Characterization of the Stress Signaling Pathway Induced by Hydrostatic Pressure in Saccharomyces cerevisiae: different transcription factors acting on mild and high pressure conditions.
Domitrovic, T.1; Fernandes M.C.1..; Boy-Marcotte, E.2; Masuda A.C1; Kurtenbach, E1.
1-Instituto de Bioquímica Médica, CSS, UFRJ, Rio e Janeiro, Brazil
2-Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France
Studies on the Hydrostatic Pressure effect on yeast cells are valuable to the comprehension of high pressure physiological adaptations, and also, to reveal novel features of stress adaptation in S cerevisiae. In response to various stresses Msn2 and Msn4 transcription factors are activated and induce a large set of genes by recognizing the stress response element (STRE) in the promoter. We have shown that the mutant strain deleted for those factors are 100X more sensitive to high hydrostatic pressure than the wild type and are unable to develop stress tolerance against 150MPa after a mild 50MPa treatment. Furthermore, RT-PCR showed that the induction of the gene HSP12, that presents STRE in the promoter region, was greatly diminished in Msn2/4 mutant cell after a 50MPa treatment. In this work, we characterized the Msn2/4 activity in various ranges of pressure by semi-quantitative RT-PCR of HSP12 and by assaying b-galactosidase activity in a strain carrying lacZ reporter gene under control of 4 STRE motifs. We verified that Msn2/4 was mostly activated by 50 MPa and this activation was controlled by cAMP, as artificially maintained high intracellular level of this second messenger prevents Msn2/4 transcriptional activity. An interesting result was the lack of b-galactosidase activity after a 100MPa treatment. This was probably not do to a failure in protein synthesis at this pressure, as HSP12p, was still induced even at 100MPa, as assayed by western-blotting. Corroborating this result, HSP12 mRNA over-expression at 100MPa could be observed even in a strain deleted for Msn2/4. As HSP12 induction was not detected after 50 MPa in the msn2/4 strain, we suggest that those factors are mainly acting at mild pressures while a still unknown regulon may be driven expression at more severe pressures. Taken together, these results, strongly indicate that Msn2/4 take part in the hydrostatic pressure stress response, and show a new characteristic for stress sensing pathway by revealing a distinct control of gene regulation among different pressure ranges. Support: CNPq and FAPERJ.
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