The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin
2005; Elsevier BV; Volume: 280; Issue: 51 Linguagem: Inglês
10.1074/jbc.m507014200
ISSN1083-351X
AutoresAntonio Santos, Mar Álvarez, M. San Mauro, C. Abrusci, Domingo Marquina,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up- (99) and down-regulated genes (47Mendoza I. Quintero F.J. Bressan R.A. Hasegawa P.M. Pardo J.M. J. Biol. Chem. 1996; 271: 23061-23067Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin. The transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up- (99) and down-regulated genes (47Mendoza I. Quintero F.J. Bressan R.A. Hasegawa P.M. Pardo J.M. J. Biol. Chem. 1996; 271: 23061-23067Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin. Killer phenomena are widespread in yeasts. Killer toxins are proteins or glycoproteins that are lethal to sensitive strains of the same species and a different variety of other yeast genera. In this line, attention has focused mainly on the characterization of killer toxins from Saccharomyces cerevisiae (K1, K2, and K28) followed more recently by the investigation of yeasts such as Kluyveromyces lactis, Zygosaccharomyces bailii, Hanseniaspora uvarum, Pichia membranifaciens, Debaryomyces hansenii, Schwanniomyces occidentalis, etc. (1Ahmed A. Sesti F. Ilan N. Shih T.M. Sturley S.L. Goldstein S.A. Cell. 1999; 99: 283-291Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 2Bevan E.A. Herring A.J. Mitchell D.J. Nature. 1973; 245: 81-86Crossref PubMed Scopus (104) Google Scholar, 3Breinig F. Tipper J.D. Schmitt J.M. Cell. 2002; 108: 395-405Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 4Chen W.B. Han Y.F. Jong S.C. Chang S.C. Appl. Environ. 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Once the protein nature of the toxin produced was established, the secreted protein was purified from the supernatant of growing cultures of P. membranifaciens in the early stationary phase. Previous biochemical studies on the PMKT 2The abbreviations used are: PMKTP. membranifaciens killer toxinHOGhigh osmolarity glycerolORFopen reading frameQ-PCRquantitative PCRAUarbitrary unitsHSPheat shock protein.2The abbreviations used are: PMKTP. membranifaciens killer toxinHOGhigh osmolarity glycerolORFopen reading frameQ-PCRquantitative PCRAUarbitrary unitsHSPheat shock protein. mechanism of killing of sensitive yeast cells indicated that PMKT is an 18-kDa protein that interacts with the (1 → 6)-β-d-glucans of the cell wall of sensitive yeasts (6Santos A. Marquina D. Leal J.A. Peinado J.M. Appl. Environ. Microbiol. 2000; 66: 1809-1813Crossref PubMed Scopus (97) Google Scholar, 10Santos A. Marquina D. Yeast. 2004; 21: 151-162Crossref PubMed Scopus (31) Google Scholar). Recently the killing mechanism of this killer toxin has been elucidated (10Santos A. Marquina D. Yeast. 2004; 21: 151-162Crossref PubMed Scopus (31) Google Scholar). Regardless of certain possible additional effects, the killer toxin of P. membranifaciens CYC 1106 acts by disrupting plasma membrane electrochemical gradients. The death of sensitive cells in the presence of killer toxin is characterized by a leakage of common physiological ions through non-regulated ion channels in the plasma membrane causing a discharge of cellular membrane potential and changes in ionic homeostasis in a way comparable to that of certain killer toxins (K1) (11Kagan B.L. Nature. 1983; 302: 709-711Crossref PubMed Scopus (88) Google Scholar). Non-selective channel formation has been suggested to be the cytotoxic mechanism of action of PMKT (10Santos A. Marquina D. Yeast. 2004; 21: 151-162Crossref PubMed Scopus (31) Google Scholar). P. membranifaciens killer toxin high osmolarity glycerol open reading frame quantitative PCR arbitrary units heat shock protein. P. membranifaciens killer toxin high osmolarity glycerol open reading frame quantitative PCR arbitrary units heat shock protein. Yeasts must cope with different adverse environmental conditions, including heat shock, oxidative stress, high osmolarity, extreme pH values, nutrient availability, and toxins from plants, fungi, or bacteria as well as heavy metals and different xenobiotics. Yeasts have therefore adapted to growth under these conditions by developing a variety of protective mechanisms ranging from general stress responses to highly specific regulatory pathways. A variety of changes in the environment activate multiple mitogen-activated protein kinase cascades, which convert these signals into appropriate metabolic responses (12Kapteyn J.C. Ter Riet B. Vink E. Blad S. De Nobel H. Van Den Ende H. Klis F.M. Mol. 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Watad A.A. Bressan R.A. Hasegawa P.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9681-9686Crossref PubMed Scopus (185) Google Scholar). The aim of this study was to determine the global gene expression responses of S. cerevisiae to the killer toxin produced by P. membranifaciens CYC 1106 with a view to gaining insight into the mechanisms and processes underlying the killing of sensitive yeast cells. Here for the first time we report evidence that the transcriptional response of S. cerevisiae to the presence of PMKT is very similar to the response of cells undergoing adaptation to ionic or osmotic changes in the cellular environment. Western analysis of the signaling through the HOG pathway revealed a Hog1p phosphorylation in response to PMKT. The analysis of arrayed ORFs was complemented and extended by the fact that a high proportion of deletion mutants of genes whose expression was observed to be affected by PMKT had altered sensitivity phenotypes. Yeast Strains and General Media—The killer strain used in this study was P. membranifaciens CYC 1106 (Complutense Yeast Collection, Complutense University of Madrid, Madrid, Spain) originally isolated from olive brines (41Marquina D. Peres C. Caldas F.V. Marques J.F. Peinado J.M. Spencer-Martins I. Lett. Appl. Microbiol. 1992; 14: 279-283Crossref Scopus (85) Google Scholar). The sensitive wild-type strain used in this study was S. cerevisiae Hansen BY4743 (MATa/MATα his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 MET15/met15Δ0 ura3Δ0/ura3Δ0) (deletion parental strains, catalog number 95400.BY4743, Invitrogen). Deletant strains were from the Saccharomyces Genome Deletion Project and are available commercially at Invitrogen (Yeast Deletion Pools-Homozygous Diploid, catalog number 95401.H1Pool). Homozygous diploids mutants were BY4743 (orf Δ::kan MX4/orf Δ::kan MX4). The basic medium used for this study was YMA medium (1% (w/v) glucose, 0.3% (w/v) yeast extract (Difco), 0.3% (w/v) malt extract (Difco), 0.5% (w/v) proteose peptone Number 3 (Difco), and 2% agar). The strains were maintained at 20 °C in YMA medium supplemented with 15% glycerol, 200 mg/liter G418 (Geneticin), and 2% agar. Time course experiments of the killing process were carried out in buffered YMB medium (YMA medium without glycerol, Geneticin, and agar) and buffered with 100 mm sodium citrate-phosphate, pH 4.0. Killer toxin activity was determined on YMA-MB agar plates (YMA medium (without glycerol and Geneticin) supplemented with 30 mg/liter methylene blue and 100 mm sodium citrate-phosphate buffer, pH 4.0). Killer Toxin Assay—We assayed for killer toxin sensitivity with a diffusion test using 6-mm-diameter antibiotic assay AA Whatman paper discs on buffered YMA-MB seeded with the selected S. cerevisiae mutant strains in parallel with the wild-type strain. Incubation was carried out at 20 °C because killer factor is rapidly inactivated at temperatures above 25 °C. The diameter of the inhibition zone was used as a measure of sensitivity to the killer toxin. For each mutant, sensitivity was tested up to three times, and the results were compared with those from the wild-type strain ((mutant inhibition area)/(wild-type inhibition area) × 100). Purification Procedure—P. membranifaciens CYC 1106 was cultured in YNB-D-Brij 58 medium (yeast nitrogen base-dextrose; Difco), 3 × 1 liter, in 2-liter Erlenmeyer flasks for 3 days at 20 °C at 150 rpm. The cells were centrifuged (4,000 × g for 10 min at 4 °C), and the supernatant was adjusted to a final glycerol concentration of 15% (v/v). The purification process was done as reported previously (9Santos A. Marquina D. Microbiology. 2004; 150: 2527-2534Crossref PubMed Scopus (79) Google Scholar). Measurement of Cell Death—S. cerevisiae BY4743 cells were grown to logarithmic phase in buffered YMB medium, collected, and subsequently resuspended in the same medium containing killer activity (1,205 AU/ml). The final cell concentration was 106 cells/ml. A control with heat-inactivated (5 min at 75 °C) killer toxin was run in parallel. Aliquots were taken periodically, and additional 10-fold dilutions were made serially to a final dilution of 10–4. Four volumes of 50 μl each were used for plating on YMA medium. The colonies were counted after 48 h of growth at 30 °C. Experimental Design and RNA Isolation—For RNA isolation, time course experiments of the killing process, using S. cerevisiae BY4743, were performed as three independent biological repeats. Approximately 200 ml of asynchronously grown cells were cultured in YMB medium (buffered with 100 mm sodium citrate-phosphate, pH 4.0) at 20 °C, shaking at 125 rpm until an A600 of 0.5 was reached. Ten milliliters of cells were harvested by centrifugation (14,000 rpm for 1 min at 4 °C) and snap-frozen in liquid nitrogen (at this point cells were collected for determination of expression profiles under basal conditions). Immediately afterward the rest of the culture was exposed to the killer toxin (1,205 AU/ml), and samples of 10 ml each were collected at 0, 15, 30, 45, 60, 90, and 120 min. Then samples for RNA extraction were harvested and frozen as above. Total RNA was extracted using the hot acid phenol method (42Lyne R. Burns G. Mata J. Penkett C.J. Rustici G. Chen D. Langford C. Vetrie D. Bähler J. BMC Genomics. 2003; 4: 27-41Crossref PubMed Scopus (181) Google Scholar). Microarray Hybridization, Scanning, and Data Acquisition—Based on the results on cell death, measured by plating, reverse transcription (Superscript, Invitrogen) was performed for 40 μg using total RNA extracted from cells after 45 min of exposure to PMKT. After the Cy3- and Cy5-dCTP-labeled cDNAs had been hybridized onto glass DNA microarrays containing 100% of all known and predicted S. cerevisiae genes, 40 μl of the mixed cDNA solution were allowed to hybridize to the microarrays at 62 °C for 12 h (Agilent hybridization chamber, Agilent Technologies, Palo Alto, CA). When the hybridization process was completed, the microarrays were washed with buffer A (0.1% SDS, 0.6× SSC) at 50 °C followed by a 5-min wash in buffer B (0.03× SSC) at 20 °C. Microarrays were scanned with Agilent scanner G2565BA (Agilent Technologies), and microarray images were analyzed with Agilent Feature Extraction software, version 7.5 (43DeRisi J.L. Iyer V.R. Brown P.O. Science. 1997; 278: 680-686Crossref PubMed Scopus (3690) Google Scholar, 44Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3888) Google Scholar). If expression ratios were >+3.0 (up-regulated) or <-2.0 (down-regulated) in four different arrays, the corresponding genes were then considered to be expressed differentially, and the average ratio from the change in expression was calculated and treated as the true ratio between the different arrays. Only genes with constant expression ratios between different experiments were considered. It must be taken into account that a large proportion of the genes (those between +3.0-fold induction and –2.0-fold repression) responded, according to our results, in an apparently random manner. It is known that when a stimulus occurs the nature of regulated transcripts changes over time, suggesting that different functions need to be activated at different time points. The origin of this variability is unknown but may be due to events that affect cells at different moments of the killing process but not simultaneously for the whole PMKT-treated yeast population. Other possibilities are small differences in toxin purity (high performance liquid chromatography-tested), media composition, or cell age. In light of this, we decided to confirm these results with quantitative real time PCR under the assumption that the constantly expressed genes were the main ones involved in the underlying process of killing. Real Time Quantitative PCR—Total RNA was obtained from cells at a time point of 45 min and then treated with DNase I (Sigma). cDNA was synthesized from samples of 1.5 μg of total RNA making use of the random primer procedure and the RETRoscript kit (Ambion, Austin, TX) following the manufacturer's instructions. Q-PCR was performed on an ABI PRISM® 7900 HT (Applied Biosystems, Warrington, UK) device using the SYBR Green PCR Master Mix (Applied Biosystems) and specific oligonucleotide primers at a concentration of 300 nm. Matching oligonucleotide primers were designed using the Primer Express software (PerkinElmer-Applied Biosystems) and Oligo software (Oligo Analizer) and synthesized by Sigma-Genosys (Sigma-Genosys, Ltd). Amplification of cDNA was performed over 40 cycles. The first cycle was performed at 95 °C for 10 min. Cycles 2–40 were performed at 95 °C for 15 s followed by 60 °C for 1 min. Each assay was performed in quadruplicate. For normalization of cDNA loading, all samples were run in parallel with a housekeeping gene (IPP1, inorganic pyrophosphatase). The specificity of PCR amplifications from the different sets of oligonucleotide primers was examined routinely by agarose gel electrophoresis. Northern Blot Analysis of the PMKT-dependent Genes—According to the results from microarrays, confirmed by Q-PCR, some osmoresponsive genes (GPD1 and GPP2) as well as CTT1 and HSP12, two general stress-responsive genes; PDR12 and TRK2, two genes highly induced at low pH; and ENA1, a gene known to be induced under high salt conditions, were studied by Northern blotting (32Rep M. Reiser V. Gartner U. Thevelein J.M. Hohmann S. Ammerer G. Ruis H. Mol. Cell. Biol. 1999; 19: 5474-5485Crossref PubMed Scopus (219) Google Scholar). To determine the extent of induction or repression of gene expression, all signals were compared with IPP1, which encodes inorganic pyrophosphatase, whose expression is not affected by osmotic stress (33Rep M. Krantz M. Thevelein M.J. Hohmann S. J. Biol. Chem. 2000; 275: 8290-8300Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Total RNA was isolated from cells (BY4743 and hog1Δ) at the selected time points (0, 15, 30, 45, 60, 90, and 120 min), separated by formaldehyde gels, and transferred onto nylon membranes (Hybond XL, Amersham Biosciences). Probes were generated by PCR from chromosomal DNA of BY4743. PCR probes of GPD1, GPP2, CTT1, HSP12, PDR12, TRK2, ENA1, and IPP1 were radiolabeled by random priming with [α-32P]ATP (MegaPrime, Amersham Biosciences), G-50-purified, and hybridized overnight at 65 °C in hybridization buffer (7% (w/v) SDS, 0.25 m sodium phosphate buffer, pH 7.5, 1 mm EDTA, and 1% (w/v) bovine serum albumin). The blots were washed (20 mm sodium phosphate, pH 7.5, 0.1% (w/v) SDS, and 1 mm EDTA), and the signal was detected either by exposure to radiosensitive film (Biomax MR, Eastman Kodak Co.). Values were normalized by comparison with IPP1 signals. Western Blotting of Hog1p and Hog1p Phosphorylation—Separation of total soluble protein (30 μg of total protein), isolated as described previously (45Davenport K.R. Sohaskey M. Kamada Y. Levin D.E. Gustin M.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 270: 30157-30161Google Scholar, 46Siderius M. Rots E. Mager W.H. Microbiology. 1997; 143: 3241-3250Crossref PubMed Scopus (48) Google Scholar), was accomplished on 12% polyacrylamide gels (BioRad), whereas subsequent Western blotting of Hog1p and phosphorylated Hog1p was performed according to standard procedures on polyvinylidene difluoride membranes (Bio-Rad). Briefly total soluble protein was isolated from S. cerevisiae BY4743 strain grown in buffered YMB medium after 0, 5, 10, 15, 30, 45, and 60 min of exposure to PMKT. Dual phosphorylation of Hog1p was determined using an anti-dually phosphorylated (Thr-174 and Tyr-176) p38 antibody (New England Biolabs, Beverly, MA). Hog1p was detected using an anti-C-terminal Hog1p antibody (Yc20, catalog number sc-6815, Santa Cruz Biotechnology, Santa Cruz, CA). Antibody binding was visualized using an ECL kit (Amersham Biosciences) after the binding of a horseradish peroxidase-conjugated secondary antibody (Sigma). Glycerol Determination—Cells (BY4743 and hog1Δ) grown overnight in YMB medium (buffered with 100 mm sodium citrate-phosphate, pH 4.0) at 20 °C were diluted to an A600 of 0.3 and grown for 4 h at 20°C. Cells were then subjected to PMKT (1,205 AU/ml). At time points (0, 0.5, 1, 1.5, 2, 2.5, 3, and 4 h) and in triplicate, 10-ml samples were taken for glycerol and dry weight determinations. Intracellular glycerol was determined by filtration with glass microfiber filters (Whatman GF/C), measuring the glycerol released by boiling the filters for 10 min. Dry weight was determined by drying filters with cells at 80 °C for 16 h. To determine the glycerol concentration of the extracellular medium during PMKT treatment, 1-ml samples of the treated cultures were centrifuged, and the supernatant was used for glycerol assays. Glycerol was determined with a glycerol measurement kit (catalog number 148270, Roche Applied Science). Genome-wide Gene Expression Profiles—To examine the gene expression response to PMKT a time course study of the killing process was carried out. After exposure of asynchronously grown cells to PMKT at times between 0 and 8 h (not shown), cell death was observed to begin after 1 h. The cell death rate in the presence of PMKT (1,205 AU/ml) was 0.22 h–1. From these death kinetics, a PMKT dosage of 1,205 AU/ml and sampling times of 0 min (untreated control) and 45 min (treated cells) after exposure to PMKT were chosen for mRNA isolation and subsequent microarray studies and Q-PCR. Genome-wide gene expression profiles of S. cerevisiae cultures grown asynchronously were examined after exposure to PMKT using whole-genome microarrays and Q-PCR. Similar results were observed for mRNA expression levels using both techniques. Validation of microarray results with Q-PCR was done to verify that array data were not the result of problems inherent to the array technology. The grouped distribution of genes that were either up- or down-regulated after exposure to PMKT (TABLES ONE and TWO) provided information pertinent to the killing mechanism of PMKT and to the response of S. cerevisiae to the toxin at the molecular level. The mRNA level of a total of 99 genes was at least 3-fold higher after the addition of PMKT (TABLE ONE). Forty-seven genes were observed to exhibit 2-fold or higher repression levels after 45 min of exposure to PMKT (TABLE TWO).TABLE ONEGenes induced by more than 3-fold after a PMKT exposureGene nameORFDescription of gene product-Fold inductionPMKT death zoneMicroarraysQ-RT-PCR%Signal transduction, gene expression, transcription, and RNA processing (31 genes)TFS1YLR178CCdc25-dependent nutrient and ammonia response cell cycle regulator15.312.5108GSP2YOR185CGTP-binding protein involved in trafficking through nuclear pores5.14.3104NAM8YHR086WProtein involved in meiotic recombination10.210.1106GIP2YER054CGlc7p-interacting protein4.33.8105SDS22YKL193CRegulatory subunit for the mitotic function of type I protein phosphatase5.54.8106YAP1aORFs falling into different categories.YML007WTranscription factor involved in halotolerance and oxidative stress response12.711.6121CUP2YGL166WCopper-dependent transcription factor6.67.2103XBP1aORFs falling into different categories.YIL101CStress-induced transcriptional repressor3.83.6WTREG1YDR028CRegulatory subunit for protein phosphatase Glc7p4.14.1102PAP1YKR002WPoly(A) polymerase required for mRNA 3′ end formation4.03.9108KSP1YHR082CSerine/threonine protein kinase5.24.3114KKQ8YKL168CWeak similarity to serine/threonine protein kinase14.111.7109PTK2YJR059WSerine/threonine protein kinase of required for polyamine uptake8.16.4104AKL1YBR059CArk family kinase-like protein; probable serine/threonine-specific protein kinase3.53.3110SCH9aORFs falling into different categories.YHR205WSerine/threonine protein kinase involved in stress response and nutrient-sensing signaling pathway10.612.5138STE20YHL007CSerine/threonine protein kinase of the pheromone pathway15.913.8114PSK1aORFs falling into different categories.YAL017WPAS kinase involved in the control of sugar metabolism and translation8.510.1123PBS2YJL128CTyrosine protein kinase of the MAP kinase kinase family19.918.5125MED2YDL005CTranscriptional regulation mediator15.314.5103ITC1YGL133WSubunit of Isw2 chromatin-remodeling complex6.68.4107SW16YLR182WTranscription factor3.32.3103TIS11YLR136CtRNA-specific adenosine deaminase 37.15.3WTUGA3YDL170WTranscriptional activator for GABA catabolic genes3.24.3104SRB2YHR041CDNA-directe
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