien and Ludwig Boltzmann-Forschungsstelle für Biochemie, A-1030 Wien, Austria.
Abstract:
Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae. They are required for the transcription of a number of genes coding for proteins with stress-protective functions. Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE). In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p. Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate. Stress-induced nuclear localization was found to be rapid, reversible, and independent of protein synthesis. Nuclear localization of Msn2p and Msn4p was shown to be correlated inversely to cAMP levels and protein kinase A (PKA) activity. A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to a SV40-NLS-GFP fusion protein. Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolished cAMP-driven nuclear export and cytoplasmic localization in unstressed cells. We propose stress and cAMP-regulated intracellular localization of Msn2p to be a key step in STRE-dependent transcription and in the general stress response.
Insights
Msn2p and Msn4p regulate stress genes in yeast by moving to the nucleus under stress. This rapid, reversible process depends on cAMP levels and protein kinase A activity, controlling gene expression.
Area of Science:
- * Molecular Biology
- * Yeast Genetics
- * Stress Response
Background:
- * Msn2p and Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae.
- * These proteins are Cys2His2 zinc finger transcription factors that bind to the stress response element (STRE).
- * STRE occupancy increases under stress, dependent on Msn2p and Msn4p presence.
Purpose of the Study:
- * To investigate the role of Msn2p and Msn4p in the general stress response of yeast.
- * To elucidate the mechanisms regulating the nuclear localization of Msn2p and Msn4p under stress conditions.
- * To determine the relationship between intracellular localization, cAMP levels, and protein kinase A (PKA) activity.
Main Methods:
- * In vivo footprinting studies to assess STRE occupation.
- * Analysis of Msn2p and Msn4p localization under various stress conditions (heat shock, osmotic stress, starvation, ethanol, sorbate).
- * Construction and testing of a SV40-NLS-GFP fusion protein with homologous regions of Msn2p/Msn4p.
- * Site-directed mutagenesis of a conserved PKA consensus site.
Main Results:
- * Msn2p and Msn4p accumulate rapidly and reversibly in the nucleus under diverse stress conditions, independent of protein synthesis.
- * Nuclear localization is inversely correlated with intracellular cAMP levels and PKA activity.
- * A specific region shared by Msn2p and Msn4p mediates stress-regulated nuclear localization.
- * Mutations in a conserved PKA consensus site disrupt cAMP-regulated localization.
Conclusions:
- * Stress-induced nuclear localization of Msn2p and Msn4p is a critical step in activating STRE-dependent gene transcription.
- * Intracellular localization of Msn2p is regulated by both stress signals and cAMP/PKA pathways.
- * This localization mechanism is central to the yeast general stress response.