A Cooperative Role for Atf1 and Pap1 in the Detoxification of the Oxidative Stress Induced by Glucose Deprivation in Schizosaccharomyces pombe
2004; Elsevier BV; Volume: 279; Issue: 40 Linguagem: Inglês
10.1074/jbc.m405509200
ISSN1083-351X
AutoresMarisa Madrid, Teresa Soto, Alejandro Franco, Vanessa Paredes, Jero Vicente‐Soler, Elena Hidalgo, Mariano Gacto, José Cansado,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoIn Schizosaccharomyces pombe, glucose concentrations below a certain threshold trigger the stress-activated protein kinase (SAPK) signal transduction pathway and promote increased transcription of Atf1-dependent genes coding for the general stress response. Removal of glucose specifically induces the nuclear accumulation of green fluorescent protein-labeled Pap1 (GFP-Pap1) and the expression of genes dependent on this transcription factor. In contrast, depletion of the nitrogen source triggers the SAPK pathway but does not activate Pap1-dependent gene transcription, indicating that carbon stress rather than growth arrest leads to an endogenous oxidative condition that favors nuclear accumulation of Pap1. The reductant agents glutathione or N-acetylcysteine suppress the nuclear accumulation of GFP-Pap1 induced by glucose deprivation without inhibiting the activation of the MAPK Sty1. In addition, cells expressing a mutant GFP-Pap1 unable to accumulate into the nucleus upon hydrogen peroxide-mediated oxidative stress failed to show this protein into the nucleus in the absence of glucose. These results support the concept of a concerted action between the SAPK pathway and the Pap1 transcription factor during glucose exhaustion by which glucose limitation induces activation of the SAPK pathway prior to the oxidative stress caused by glucose deprivation. The ensuing induction of Atf1-dependent genes (catalase) decreases the level of hydroperoxides allowing Pap1 nuclear accumulation and function. Congruent with this interpretation, glucose-depleted cells show higher adaptive response to exogenous oxidative stress than those maintained in the presence of glucose. In Schizosaccharomyces pombe, glucose concentrations below a certain threshold trigger the stress-activated protein kinase (SAPK) signal transduction pathway and promote increased transcription of Atf1-dependent genes coding for the general stress response. Removal of glucose specifically induces the nuclear accumulation of green fluorescent protein-labeled Pap1 (GFP-Pap1) and the expression of genes dependent on this transcription factor. In contrast, depletion of the nitrogen source triggers the SAPK pathway but does not activate Pap1-dependent gene transcription, indicating that carbon stress rather than growth arrest leads to an endogenous oxidative condition that favors nuclear accumulation of Pap1. The reductant agents glutathione or N-acetylcysteine suppress the nuclear accumulation of GFP-Pap1 induced by glucose deprivation without inhibiting the activation of the MAPK Sty1. In addition, cells expressing a mutant GFP-Pap1 unable to accumulate into the nucleus upon hydrogen peroxide-mediated oxidative stress failed to show this protein into the nucleus in the absence of glucose. These results support the concept of a concerted action between the SAPK pathway and the Pap1 transcription factor during glucose exhaustion by which glucose limitation induces activation of the SAPK pathway prior to the oxidative stress caused by glucose deprivation. The ensuing induction of Atf1-dependent genes (catalase) decreases the level of hydroperoxides allowing Pap1 nuclear accumulation and function. Congruent with this interpretation, glucose-depleted cells show higher adaptive response to exogenous oxidative stress than those maintained in the presence of glucose. Glucose is a powerful signaling molecule that promotes major metabolic changes in cells (1Rolland F. Winderickx J. Thevelein J.M. Trends Biochem. Sci. 2001; 26: 310-317Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Glucose metabolism produces compounds directly related to the detoxification of intracellular hydroperoxides formed as byproducts by ongoing metabolic processes (2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (296) Google Scholar). In human tumor cells, which typically show strong glycolysis and a reduced rate of respiration, deprivation of glucose causes a strong metabolic oxidative stress characterized by increased steady state levels of intracellular hydroperoxides and glutathione disulfide (3Lee Y.J. Galoforo S.S. Berns C.M. Chen J.C. Davis B.H. Sim J.E. Corry P.M. Spitz D.R. J. Biol. Chem. 1998; 273: 5294-5299Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 4Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Strong evidence indicates that the absence of glucose also triggers signaling cascades that activate transcription factors and the expression of stress-related genes attempting to redirect cellular functions (2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (296) Google Scholar). Remarkably, glucose metabolism in some fermenting yeasts is quite similar to that of tumor cells. In particular, the Crabtree-positive fission yeast S. pombe ferments glucose under aerobic conditions. Unlike Saccharomyces cerevisiae, this yeast lacks enzymes of the glyoxylate cycle that maintain diauxic growth after glucose depletion and utilizes very few non-sugar carbon sources (5Flores C. Rodriguez C. Petit T. Gancedo C. FEMS Microbiol. Rev. 2000; 24: 507-529Crossref PubMed Google Scholar, 6Van Dijken J.P. Weusthuis R.A. Peonk J.T. Antonie Van Leeuwenhoek. 1993; 63: 343-352Crossref PubMed Scopus (165) Google Scholar). Thus, as soon as glucose disappears and respiration of the fermentation products is impaired, cultures of S. pombe may suffer nutritional stress. The mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; DEM, diethyl maleate; Ha, hemagglutinin; Ha6H, epitope comprising hemagglutinin antigen plus six histidine residues; GFP, green fluorescent protein; EMM, Edinburgh minimal medium; GSH, glutathione; NAC, N-acetyl-l-cysteine; kbp, kilobase pair; ASK1, apoptosis signal-regulating kinase 1. signaling pathways are critical for the sensing and response of eukaryotic cells to changes in the external environment (7Treisman R. Curr. Opin. Cell Biol. 1998; 8: 205-215Crossref Scopus (1167) Google Scholar, 8Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2931) Google Scholar). These MAPK cascades are highly conserved through evolution and serve to transduce signals to the nucleus, resulting in new patterns of gene expression (9Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4287) Google Scholar, 10Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (538) Google Scholar). The identification of a highly conserved stress-activated protein kinase (SAPK) pathway in S. pombe allows us to analyze the mechanisms by which SAPKs are activated in a system more amenable than higher eukaryotic cells (11Warbrick E. Fantes P. EMBO J. 1991; 10: 4291-4299Crossref PubMed Scopus (118) Google Scholar, 12Millar J.B.A. Buck V. Wilkinson M.G. Genes Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (315) Google Scholar, 13Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (400) Google Scholar, 14Kato T. Okazaki K. Murakami H. Stettler S. Fantes P. Okayama H. FEBS Lett. 1996; 378: 207-212Crossref PubMed Scopus (155) Google Scholar, 15Degols G. Shiozaki K. Russell P. Mol. Cell. Biol. 1996; 16: 2870-2877Crossref PubMed Scopus (262) Google Scholar). In this yeast, the central element of the SAPK cascade is the MAPK Sty1 (also known as Spc1 or Phh1), which is highly homologous to mammalian p38 kinase and becomes activated by a similar series of stresses (12Millar J.B.A. Buck V. Wilkinson M.G. Genes Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (315) Google Scholar, 13Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (400) Google Scholar, 15Degols G. Shiozaki K. Russell P. Mol. Cell. Biol. 1996; 16: 2870-2877Crossref PubMed Scopus (262) Google Scholar, 16Degols G. Russell P. Mol. Cell. Biol. 1997; 17: 3353-3356Crossref Google Scholar, 17Shieh J.C. Wilkinson M.G. Buck V. Morgan B. Makino K. Millar J.B.A. Genes Dev. 1997; 11: 1008-1022Crossref PubMed Scopus (159) Google Scholar). MAPK Sty1 is directly phosphorylated by MAPK kinase Wis1; however, the transmission pathway of the stress signal to Wis1 is dual, and either MAPK kinase kinase Wak1 (also known as Wis4 or Wik1) or MAPK kinase kinase Win1 is responsible for Wis1 phosphorylation (18Price M.A. Cruzalegui F.H. Treisman R. EMBO J. 1996; 15: 6552-6563Crossref PubMed Scopus (303) Google Scholar). A response regulator protein, Mcs4, associates with Wak1, and probably with Win1, to regulate MAPK kinase kinase activity in response to several stimuli (17Shieh J.C. Wilkinson M.G. Buck V. Morgan B. Makino K. Millar J.B.A. Genes Dev. 1997; 11: 1008-1022Crossref PubMed Scopus (159) Google Scholar, 19Buck V. Quinn J. Soto T. Martin H. Saldanha J. Makino K. Morgan B.A. Millar J.B.A. Mol. Biol. Cell. 2001; 12: 407-419Crossref PubMed Scopus (143) Google Scholar). In S. pombe different transcription factors function downstream of the MAPK Sty1 cascade, among which Atf1 and Pap1 have been studied extensively. Atf1 (also known as Gad7 or Mts1) is a mammalian ATF-2 homologue b-ZIP protein that associates to and is phosphorylated by Sty1 following different stresses (20Takeda T. Toda T. Kominami K. Kanoshu A. Yanagida M. Jones N. EMBO J. 1995; 14: 6193-6208Crossref PubMed Scopus (232) Google Scholar, 21Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (371) Google Scholar, 22Wilkinson M.G. Samuels M. Takeda T. Toone M.W. Shieh J. Toda T. Millar J.B.A. Jones N. Genes Dev. 1996; 10: 2289-2301Crossref PubMed Scopus (317) Google Scholar). In fact, Sty1 is the only known kinase involved in Atf1 phosphorylation during stress. Transcription of a wide array of stress-response genes like gpx1+ (coding for glutathione peroxidase), ntp1+ (neutral trehalase), ctt1+ (cytoplasmic catalase), fbp1+ (fructose-1,6-bisphosphatase), or ste11+ (a high mobility group protein involved in the regulation of sexual differentiation) is controlled by Sty1 through Atf1 (21Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (371) Google Scholar, 22Wilkinson M.G. Samuels M. Takeda T. Toone M.W. Shieh J. Toda T. Millar J.B.A. Jones N. Genes Dev. 1996; 10: 2289-2301Crossref PubMed Scopus (317) Google Scholar). Another transcription factor, Pap1, encoded by the pap1+ gene, has been isolated as required for survival to oxidative stress and, like its S. cerevisiae homologue YAP1, shows high homology to mammalian c-Jun (23Toda T. Shimanuki M. Yanagida M. Genes Dev. 1991; 5: 60-73Crossref PubMed Scopus (302) Google Scholar). S. pombe cells deleted in pap1+ show high sensitivity to oxidative stress but not to osmotic stress or nutrient deprivation (24Toone W.M. Kige S. Samuels S. Morgan B.A. Toda T. Jones N. Genes Dev. 1998; 12: 1453-1463Crossref PubMed Scopus (270) Google Scholar). Moreover, Pap1 activity is regulated at the level of cellular localization. In glucose-growing cells Pap1 localizes to the cytoplasm but accumulates in the cell nucleus upon oxidative stress with hydrogen peroxide or the glutathione-depleting agent diethyl maleate (DEM). Hydrogen peroxide reversibly oxidizes two cysteine residues in Pap1 (at positions 278 and 501), whereas DEM induces a non-reversible modification (25Castillo E.A. Ayté J. Chiva C. Moldón A. Carrascal M. Abián J. Jones N. Hidalgo E. Mol. Microbiol. 2002; 45: 243-254Crossref PubMed Scopus (78) Google Scholar). As a result, modified Pap1 is unable to interact in both cases with the exportin Crm1 through the nuclear export signal located at the Pap1 carboxyl terminus (25Castillo E.A. Ayté J. Chiva C. Moldón A. Carrascal M. Abián J. Jones N. Hidalgo E. Mol. Microbiol. 2002; 45: 243-254Crossref PubMed Scopus (78) Google Scholar). This favors its nuclear accumulation and the increased transcription of essential genes for defense against oxidative stress, like ctt1+ (catalase), trr1+ (thioredoxin reductase), or sod1+ (superoxide dismutase) (24Toone W.M. Kige S. Samuels S. Morgan B.A. Toda T. Jones N. Genes Dev. 1998; 12: 1453-1463Crossref PubMed Scopus (270) Google Scholar, 26Mutoh N. Nakagawa C.W. Yamada K. Curr. Genet. 2002; 41: 82-88Crossref PubMed Scopus (18) Google Scholar). In contrast to Atf1, Pap1 is neither phosphorylated nor a substrate for Sty1 upon stress conditions; however, Sty1 presence/function is needed for nuclear accumulation of Pap1 in response to high concentrations of hydrogen peroxide, but not at low concentrations of the prooxidant (27Quinn J. Findlay V.J. Dawson K. Millar J.B.A. Jones N. Morgan B.A. Toone W.M. Mol. Biol. Cell. 2002; 13: 805-816Crossref PubMed Scopus (171) Google Scholar, 28Vivancos A.P. Castillo E.A. Jones N. Ayté J. Hidalgo E. Mol. Microbiol. 2004; (in press)PubMed Google Scholar). Several results have been reported previously that carbon starvation is an environmental stress able to activate the MAPK Sty1 in S. pombe (29Stettler S. Warbrick E. Prochnik S. Mackie S. Fantes P. J. Cell Sci. 1996; 109: 1927-1935Crossref PubMed Google Scholar, 30Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1997; 8: 409-419Crossref PubMed Scopus (113) Google Scholar). However, the contribution of the SAPK pathway to the cellular responses under glucose depletion after fermentative growth has not been investigated in detail. Transfer of cells from a glucose-rich to a glucose-free culture medium without an alternative carbon source may help to reveal characteristic responses normally masked under conditions of sugar availability or cell growth. Following such an approach we have analyzed the stress signal induced by glucose deprivation in cultures maintained under similar osmotic contexts to avoid the influence of stressing conditions unrelated to the absence of glucose. In this work we demonstrate that glucose limitation in S. pombe not only promotes activation of the SAPK signaling pathway that results in increased expression of Atf1-dependent stress-related genes but also induces an oxidative stress that favors the concerted expression of additional genes depending on the transcription factor Pap1. These results may help us to understand the mechanisms underlying the comparative resistance of glucose-depleted cells against external oxidative conditions. Strains, Plasmids, and Growth Conditions—The S. pombe strains employed in this study were the wild type strain TK003 (h–leu1–32) (14Kato T. Okazaki K. Murakami H. Stettler S. Fantes P. Okayama H. FEBS Lett. 1996; 378: 207-212Crossref PubMed Scopus (155) Google Scholar) and the mutants TK107 (h–leu1–32 ura4-D18 Δsty1:: ura4+) (14Kato T. Okazaki K. Murakami H. Stettler S. Fantes P. Okayama H. FEBS Lett. 1996; 378: 207-212Crossref PubMed Scopus (155) Google Scholar), NT146 (h–leu1–32 ura4-D18 Δatf1:: ura4+) (20Takeda T. Toda T. Kominami K. Kanoshu A. Yanagida M. Jones N. EMBO J. 1995; 14: 6193-6208Crossref PubMed Scopus (232) Google Scholar), TP108-3c [h–leu1–32 ura4-D18 Δpap1:: ura4+) (23Toda T. Shimanuki M. Yanagida M. Genes Dev. 1991; 5: 60-73Crossref PubMed Scopus (302) Google Scholar), and CA334 (h–leu1–32 ura4-D18 Δpap1:: ura4 Δatf1+:: ura4+) (31Nguyen A.N. Lee A. Place W. Shiozaki K. Mol. Biol. Cell. 2000; 11: 1169-1181Crossref PubMed Scopus (139) Google Scholar). Strains JM1521 (h+ade6-M210 his7–366 leu1–32 ura4-D18 sty1+:Ha6H(ura4+)) and JM1821 (h–ade6-M216 leu1–32 ura4-D18 atf1+:Ha6H (ura4+)) harbor a genomic copy of sty1+ or atf1+ tagged with two copies of the Ha epitope and six histidine residues, respectively (32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar). To visualize the localization of a GFP-Pap1 fusion, we used strain EHH14, which harbors an integrated copy of the wild type GFP-Pap1 chimeric gene under the control of the thiamine-repressible promoter nmt1 (25Castillo E.A. Ayté J. Chiva C. Moldón A. Carrascal M. Abián J. Jones N. Hidalgo E. Mol. Microbiol. 2002; 45: 243-254Crossref PubMed Scopus (78) Google Scholar). Strain EHH14.C278A encodes a mutated version of GFP-Pap1 in the cysteine residue 278 of Pap1, which is critical for protein oxidation mediated by hydrogen peroxide (25Castillo E.A. Ayté J. Chiva C. Moldón A. Carrascal M. Abián J. Jones N. Hidalgo E. Mol. Microbiol. 2002; 45: 243-254Crossref PubMed Scopus (78) Google Scholar). Plasmid p41GFT-Pap1 expresses an amino-terminal GFP-fused version of Pap1 under the control of an attenuated version (41X) of the nmt1 promoter (24Toone W.M. Kige S. Samuels S. Morgan B.A. Toda T. Jones N. Genes Dev. 1998; 12: 1453-1463Crossref PubMed Scopus (270) Google Scholar). S. pombe strains were routinely grown with shaking at 28 °C in EMM2 (32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar) with 7% glucose (repressing conditions) to a final A600 of 0.5, recovered by filtration, and resuspended in the same medium without glucose but with glycerol, sorbitol, glucose plus glycerol, or glucose plus sorbitol, depending on the particular experiment (see below). When indicated, reduced glutathione (GSH, 0.16 mm) or N-acetyl-l-cysteine (NAC, 30 mm) was added (2Spitz D.R. Sim J.E. Ridnour L.A. Galoforo S.S. Lee Y.J. Ann. N. Y. Acad. Sci. 2000; 899: 349-362Crossref PubMed Scopus (296) Google Scholar, 33Wiatrowski H.A. Carlson M. Eukaryot. Cell. 2003; 2: 19-26Crossref PubMed Scopus (34) Google Scholar). Culture media were supplemented with adenine, leucine, histidine, or uracil (100 mg/liter, all obtained from Sigma) depending on the requirements for each particular strain. Transformation of yeast strains was performed by the lithium acetate method as described elsewhere (32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar). Purification and Detection of Activated Sty1-Ha6H and Atf1-Ha6H Proteins following Glucose Deprivation—Yeast cells grown in EMM2 with 7% glucose to an A600 of 0.5 (actual glucose concentration = 6%, determined by the glucose oxidase method) were recovered by filtration and resuspended in the same medium devoid of glucose. EMM2 without glucose was osmotically equilibrated with 3% glycerol, 2.8% glycerol plus 0.5% glucose, 2.5% glycerol plus 1% glucose, 6% sorbitol, 5.5% sorbitol plus 0.5% glucose, or 5% sorbitol plus 1% glucose. At different times, the cells from 30 ml of culture were harvested by centrifugation at 4 °C, and yeast pellets were immediately frozen in liquid nitrogen. Under these conditions, the previously described Sty1 phosphorylation resulting from centrifugation (30Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1997; 8: 409-419Crossref PubMed Scopus (113) Google Scholar, 32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar) was not observed in unstressed cells. To analyze Sty1, total cell homogenates were prepared under native conditions employing chilled acid-washed glass beads and lysis buffer (10% glycerol, 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Nonidet P-40, supplemented with specific protease and phosphatase inhibitor cocktails (Sigma)). The lysates were removed and cleared by centrifugation at 13,000 × g for 15 min. Ha6H-tagged Sty1 was purified by using nickel-nitrilotriacetic acid-agarose beads (Qiagen Inc.) as reported previously (32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar). The purified proteins were resolved in 10% SDS-polyacrylamide gels, transferred to nitrocellulose filters (Amersham Biosciences), and incubated with either mouse anti-Ha (Roche Applied Science, clone 12CA5) or mouse antiphospho-p38 (New England Biolabs) antibodies. The immunoreactive bands were revealed with an anti-mouse horseradish peroxidase-conjugated secondary antibody (Sigma) and the ECL system (Amersham Biosciences). For Atf1-Ha6H purification, the pelleted cells were lysed into denaturing lysis buffer (6 m guanidine HCl, 0.1 m sodium phosphate, 50 mm Tris HCl, pH 8.0), and the Atf1 protein was isolated by affinity precipitation on nickel-nitrilotriacetic acid-agarose beads as described previously (34Shiozaki K. Russell P. Methods Enzymol. 1997; 283: 503-520Google Scholar). The purified proteins were resolved in 6% SDS-polyacrylamide gels, transferred to nitrocellulose filters (Amersham Biosciences), and incubated with a mouse anti-Ha antibody (12CA5). RNA Isolation and Hybridization—Yeast cells grown in EMM2 with 7% glucose to an A600 of 0.5 were recovered by filtration and resuspended in the same medium with 3% glycerol. Volumes of 50 ml of the cultures were recovered at different times, and total RNA preparations were obtained as described by Franco et al. (35Franco A. Soto T. Vicente-Soler J. Guillén P.V. Cansado J. Gacto M. J. Bacteriol. 2000; 182: 5880-5884Crossref PubMed Scopus (16) Google Scholar) and resolved through 1.5% agarose-formaldehyde gels. Northern (RNA)-hybridization analyses were performed as described by Sambrook et al. (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1998Google Scholar). The probes employed were amplified by PCR and included the following: a 0.7-kbp fragment of the apt1+ gene (23Toda T. Shimanuki M. Yanagida M. Genes Dev. 1991; 5: 60-73Crossref PubMed Scopus (302) Google Scholar) that was amplified with the 5′-oligonucleotide CCCAGTATGTCTACC and the 3′-oligonucleotide AAGTCTTACTTGCGG, a 0.4-kbp fragment of the gpx1+ gene (37Yamada K. Nakagawa C.W. Mutoh N. Yeast. 1999; 15: 1125-1132Crossref PubMed Scopus (36) Google Scholar) amplified with the 5′-oligonucleotide TTCTACGACTTGGCT and the 3′-oligonucleotide ACACTCTCGATATCG, a 0.9-kbp fragment of the trr1+ gene (38Benko Z. Miklos I. Carr A.M. Sipiczki M. Curr. Genet. 1997; 31: 481-487Crossref PubMed Scopus (15) Google Scholar) amplified with the 5′-oligonucleotide GTGACTCACAACAAG and the 3′-oligonucleotide TAATCGGTATCTTCC, a 2.1-kbp fragment of the pyp2+ gene (12Millar J.B.A. Buck V. Wilkinson M.G. Genes Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (315) Google Scholar) amplified with the 5′-oligonucleotide CCGAGAGCGTTTCTTGGA and the 3′-oligonucleotide AAGGGCTTGGAAGCCTGG, and a 1-kbp fragment of the fbp1+ gene (39Hoffman C.S. Winston F. Genetics. 1990; 124: 807-816Crossref PubMed Google Scholar) amplified with the 5′-oligonucleotide CTTCCAAGCCAAATACTG and the 3′-oligonucleotide GATCTCGACGAAATCGAC. Probes for ctt1+ and leu1+ were prepared as reported previously (32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar). To establish quantitative conclusions, the level of mRNAs was determined in a PhosphorImager (Amersham Biosciences) and compared with the internal control (leu1+ mRNA). Fluorescence Microscopy—To localize the GFP-Pap1 fusion, yeast cultures were grown in EMM2 with 7% glucose (with or without GSH and NAC) to an A600 of 0.5, recovered by filtration, and resuspended in the same medium with 6% glucose but lacking the nitrogen source (EMM2-N), 3% glycerol, 6% sorbitol, or 0.01–0.1% glucose plus 3% glycerol. Treatment with DEM was performed by adding the compound to glucose-growing cells at a final concentration of 4 mm. Small aliquots (10 μl) of the yeast cultures were loaded onto poly-l-lysine-coated slides, and the remaining suspension was withdrawn by aspiration. For nuclear staining, 3 μl of Hoechst in 50% glycerol was added. Fluorescence microscopy was performed on a Leica DM 4000B microscope with a ×100 objective. Images were captured with a cooled Leica DC 300F camera and IM50 software and then imported into Adobe PhotoShop 6.0 (Adobe Systems, Mountain View, CA). Cell Viability Assays and Analytical Determinations—Yeast strains grown in YES medium (2% glucose and 0.6% yeast extract) with 7% glucose to an A600 of 0.5 were recovered by filtration, resuspended for 1hat28 °C in the same medium with either 6% glucose or 3% glycerol, and treated for 1 h with 80 mm H2O2. The samples were diluted and spread in triplicate onto plates containing YES solid medium, and cell viability was measured by their ability to form colonies on this medium after incubation at 28 °C for 5 days. Results represent the mean values ± S.D. from three different experiments. Glucose concentration in the growth media was assayed by the glucose oxidase method (40Carrillo C. Vicente-Soler J. Gacto M. Microbiology. 1994; 140: 1467-1472Crossref PubMed Scopus (27) Google Scholar). Protein determination was performed according to Lowry et al. (41Lowry O.H. Rosegrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). SAPK Activation during Glucose Depletion—We first analyzed the kinetics of Sty1 activation due to glucose exhaustion. An exponentially glucose-growing culture of strain JM1521, which harbors a genomic copy of sty1+ tagged with two copies of the Ha epitope and six histidine residues (32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar), was shifted to a similar medium containing a non-assimilated carbon source (glycerol or sorbitol) instead of glucose. It should be noted that S. pombe does not feed on glycerol alone unless glucose is present for initial growth (42Higuchi T. Watanabe Y. Yamamoto M. Mol. Cell. Biol. 2002; 22: 1-11Crossref PubMed Scopus (101) Google Scholar) 2M. Madrid, T. Soto, A. Franco, V. Paredes, J. Vicente, E. Hidalgo, M. Gacto, and J. Cansado, unpublished observations. and that sorbitol is not a carbon source for this yeast (43Burnett J.A. Payne R.V. Yarrow D. Yeasts: Characteristics and Identification. Cambridge University Press, Cambridge, UK1983: 484Google Scholar) either. These compounds were used to prevent drastic osmotic changes in the non-glucose-containing medium and, hence, to avoid any potential disturbance of the SAPK pathway unrelated to glucose deprivation (44Bone N. Millar J.B. Toda T. Armstrong J. Curr. Biol. 1998; 8: 135-144Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Samples were collected at different times from the new medium. Sty1-HA6H protein was purified by affinity chromatography, and its activation was analyzed by Western immunoblotting using antiphospho-p38 antibodies. As shown in Fig. 1A and confirming previous reports (30Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1997; 8: 409-419Crossref PubMed Scopus (113) Google Scholar), the absence of glucose provoked a clear peak of activation of Sty1, with maximal phosphorylation at 5 min, followed by a rather slight decrease. The occurrence of a rapid Sty1-mediated response during the transition from glucose to non-glucose conditions led us to assess the phosphorylation status of the downstream transcription factor Atf1. This was performed by employing strain JM1821, which carries a genomic copy of the atf1+ gene tagged with two copies of the Ha epitope and six histidine residues (32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar). Earlier studies demonstrated that Atf1 of unstressed cells migrates in gel as a single protein of ∼85 kDa that undergoes a phosphorylation-dependent band shift under different stresses (32Soto T. Beltrán F.F. Paredes V. Madrid M. Millar J.B.A. Vicente-Soler J. Cansado J. Gacto M. Eur. J. Biochem. 2002; 269: 1-10Crossref Scopus (1) Google Scholar). As shown in Fig. 1A, glucose deprivation induced a Sty1-dependent band shift in the migration of Atf1 because of in vivo phosphorylation, whose initial kinetics matched closely that observed for Sty1 phosphorylation. Essentially identical results were obtained when sorbitol was used to balance osmolarity (Fig. 1D), confirming that the activation of the SAPK pathway is exclusively due to glucose limitation. To clarify whether SAPK activation was triggered only after a complete exhaustion of glucose, we further analyzed the glucose deprivation-induced phosphorylation of Sty1/Atf1. As shown in Fig. 1, B and E, a switch from high glucose-containing medium to osmotically equilibrated medium (containing glycerol or sorbitol) with glucose concentrations of 1% (w/v) or higher (not shown) did not induce Sty1/Atf1 phosphorylation. However, lower glucose concentrations (0.5% or less) prompted a rapid and transient increase in both Sty1 and Atf1 phosphorylation (Fig. 1, C and F) that was almost identical to that observed after complete glucose depletion (Fig. 1, A and D). Similar results were obtained when yeast cultures reached the early stationary phase of growth. 3E. Hidalgo, unpublished results. Hence, these data demonstrate that the activation of the SAPK pathway in S. pombe by a downshift in glucose concentration takes place even in the presence of a certain amount of this carbon source. Different Stress-responsive Genes Are Induced through the SAPK Pathway by Glucose Depletion—Because a decreased glucose concentration fully activates the SAPK pathway in S. pombe, we determined the expression of a set of genes previously described as totally or partially dependent on Sty1 through its main downstream effector Atf1. Cells from glucose-growing cultures were transferred to
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