TY - JOUR
AU1 - Snoek, Ishtar S.I.
AU2 - Tai, Siew L.
AU3 - Pronk, Jack T.
AU4 - Steensma, H. Yde
AU5 - Daran, Jean-Marc
AB - Abstract Despite the scientific and applied interest in the anaerobic metabolism of Saccharomyces cerevisiae, not all genes whose transcription is upregulated under anaerobic conditions have yet been linked to known transcription factors. Experiments with a reporter construct in which the promoter of the anaerobically upregulated TIR1 gene was fused to lacZ revealed a loss of anaerobic upregulation in an snf7Δ mutant. Anaerobic upregulation was restored by expression of a truncated allele of RIM101 that encodes for a constitutively active Rim101p. Analysis of lacZ expression in several deletion mutants confirmed that the effect of Snf7p on anaerobic upregulation of TIR1 involved Rim101p. Further studies with deletion mutants in NRG1, NRG2 and SMP1, which were previously shown to be regulated by Rim101p, could not totally elucidate the TIR1 regulation, suggesting the involvement of a more complex regulation network. However, the aerobic repression mechanism of TIR1 involved the general repressor Ssn6p–Tup1p. Transcriptome analysis in anaerobic chemostat cultures revealed that 26 additional genes exhibited an Snf7p/Rim101p-dependent anaerobic upregulation, among which, besides TIR1, are four other anaerobic genes SML1, MUC1, AAC3 and YBR300C. These results provide new evidence on the implication of the Rim101p cascade in the transcriptional regulation of anaerobic metabolism in S. cerevisiae. Saccharomyces cerevisiae, anaerobiosis, gene regulation, RIM101, SNF7, chemostat-based transcriptome analysis Introduction The genus Saccharomyces is unique among yeasts because of its ability to grow fast under strict anaerobic conditions (Visser, 1990), provided that the growth medium is supplemented with a source of unsaturated fatty acids and sterols, whose synthesis requires molecular oxygen (Rosenfeld & Beauvoit, 2003). Hitherto, the molecular basis for this ability, which is of considerable industrial relevance, remains incompletely understood. Sixty-five Saccharomyces cerevisiae genes showed a consistently higher transcript level under anaerobic conditions when aerobic and anaerobic growth was compared under four different nutrient limitation regimes (Tai, 2005). Several regulatory networks have been implicated in the transcriptional upregulation of yeast genes in the absence of oxygen. ROX1 is one of the targets of Hap1p, which is activated by oxygen-bound heme (Zhang & Guarente, 1994). Its product, Rox1p, represses the expression of hypoxic genes in the presence of oxygen (Deckert, 1995a, b). Repression also seems to rely on Mot3p, Mox1p, Mox2p and the Tup1/Ssn6 complex (Abramova, 2001). The transcription factors UPC2 and ECM22 are involved in the anaerobic induction of the genes involved in sterol import (Crowley, 1998; Shianna, 2001; Wilcox, 2002; ter Linde, 2003). Although these regulators control a substantial number of anaerobically upregulated genes, the transcriptional upregulation of many other ‘anaerobic’ genes has not been attributed to specific regulators. We recently performed a screen for transcriptional regulators of anaerobic genes in which the promoter of the TIR1 gene was fused to a lacZ reporter gene (Snoek, 2007). TIR1, which encodes a cell wall mannoprotein, exhibits very high transcript levels under anaerobic conditions and very low levels under aerobic conditions, although its role during anaerobic growth is unknown. In the screening experiments, a functional SNF7 gene was shown to be required for the anaerobic induction of TIR1. Although SNF7 has not been implicated previously in the regulation of ‘anaerobic’ genes, its molecular biology and role in cellular regulation have been investigated extensively in other contexts. Snf7p is a component of the subcomplex endosomal sorting complex required for transport (ESCRT)-III, which, together with ESCRT-0, -I, -II and Vps4 AAA-ATPase, forms the ESCRT. The ESCRT complex has been implicated previously in several and unrelated processes including the multivesicular body (MVB) biogenesis, which plays a critical role in the decision between recycling and degradation of membrane proteins (Babst, 2002; Bowers, 2004). Besides, it is now clearly demonstrated that Snf7p participates in the activation of the transcription factor Rim101p. Together with Rim20p, Rim13p and probably Rim8p, Snf7p participates in the proteolytic activation of the transcriptional regulator Rim101p. Initially involved in an alkaline pH response (Lamb, 2001; Penalva & Arst, 2002; Xu, 2004), Rim101p has been implicated in SUC2 expression (Weiss, 2008), cell wall assembly (Castrejon, 2006), adaptative response to weak acid stress (Mira, 2009) and the response to toxins (Ikeda, 2008). The aim of the present study is to investigate the role of SNF7 in the regulation of TIR1, to assess whether this role involves the Rim101p pathway and to investigate whether Snf7p and/or Rim101p are also involved in the regulation of other ‘anaerobic’ S. cerevisiae genes. After analyzing the transcriptional regulation of TIR1 in several genetic backgrounds, a chemostat-based transcriptome analysis was performed on snf7Δ and rim101Δ strains as well as on the isogenic reference strain. Sets of genes that were differentially expressed in the snf7 and rim101 deletion strains were then compared with sets of genes that were previously shown to be transcriptionally upregulated in anaerobic chemostat cultures of S. cerevisiae (Piper, 2002; Tai, 2005). Materials and methods Strains, plasmids and media The strains and plasmids used in this study are listed in Table 1 and the oligonucleotide primers are listed in Supporting Information, Table S1. Deletion mutants in the BY4741 genetic background were obtained from Open Biosystems (Huntsville, AL) (Winzeler, 1999). The geneticin (G418) resistance cassettes were amplified using the pUG6 vector as a template (Guldener, 1996). snf7Δ and rim101Δ deletion cassettes amplified using specific primer pairs (Table S1) were transformed into CEN.PK113-7D (provided by Dr P. Kötter, Frankfurt, Germany), resulting in strains GG3201 and IMK229, respectively. The phleomycin-resistant cassette was amplified using the pUG66 vector as a template (Gueldener, 2002). The nrg2Δ deletion cassette amplified with specific primers was transformed into BY4741-nrg1Δ, resulting in IMK233. The NRG1 promoter replacement cassette was generated using the vector pUG6-TPIpr. The TPI1 promoter was PCR amplified using primers TPI1 pro fw and TPI1 pro rev (Table S1). The fragment was digested with SfiI and SacII and cloned in pUG6 (Guldener, 1996) digested with the same restriction enzymes. After ligation, the resulting plasmid pUG6-TPI1pr was used as a template to generate the NRG1 promoter replacement cassette, which was subsequently transformed into BY4741 and CEN.PK113-7D, resulting in IMI003 and IMI004, respectively. Table 1 Strains and plasmids used in this study Strains  Genotypes  References  CEN.PK113-7D  MATa MAL2-8c SUC2  P. Kötter  BY4741  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0  Winzeler (1999)  IMZ162  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pTIR (natMX AprpTIR-lacZ)  This study  BY4743  MATa/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0  Winzeler (1999)  GG3201  MATa MAL2-8c SUC2 snf7∷loxP-kanr-loxP  Snoek (2007)  IMK229  MATa MAL2-8c SUC2 rim101∷loxP-kanr-loxP  This study  IMK233  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 nrg1∷loxP-Kanr-LoxP nrg2∷bler  This study  IMZ163  MATa MAL2-8cSUC2 pTIR (URA3 AprpTIR-lacZ natMX)  This study  BY4741-vsp24Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 vps24Δ∷KanMX  Winzeler (1999)  IMZ164  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 vps24Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-doa4Δ  MATahis3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0doa4Δ∷KanMX  Winzeler (1999)  IMZ165  MATahis3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0doa4Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-smp1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 smp1Δ∷KanMX  Winzeler (1999)  IMZ166  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 smp1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp4Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps4Δ∷KanMX  Winzeler (1999)  IMZ167  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps4Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim8Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim8Δ∷KanMX  Winzeler (1999)  IMZ168  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim8Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-snf7Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX  Winzeler (1999)  IMZ169  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps2Δ∷KanMX  Winzeler (1999)  IMZ170  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim20Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim20Δ∷KanMX  Winzeler (1999)  IMZ171  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim20Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim101Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim101Δ∷KanMX  Winzeler (1999)  IMZ172  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim101Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-upc2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0upc2Δ∷KanMX  Winzeler (1999)  IMZ173  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 upc2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-ssn6Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 ssn6Δ∷KanMX  Winzeler (1999)  IMZ174  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0ssn6Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-tup1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0tup1Δ∷KanMX  Winzeler (1999)  IMZ175  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 tup1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rox1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rox1Δ∷KanMX  Winzeler (1999)  IMZ176  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rox1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-bro1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0bro1Δ∷KanMX  Winzeler (1999)  IMZ177  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0bro1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim13Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim13Δ∷KanMX  Winzeler (1999)  IMZ178  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim13Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp20Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps20Δ∷KanMX  Winzeler (1999)  IMZ179  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps20Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-nrg1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX  Winzeler (1999)  IMZ180  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-slt2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 slt2Δ∷KanMX  Winzeler (1999)  IMZ181  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0slt2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-nrg2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg2Δ∷KanMX  Winzeler (1999)  IMZ182  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-mot3Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0mot3Δ∷KanMX  Winzeler (1999)  IMZ183  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0mot3Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  IMZ184  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX nrg2Δ∷bler pTIR (natMX AprpTIR-lacZ)  This study  IMI003  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0loxP-kanMX-loxP∷TPI1pro-NRG1  This study  IMZ185  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0loxP-kanMX-loxP∷TPI1pro-NRG1 pTIR (natMX AprpTIR-lacZ)  This study  IMI004  MATa MAL2-8cSUC2 loxP-KanMX-loxP∷TPI1pro∷NRG1  This study  IMZ186  MATa MAL2-8cSUC2 loxP-KanMX-loxP∷TPI1pro-NRG1 pTIR (natMX AprpTIR-lacZ)  This study  IMZ187  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pTIR (natMX AprpTIR-lacZ) pUDeRIM101-531 (LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter)  This study  IMZ188  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim101Δ∷KanMX pTIR (natMX AprpTIR-lacZ) pUDeRIM101-531 (LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter)  This study  IMZ189  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pTIR (natMX AprpTIR-lacZ) pRS315-SNF7 (ARSH4, CEN6, LEU2, Apr, ORI C SNF7)  This study  IMZ190  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pDAN (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ191  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pDAN (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ192  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pANB (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ193  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pANB (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    pRUL302  ARSH4 CEN6 URA3 AprORI C LacZ∷CYC1ter  ter Linde (2003)  pRUL415  ARSH4 CEN6 natMX AprORI C LacZ∷CYC1ter  This study  pPDAN  ARSH4 CEN6 natMX AprORI C DAN1pro(−1232 to +23) -lacZ-CYC1ter  This study  pPTIR  ARSH4 CEN6 natMX AprORI C TIR1pro (−1429 to +5) -lacZ-CYC1ter  This study  pPANB  ARSH4 CEN6 natMX AprORI C ANB1pro (−1505 to +5)-lacZ-CYC1ter  This study  pRS315  ARSH4, CEN6, LEU2, Apr, ORI C  Sikorski & Hieter (1989)  pRS315-SNF7  ARSH4, CEN6, LEU2, Apr, ORI C SNF7  This study  pUG6  ORIC, Apr, loxP-kanMX-loxP    pUG6-TPI1 pr  ORIC, Apr, loxP-kanMX-loxP∷TPI1pr    pKlNAT  ARSH4, CEN6, natMX, Apr, ORI C, KlCEN2, KARS  Steensma & Ter Linde (2001)  pENTR/D-TOPO  ORI, KanMX,  Invitrogen  pAG425GPD-ccdB  LEU2, Apr, 2μ, TDH3pro-ccdB-CYC1ter  Alberti (2007)  pUDeRIM101-531  LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter  This study  Strains  Genotypes  References  CEN.PK113-7D  MATa MAL2-8c SUC2  P. Kötter  BY4741  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0  Winzeler (1999)  IMZ162  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pTIR (natMX AprpTIR-lacZ)  This study  BY4743  MATa/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0  Winzeler (1999)  GG3201  MATa MAL2-8c SUC2 snf7∷loxP-kanr-loxP  Snoek (2007)  IMK229  MATa MAL2-8c SUC2 rim101∷loxP-kanr-loxP  This study  IMK233  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 nrg1∷loxP-Kanr-LoxP nrg2∷bler  This study  IMZ163  MATa MAL2-8cSUC2 pTIR (URA3 AprpTIR-lacZ natMX)  This study  BY4741-vsp24Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 vps24Δ∷KanMX  Winzeler (1999)  IMZ164  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 vps24Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-doa4Δ  MATahis3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0doa4Δ∷KanMX  Winzeler (1999)  IMZ165  MATahis3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0doa4Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-smp1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 smp1Δ∷KanMX  Winzeler (1999)  IMZ166  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 smp1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp4Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps4Δ∷KanMX  Winzeler (1999)  IMZ167  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps4Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim8Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim8Δ∷KanMX  Winzeler (1999)  IMZ168  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim8Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-snf7Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX  Winzeler (1999)  IMZ169  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps2Δ∷KanMX  Winzeler (1999)  IMZ170  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim20Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim20Δ∷KanMX  Winzeler (1999)  IMZ171  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim20Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim101Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim101Δ∷KanMX  Winzeler (1999)  IMZ172  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim101Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-upc2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0upc2Δ∷KanMX  Winzeler (1999)  IMZ173  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 upc2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-ssn6Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 ssn6Δ∷KanMX  Winzeler (1999)  IMZ174  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0ssn6Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-tup1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0tup1Δ∷KanMX  Winzeler (1999)  IMZ175  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 tup1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rox1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rox1Δ∷KanMX  Winzeler (1999)  IMZ176  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rox1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-bro1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0bro1Δ∷KanMX  Winzeler (1999)  IMZ177  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0bro1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim13Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim13Δ∷KanMX  Winzeler (1999)  IMZ178  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim13Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp20Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps20Δ∷KanMX  Winzeler (1999)  IMZ179  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps20Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-nrg1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX  Winzeler (1999)  IMZ180  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-slt2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 slt2Δ∷KanMX  Winzeler (1999)  IMZ181  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0slt2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-nrg2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg2Δ∷KanMX  Winzeler (1999)  IMZ182  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-mot3Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0mot3Δ∷KanMX  Winzeler (1999)  IMZ183  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0mot3Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  IMZ184  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX nrg2Δ∷bler pTIR (natMX AprpTIR-lacZ)  This study  IMI003  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0loxP-kanMX-loxP∷TPI1pro-NRG1  This study  IMZ185  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0loxP-kanMX-loxP∷TPI1pro-NRG1 pTIR (natMX AprpTIR-lacZ)  This study  IMI004  MATa MAL2-8cSUC2 loxP-KanMX-loxP∷TPI1pro∷NRG1  This study  IMZ186  MATa MAL2-8cSUC2 loxP-KanMX-loxP∷TPI1pro-NRG1 pTIR (natMX AprpTIR-lacZ)  This study  IMZ187  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pTIR (natMX AprpTIR-lacZ) pUDeRIM101-531 (LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter)  This study  IMZ188  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim101Δ∷KanMX pTIR (natMX AprpTIR-lacZ) pUDeRIM101-531 (LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter)  This study  IMZ189  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pTIR (natMX AprpTIR-lacZ) pRS315-SNF7 (ARSH4, CEN6, LEU2, Apr, ORI C SNF7)  This study  IMZ190  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pDAN (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ191  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pDAN (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ192  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pANB (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ193  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pANB (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    pRUL302  ARSH4 CEN6 URA3 AprORI C LacZ∷CYC1ter  ter Linde (2003)  pRUL415  ARSH4 CEN6 natMX AprORI C LacZ∷CYC1ter  This study  pPDAN  ARSH4 CEN6 natMX AprORI C DAN1pro(−1232 to +23) -lacZ-CYC1ter  This study  pPTIR  ARSH4 CEN6 natMX AprORI C TIR1pro (−1429 to +5) -lacZ-CYC1ter  This study  pPANB  ARSH4 CEN6 natMX AprORI C ANB1pro (−1505 to +5)-lacZ-CYC1ter  This study  pRS315  ARSH4, CEN6, LEU2, Apr, ORI C  Sikorski & Hieter (1989)  pRS315-SNF7  ARSH4, CEN6, LEU2, Apr, ORI C SNF7  This study  pUG6  ORIC, Apr, loxP-kanMX-loxP    pUG6-TPI1 pr  ORIC, Apr, loxP-kanMX-loxP∷TPI1pr    pKlNAT  ARSH4, CEN6, natMX, Apr, ORI C, KlCEN2, KARS  Steensma & Ter Linde (2001)  pENTR/D-TOPO  ORI, KanMX,  Invitrogen  pAG425GPD-ccdB  LEU2, Apr, 2μ, TDH3pro-ccdB-CYC1ter  Alberti (2007)  pUDeRIM101-531  LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter  This study  * Institut für Mikrobiologie des J.W. Goethe Universiteit. View Large Table 1 Strains and plasmids used in this study Strains  Genotypes  References  CEN.PK113-7D  MATa MAL2-8c SUC2  P. Kötter  BY4741  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0  Winzeler (1999)  IMZ162  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pTIR (natMX AprpTIR-lacZ)  This study  BY4743  MATa/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0  Winzeler (1999)  GG3201  MATa MAL2-8c SUC2 snf7∷loxP-kanr-loxP  Snoek (2007)  IMK229  MATa MAL2-8c SUC2 rim101∷loxP-kanr-loxP  This study  IMK233  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 nrg1∷loxP-Kanr-LoxP nrg2∷bler  This study  IMZ163  MATa MAL2-8cSUC2 pTIR (URA3 AprpTIR-lacZ natMX)  This study  BY4741-vsp24Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 vps24Δ∷KanMX  Winzeler (1999)  IMZ164  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 vps24Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-doa4Δ  MATahis3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0doa4Δ∷KanMX  Winzeler (1999)  IMZ165  MATahis3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0doa4Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-smp1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 smp1Δ∷KanMX  Winzeler (1999)  IMZ166  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 smp1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp4Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps4Δ∷KanMX  Winzeler (1999)  IMZ167  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps4Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim8Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim8Δ∷KanMX  Winzeler (1999)  IMZ168  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim8Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-snf7Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX  Winzeler (1999)  IMZ169  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps2Δ∷KanMX  Winzeler (1999)  IMZ170  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim20Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim20Δ∷KanMX  Winzeler (1999)  IMZ171  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim20Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim101Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim101Δ∷KanMX  Winzeler (1999)  IMZ172  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim101Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-upc2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0upc2Δ∷KanMX  Winzeler (1999)  IMZ173  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 upc2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-ssn6Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 ssn6Δ∷KanMX  Winzeler (1999)  IMZ174  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0ssn6Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-tup1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0tup1Δ∷KanMX  Winzeler (1999)  IMZ175  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 tup1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rox1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rox1Δ∷KanMX  Winzeler (1999)  IMZ176  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rox1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-bro1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0bro1Δ∷KanMX  Winzeler (1999)  IMZ177  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0bro1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim13Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim13Δ∷KanMX  Winzeler (1999)  IMZ178  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim13Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp20Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps20Δ∷KanMX  Winzeler (1999)  IMZ179  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps20Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-nrg1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX  Winzeler (1999)  IMZ180  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-slt2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 slt2Δ∷KanMX  Winzeler (1999)  IMZ181  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0slt2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-nrg2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg2Δ∷KanMX  Winzeler (1999)  IMZ182  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-mot3Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0mot3Δ∷KanMX  Winzeler (1999)  IMZ183  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0mot3Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  IMZ184  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX nrg2Δ∷bler pTIR (natMX AprpTIR-lacZ)  This study  IMI003  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0loxP-kanMX-loxP∷TPI1pro-NRG1  This study  IMZ185  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0loxP-kanMX-loxP∷TPI1pro-NRG1 pTIR (natMX AprpTIR-lacZ)  This study  IMI004  MATa MAL2-8cSUC2 loxP-KanMX-loxP∷TPI1pro∷NRG1  This study  IMZ186  MATa MAL2-8cSUC2 loxP-KanMX-loxP∷TPI1pro-NRG1 pTIR (natMX AprpTIR-lacZ)  This study  IMZ187  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pTIR (natMX AprpTIR-lacZ) pUDeRIM101-531 (LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter)  This study  IMZ188  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim101Δ∷KanMX pTIR (natMX AprpTIR-lacZ) pUDeRIM101-531 (LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter)  This study  IMZ189  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pTIR (natMX AprpTIR-lacZ) pRS315-SNF7 (ARSH4, CEN6, LEU2, Apr, ORI C SNF7)  This study  IMZ190  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pDAN (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ191  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pDAN (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ192  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pANB (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ193  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pANB (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    pRUL302  ARSH4 CEN6 URA3 AprORI C LacZ∷CYC1ter  ter Linde (2003)  pRUL415  ARSH4 CEN6 natMX AprORI C LacZ∷CYC1ter  This study  pPDAN  ARSH4 CEN6 natMX AprORI C DAN1pro(−1232 to +23) -lacZ-CYC1ter  This study  pPTIR  ARSH4 CEN6 natMX AprORI C TIR1pro (−1429 to +5) -lacZ-CYC1ter  This study  pPANB  ARSH4 CEN6 natMX AprORI C ANB1pro (−1505 to +5)-lacZ-CYC1ter  This study  pRS315  ARSH4, CEN6, LEU2, Apr, ORI C  Sikorski & Hieter (1989)  pRS315-SNF7  ARSH4, CEN6, LEU2, Apr, ORI C SNF7  This study  pUG6  ORIC, Apr, loxP-kanMX-loxP    pUG6-TPI1 pr  ORIC, Apr, loxP-kanMX-loxP∷TPI1pr    pKlNAT  ARSH4, CEN6, natMX, Apr, ORI C, KlCEN2, KARS  Steensma & Ter Linde (2001)  pENTR/D-TOPO  ORI, KanMX,  Invitrogen  pAG425GPD-ccdB  LEU2, Apr, 2μ, TDH3pro-ccdB-CYC1ter  Alberti (2007)  pUDeRIM101-531  LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter  This study  Strains  Genotypes  References  CEN.PK113-7D  MATa MAL2-8c SUC2  P. Kötter  BY4741  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0  Winzeler (1999)  IMZ162  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pTIR (natMX AprpTIR-lacZ)  This study  BY4743  MATa/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0  Winzeler (1999)  GG3201  MATa MAL2-8c SUC2 snf7∷loxP-kanr-loxP  Snoek (2007)  IMK229  MATa MAL2-8c SUC2 rim101∷loxP-kanr-loxP  This study  IMK233  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 nrg1∷loxP-Kanr-LoxP nrg2∷bler  This study  IMZ163  MATa MAL2-8cSUC2 pTIR (URA3 AprpTIR-lacZ natMX)  This study  BY4741-vsp24Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 vps24Δ∷KanMX  Winzeler (1999)  IMZ164  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 vps24Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-doa4Δ  MATahis3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0doa4Δ∷KanMX  Winzeler (1999)  IMZ165  MATahis3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0doa4Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-smp1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 smp1Δ∷KanMX  Winzeler (1999)  IMZ166  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 smp1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp4Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps4Δ∷KanMX  Winzeler (1999)  IMZ167  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps4Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim8Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim8Δ∷KanMX  Winzeler (1999)  IMZ168  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim8Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-snf7Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX  Winzeler (1999)  IMZ169  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps2Δ∷KanMX  Winzeler (1999)  IMZ170  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim20Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim20Δ∷KanMX  Winzeler (1999)  IMZ171  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim20Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim101Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 rim101Δ∷KanMX  Winzeler (1999)  IMZ172  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim101Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-upc2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0upc2Δ∷KanMX  Winzeler (1999)  IMZ173  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 upc2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-ssn6Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 ssn6Δ∷KanMX  Winzeler (1999)  IMZ174  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0ssn6Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-tup1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0tup1Δ∷KanMX  Winzeler (1999)  IMZ175  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 tup1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rox1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rox1Δ∷KanMX  Winzeler (1999)  IMZ176  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rox1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-bro1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0bro1Δ∷KanMX  Winzeler (1999)  IMZ177  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0bro1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-rim13Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim13Δ∷KanMX  Winzeler (1999)  IMZ178  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim13Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-vsp20Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps20Δ∷KanMX  Winzeler (1999)  IMZ179  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0vps20Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-nrg1Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX  Winzeler (1999)  IMZ180  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-slt2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 slt2Δ∷KanMX  Winzeler (1999)  IMZ181  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0slt2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-nrg2Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg2Δ∷KanMX  Winzeler (1999)  IMZ182  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg2Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  BY4741-mot3Δ  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0mot3Δ∷KanMX  Winzeler (1999)  IMZ183  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0mot3Δ∷KanMX pTIR (natMX AprpTIR-lacZ)  This study  IMZ184  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0nrg1Δ∷KanMX nrg2Δ∷bler pTIR (natMX AprpTIR-lacZ)  This study  IMI003  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0loxP-kanMX-loxP∷TPI1pro-NRG1  This study  IMZ185  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0loxP-kanMX-loxP∷TPI1pro-NRG1 pTIR (natMX AprpTIR-lacZ)  This study  IMI004  MATa MAL2-8cSUC2 loxP-KanMX-loxP∷TPI1pro∷NRG1  This study  IMZ186  MATa MAL2-8cSUC2 loxP-KanMX-loxP∷TPI1pro-NRG1 pTIR (natMX AprpTIR-lacZ)  This study  IMZ187  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pTIR (natMX AprpTIR-lacZ) pUDeRIM101-531 (LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter)  This study  IMZ188  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0rim101Δ∷KanMX pTIR (natMX AprpTIR-lacZ) pUDeRIM101-531 (LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter)  This study  IMZ189  MATa his3Δleu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pTIR (natMX AprpTIR-lacZ) pRS315-SNF7 (ARSH4, CEN6, LEU2, Apr, ORI C SNF7)  This study  IMZ190  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pDAN (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ191  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pDAN (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ192  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0 pANB (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    IMZ193  MATa his3Δ1 leu2Δ0 lys2Δ0 met15Δ0 ura3Δ0snf7Δ∷KanMX pANB (natMX DAN1pro(−1232 to +23) -lacZ-CYC1ter)    pRUL302  ARSH4 CEN6 URA3 AprORI C LacZ∷CYC1ter  ter Linde (2003)  pRUL415  ARSH4 CEN6 natMX AprORI C LacZ∷CYC1ter  This study  pPDAN  ARSH4 CEN6 natMX AprORI C DAN1pro(−1232 to +23) -lacZ-CYC1ter  This study  pPTIR  ARSH4 CEN6 natMX AprORI C TIR1pro (−1429 to +5) -lacZ-CYC1ter  This study  pPANB  ARSH4 CEN6 natMX AprORI C ANB1pro (−1505 to +5)-lacZ-CYC1ter  This study  pRS315  ARSH4, CEN6, LEU2, Apr, ORI C  Sikorski & Hieter (1989)  pRS315-SNF7  ARSH4, CEN6, LEU2, Apr, ORI C SNF7  This study  pUG6  ORIC, Apr, loxP-kanMX-loxP    pUG6-TPI1 pr  ORIC, Apr, loxP-kanMX-loxP∷TPI1pr    pKlNAT  ARSH4, CEN6, natMX, Apr, ORI C, KlCEN2, KARS  Steensma & Ter Linde (2001)  pENTR/D-TOPO  ORI, KanMX,  Invitrogen  pAG425GPD-ccdB  LEU2, Apr, 2μ, TDH3pro-ccdB-CYC1ter  Alberti (2007)  pUDeRIM101-531  LEU2, Apr, 2μ, TDH3pro-RIM101-531-CYC1ter  This study  * Institut für Mikrobiologie des J.W. Goethe Universiteit. View Large The Streptomyces noursei nourseothricin resistance gene nat1 from pKlNat (Steensma & Ter Linde, 2001) was excised using BglII and StuI, and ligated into the BglII and SmaI sites of pRUL302 (ter Linde, 2003), resulting in plasmid pRUL415. Promoter regions of the genes DAN1 (−1232 to +23 bp from the ATG start codon), TIR1 (−1429 to +5 bp from the ATG start codon) and ANB1 (−1505 to +5 bp from the ATG start codon) were PCR amplified from genomic DNA of the BY4743 strain with specific primers (Table S1) and cloned between the HindIII and the BamHI sites of pRUL415, in front of the LacZ gene, yielding plasmids pDAN, pTIR and pANB, respectively (Table 1). pDAN, pTIR and pANB were transformed into the reference BY4741 strain (IMZ192, IMZ162 and IMZ193, respectively) and deletion mutant strains (from IMZ164 to IMZ189) (Table 1). pTIR was transformed in CEN.PK113-7D, yielding the strain IMZ163 (Table 1). The SNF7 gene, including its native promoter and terminator (−504 to +1149 bp from the start codon, PCR amplified with the primers indicated in Table S1), was cloned using the BamHI and SalI sites of pRS315 (Sikorski & Hieter, 1989). The plasmid pRS315-SNF7 was transformed in BY4741-snf7Δ, yielding IMZ189. A truncated version of RIM101, allele RIM101-531 (Hayashi, 2005), was PCR amplified with primers RIM101-531forward and RIM101-531STOPreverse (Table S1), and cloned into pENTR/D-TOPO (Invitrogen, Breda, The Netherlands). The RIM101-531 allele was further recombined by with BP clonase (Invitrogen) in the advanced Gateway™ destination expression vector pAG415GPD-ccdB (Alberti, 2007), yielding the expression vector pUDeRIM101-531. Subsequently, pUDeRIM101-531 was transformed into BY4741-rim101Δ and BY4741-snf7Δ, resulting in strains IMZ188 and IMZ187, respectively. Individual yeast strains were transformed using the standard lithium acetate method (Gietz & Schiestl, 2007a). For the screening experiment in which the possible role of SNF7 in the regulation of anaerobically induced genes was identified (Snoek, 2007), the collection of haploid S. cerevisiae gene-deletion mutants was used, in which all nonessential ORFs in S. cerevisiae BY4741 are replaced by a KanMX cassette (Winzeler, 1999). Strains from the collection were transformed in 96-well microtiter plates with an adapted high-throughput version of the lithium acetate transformation method (Gietz & Schiestl, 2007b). Shake-flask cultures were grown either on YPD (Difco peptone 2%, Difco yeast extract 1%, glucose 2%), when necessary provided with 150 μg mL−1 G418, 100 μg mL−1 of nourseothricin or 100 μg mL−1 of phleomycin, or on a chemically defined medium (Burke, 2000). When required, the chemically defined medium was supplemented with l-lysine (30 μg mL−1), l-leucine (30 μg mL−1), l-histidine (20 μg mL−1) or uracil (30 μg mL−1). For anaerobic growth, 420 μg mL−1 Tween-80 and 10 μg mL−1 ergosterol were added to the media (Verduyn, 1990). Anaerobic cultures were incubated in a Bactron Anaerobic Environmental Chamber (Sheldon Manufacturing Inc., Cornelius, OR). Escherichia coli was grown in Luria–Bertani medium (Sambrook, 1989). If plasmids were present, ampicillin was added to 60 μg mL−1. When indicated, media were solidified by the addition of 1.5% agar (Difco, Lawrence, KS). Chemostat fermentation and microarray experiments Anaerobic glucose-limited chemostat cultures were grown at a dilution rate of 0.10 h−1 on a synthetic medium supplemented with the anaerobic growth factors Tween-80 and ergosterol as described previously (Tai, 2005). Independent triplicate fermentations were run for the snf7Δ strain and the reference strain CEN.PK113-7D; the rim101Δ strain was grown in independent duplicate cultures. In brief, 2-L Applikon fermenters with a working volume of 1 L were used at a temperature of 30 °C and a stirrer speed of 800 r.p.m. Culture pH was maintained at 5.0 by the automatic addition of 2 M KOH. Oxygen and carbon dioxide (CO2) concentrations in the off-gas were measured using an NGA 2000 Rosemount gas analyzer. Substrate and metabolite concentrations in the culture supernatants and media were analyzed using HPLC on an Aminex HPX-87H column (Biorad, Hercules, CA) with 5 mM H2SO4 as the mobile phase. Culture dry weights were determined via filtration (Postma, 1989). For the culture of GG3201 (snf7Δ) and its isogenic reference CEN.PK113-7D, rapid broth quenching in liquid nitrogen, RNA isolation and transcriptome analysis with Affymetrix microarrays were performed as described previously in Daran-Lapujade (2004), while for the IMK229 (rim101Δ) mutant and its isogenic reference CEN.PK113-7D, rapid broth quenching in liquid nitrogen, RNA isolation and transcriptome analysis with Affymetrix microarrays were performed as described in De Nicola (2007). The main difference between the two protocols is that the protocol used for the snf7Δ strain includes a poly A-mRNA isolation step (with cDNA synthesis being performed on purified mRNA), while the protocol used for the rim101Δ strain excludes the purification step (with cDNA synthesis being performed on total RNA). The results were analyzed using the statistical analysis of microarrays tool (sam, version 2.0) (Tusher, 2001), with a false-discovery rate of 1% and a minimum fold change of 2.00. The complete transcript data set used and produced in this study can be downloaded from the Genome Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/index.cgi) under the series accession number GSE18128. Groups of coresponsive genes were investigated for over-representation of functional annotation categories [Gene Ontology (GO) annotation (Hong, 2008)] as described previously (Kresnowati, 2006; Knijnenburg, 2007). Promoter analysis was performed using the web-based software regulatory sequence analysis (rsa) tools (van Helden, 1998, 2000). The promoters (from −800 to −1) of each set of coregulated genes were analyzed for overrepresented hexanucleotides. β-Galactosidase assay A high-throughput, microtiter plate β-galactosidase assay was used in the screening that identified the possible role of SNF7 in the regulation of anaerobically induced genes (Snoek, 2007). For quantitative analysis of β-galactosidase activities, 50 mL of overnight shake-flask cultures (grown either aerobically or anaerobically on a glucose chemically defined medium) were harvested by centrifugation (10 min, 5000 g, 4 °C). The pellet was washed with 10 mM phosphate buffer at pH 7.5, containing 2 mM EDTA, and resuspended in 4 mL of the same buffer. Cell extracts were prepared using a FastPrep kit (Thermo Electron Corporation, Waltham, MA) and used for spectrophotometric analysis of β-galactosidase activity (ter Linde, 2003). Assays were performed at 30 °C and specific activities were calculated based on an absorption coefficient of o-nitrophenol at 420 nm of 4.6 mM−1 cm−1. Protein concentrations in the cell extracts were estimated using the Lowry method (Lowry, 1951). Phenotypic analysis Sodium dodecyl sulfate (SDS) sensitivity assays were performed by growing the cells overnight in a synthetic medium with the suitable auxotrophic requirement for each strain. After 18 h, the cells were diluted to an OD660 nm of 0.2 in a fresh synthetic medium. Serial dilutions were made and 2 μL of each dilution was spotted onto YPD plates with the anaerobic growth factors Tween-80 and ergosterol. The SDS concentration ranged from 0% to 0.04% (w/v). Plates were incubated aerobically or anaerobically (in Anaerocult IS sacks, Merck, Germany) for 72 h at 30 °C. Results Involvement of SNF7 in the transcriptional regulation of three ‘anaerobic’ yeast genes We first implicated SNF7 in the regulation of anaerobically upregulated genes during a partial screening (Snoek, 2007) of the BY4741-based haploid S. cerevisiae deletion collection (Winzeler, 1999). After transformation with a plasmid carrying a lacZ reporter gene fused to the promoter of the TIR1 gene, lacZ expression was not observed in anaerobic cultures of an snf7Δ mutant (Snoek, 2007), whereas the transcription of TIR1 [which encodes a cell wall mannoprotein (Kitagaki, 1997)] is strongly upregulated under anaerobic conditions in wild-type S. cerevisiae (Abramova, 2001; Cohen, 2001). To verify the results of this preliminary screening experiment, which was performed in microtiter plates, LacZ expression was analyzed in anaerobic shake-flask cultures. Deletion of SNF7 completely abolished lacZ expression (Table 2). Furthermore, transcriptome analysis in anaerobic, glucose-limited chemostat cultures showed a 25-fold reduction of TIR1 transcript levels in an snf7Δ mutant (strain GG3201) relative to the isogenic CEN.PK113-7D reference strain (Table 2). These results confirmed that SNF7 strongly influences the anaerobic upregulation of TIR1 transcription in two different genetic backgrounds (BY4741 and CEN.PK113-7D). The ANB1 and DAN1 genes [encoding translation elongation factor eIF-5A (Mehta, 1990) and a cell wall mannoprotein (Mrsa, 1999), respectively] are also transcriptionally induced under anaerobic conditions in wild-type S. cerevisiae strains (Schwelberger, 1993; Abramova, 2001). Experiments with lacZ constructs in batch cultures showed a severe reduction of anaerobic upregulation of these two genes in an snf7Δ strain (strains IMZ191 and IMZ193) (Table 2). However, only a moderate effect was observed on the transcript levels in anaerobic, glucose-limited chemostat cultures (Table 2). Table 2 Expression values as measured by a β-galactosidase assay of promoter fusion constructs in shake-flask cultures in strains IMZ162 (pTIR), IMZ169 (snf7Δ pTIR), IMZ190 (pDAN), IMZ191 (snf7Δ pDAN), IMZ192 (pANB1) and IMZ193 (snf7Δ pANB) and by microarray analysis of chemostat cultures, for the TIR1, DAN1 and ANB1 genes in the Δsnf7 strain (GG3201), under anaerobic conditions and the isogenic reference strain (CEN.PK113-7D) under aerobic and anaerobic conditions   SNF7 aerobic  SNF7 anaerobic  snf7Δ anaerobic          Strain plasmid  β-Galactosidase (U mg−1 protein)  mRNA (AU)  β-Galactosidase (U mg−1 protein)  mRNA (AU)  β-Galactosidase (U mg−1 protein)  mRNA (AU)  pTIR1  ND  100 ± 21  0.38 ± 0.02  4249 ± 292  ND  172 ± 9  pDAN1  ND  21 ± 12  0.37 ± 0.01  3125 ± 140  0.18 ± 0.01  1886 ± 462  pANB1  ND  22 ± 3  0.16 ± 0.01  2628 ± 159  0.06 ± 0.00  1383 ± 529    SNF7 aerobic  SNF7 anaerobic  snf7Δ anaerobic          Strain plasmid  β-Galactosidase (U mg−1 protein)  mRNA (AU)  β-Galactosidase (U mg−1 protein)  mRNA (AU)  β-Galactosidase (U mg−1 protein)  mRNA (AU)  pTIR1  ND  100 ± 21  0.38 ± 0.02  4249 ± 292  ND  172 ± 9  pDAN1  ND  21 ± 12  0.37 ± 0.01  3125 ± 140  0.18 ± 0.01  1886 ± 462  pANB1  ND  22 ± 3  0.16 ± 0.01  2628 ± 159  0.06 ± 0.00  1383 ± 529  Averages and SDs of the microarray data are from independent triplicate cultures and expressed in Affymetrix fluorescence unit (AU). The β-galactosidase activities are from independent duplicate cultures and expressed in U mg−1 protein (a unit being defined as μmol of ONP released per minute at 30°C and pH 7.5). ND, not detectable. View Large Table 2 Expression values as measured by a β-galactosidase assay of promoter fusion constructs in shake-flask cultures in strains IMZ162 (pTIR), IMZ169 (snf7Δ pTIR), IMZ190 (pDAN), IMZ191 (snf7Δ pDAN), IMZ192 (pANB1) and IMZ193 (snf7Δ pANB) and by microarray analysis of chemostat cultures, for the TIR1, DAN1 and ANB1 genes in the Δsnf7 strain (GG3201), under anaerobic conditions and the isogenic reference strain (CEN.PK113-7D) under aerobic and anaerobic conditions   SNF7 aerobic  SNF7 anaerobic  snf7Δ anaerobic          Strain plasmid  β-Galactosidase (U mg−1 protein)  mRNA (AU)  β-Galactosidase (U mg−1 protein)  mRNA (AU)  β-Galactosidase (U mg−1 protein)  mRNA (AU)  pTIR1  ND  100 ± 21  0.38 ± 0.02  4249 ± 292  ND  172 ± 9  pDAN1  ND  21 ± 12  0.37 ± 0.01  3125 ± 140  0.18 ± 0.01  1886 ± 462  pANB1  ND  22 ± 3  0.16 ± 0.01  2628 ± 159  0.06 ± 0.00  1383 ± 529    SNF7 aerobic  SNF7 anaerobic  snf7Δ anaerobic          Strain plasmid  β-Galactosidase (U mg−1 protein)  mRNA (AU)  β-Galactosidase (U mg−1 protein)  mRNA (AU)  β-Galactosidase (U mg−1 protein)  mRNA (AU)  pTIR1  ND  100 ± 21  0.38 ± 0.02  4249 ± 292  ND  172 ± 9  pDAN1  ND  21 ± 12  0.37 ± 0.01  3125 ± 140  0.18 ± 0.01  1886 ± 462  pANB1  ND  22 ± 3  0.16 ± 0.01  2628 ± 159  0.06 ± 0.00  1383 ± 529  Averages and SDs of the microarray data are from independent triplicate cultures and expressed in Affymetrix fluorescence unit (AU). The β-galactosidase activities are from independent duplicate cultures and expressed in U mg−1 protein (a unit being defined as μmol of ONP released per minute at 30°C and pH 7.5). ND, not detectable. View Large Anaerobic upregulation of TIR1 involves functions ‘downstream’ Snf7p/Vps20p Snf7p participates in the ESCRT pathway as part of the ESCRT-III complex (Babst, 2002). To investigate a possible role of this complex in the anaerobic upregulation of TIR1, the mutant strains corresponding to three other components of the ESCRT-III subcomplex (vps2Δ, vps20Δ, vps24Δ), the mutants corresponding to the two fungal homologues of the mammalian protein Alix interacting with Snf7p (bro1Δ, rim20Δ) as well as a mutant of the Vps/Vta1 complex (vps4Δ) and a ubiquitin-specific protease that acts in recycling ubiquitin from protein substrates targeted to the proteasome and vacuole interacting with Bro1p (doa4Δ) were selected from the yeast deletion collection (Winzeler, 1999). The seven strains were transformed with the pTIR plasmid (yielding strains IMZ164, IMZ165, IMZ167, IMZ169, IMZ170, IMZ177 and IMZ179; Table 1) and β-galactosidase activities were determined in anaerobic shake-flask cultures. A clear distinction between the components of the ESCRT complex could be drawn. Like SNF7 deletion, the vps20Δ strain (IMZ179) exhibited a complete reduction of β-galactosidase activity, while vps2Δ (IMZ170) and vps24Δ (IMZ164) mutants exhibited a β-galactosidase twofold higher relative to the reference strain (IMZ162) (Fig. 1). The higher β-galactosidase activity of the vps2Δ and vps24Δ strains relative to the control IMZ162 suggested that Snf7p/Rim101p-dependent TIR1 expression might be titrated by the ESCRT-III subunits (Vps2p and Vps24p) or could result from the constitutive activation of Rim101p in these mutants (Hayashi, 2005). Fig. 1 View large Download slide Induction of TIR1 promoter-driven β-galactosidase activity. (a) β-Galactosidase activity was measured in reference strain IMZ162 and in strains carrying deletion in the genes SNF7, VPS2, VPS20, VPS24, BRO1, RIM20, VPS4, DOA4, RIM101, RIM8 and RIM13. (b) β-Galactosidase activity was measured in reference strain IMZ162 and in strains IMZ169 (snf7Δ), IMZ172 (rim101Δ), IMZ188 (rim101ΔTDH3pr-RIM101-531) and IMZ187 (snf7ΔTDH3pr-RIM101-531). Exponential-phase cells growing on glucose under aerobic and anaerobic conditions were harvested and β-galactosidase activity was measured. β-Galactosidase activities are expressed as μM of o-nitrophenol (ONP) formed per min and per mg of protein. The activity displayed is the average of two independent biological samples and its mean deviation. Fig. 1 View large Download slide Induction of TIR1 promoter-driven β-galactosidase activity. (a) β-Galactosidase activity was measured in reference strain IMZ162 and in strains carrying deletion in the genes SNF7, VPS2, VPS20, VPS24, BRO1, RIM20, VPS4, DOA4, RIM101, RIM8 and RIM13. (b) β-Galactosidase activity was measured in reference strain IMZ162 and in strains IMZ169 (snf7Δ), IMZ172 (rim101Δ), IMZ188 (rim101ΔTDH3pr-RIM101-531) and IMZ187 (snf7ΔTDH3pr-RIM101-531). Exponential-phase cells growing on glucose under aerobic and anaerobic conditions were harvested and β-galactosidase activity was measured. β-Galactosidase activities are expressed as μM of o-nitrophenol (ONP) formed per min and per mg of protein. The activity displayed is the average of two independent biological samples and its mean deviation. Deletion of the fungal homologues of the mammalian protein Alix, BRO1 and RIM20, yielded two separate phenotypes. The bro1Δ strain (IMZ177) still exhibited LacZ activity. In contrast, no such activity was measured in a rim20Δ strain (IMZ171) (Fig. 1a). Snf7p and Vps20p form a subcomplex within the ESCRT-III whose function depends on Bro1p and Rim20p. Under anaerobic conditions, TIR1 expression thus depends on Snf7p, Vps20p and Rim20p, but not on Vps24p, Vps4p and Bro1p. These results indicated that TIR1 expression is dependent on the Rim101p pathway. The β-galactosidase activity was also measured under aerobic conditions, and similar to the reference strain, the deletion mutants did not express detectable levels of β-galactosidase in aerobic cultures (data not shown). The effect of Snf7p on the anaerobic upregulation of TIR1 involves the Rim101p pathway The loss of β-galactosidase activity from a TIR1 promoter construct in a rim20Δ strain already suggested that induction of TIR1 expression under anaerobic conditions may involve the Rim101p pathway. To investigate this hypothesis, strains with deletions in RIM101 (encoding a transcriptional repressor involved in response to pH and in cell wall construction), RIM13 (encoding a calpain-like protease) and RIM8 (encoding a protein of unknown function involved in the proteolytic activation of Rim101p in response to alkaline pH) were selected from the yeast deletion library, transformed with pTIR (yielding strains IMZ172, IMZ178 and IMZ168, respectively), grown aerobically and anaerobically and tested for β-galactosidase activity. No β-galactosidase activity was detected in any of these mutants, either in aerobically or in anaerobically grown cultures (Fig. 1a). To confirm that the TIR1 expression was dependent on the proteolytic activation of Rim101p, a RIM101 allele encoding a C-terminally truncated version of the protein (lacking amino acids 531–626) of Rimp101 was generated (Futai, 1999; Hayashi, 2005). The truncated Rim101p allele RIM101-531 was cloned and expressed under the control of a TDH3 constitutive promoter. The plasmid obtained, PUDeRIM101-531 (Table 1), was transformed into the rim101Δ and snf7Δ strains harboring the pTIR plasmid (yielding strains IMZ188 and IMZ187). The expression of this RIM101-531 allele complemented the RIM101 deletion and was able to induce TIR1 promoter-driven β-galactosidase activity in an snf7Δ mutant under anaerobic conditions (Fig. 1b). However, the level of LacZ induction was higher in an snf7Δ than in a rim101Δ. We could hypothesize that the presence of Snf7p in the rim101Δ strain may still interact with the truncated Rim101p and subsequently affect its function. Besides, no activity was observed aerobically. These results demonstrate that, under anaerobic conditions, the expression of TIR1 is dependent on Snf7p and on the Rim101p signalling pathway. Chemostat-based transcriptome analysis of snf7Δ and rim101Δ mutants: global responses Genome-wide transcriptional responses to the inactivation of SNF7 and RIM101 were analyzed in anaerobic, glucose-limited chemostat cultures (dilution rate of 0.10 h−1) of the prototrophic reference strain S. cerevisiae CEN.PK113-7D and the isogenic snf7Δ (GG3201) and rim101Δ (IMK229) strains. Biomass yields in triplicate chemostat cultures of the reference strain CEN.PK113-7D and the isogenic snf7Δ mutant and in duplicate cultures of the rim101Δ strain were not significantly different (0.09 ± 0.01; 0.10 ± 0.02; 0.10 ± 0.003 g biomass g−1 glucose, respectively) and the biomass-specific rates of CO2 production (10.3 ± 0.4; 9.3 ± 0.2; 9.1 ± 0.7 mmol g−1 biomass, respectively) were also similar. Transcriptomes of triplicate glucose-limited anaerobic chemostat cultures of the snf7Δ deletion strain were compared with those of the reference strain CEN.PK113-7D obtained under identical conditions of growth and sample preparation (Tai, 2005). The relative average coefficient of variation of the snf7Δ arrays was 24% and 9% for those of the CEN.PK113-7D reference strain (both run in triplicate). Similarly, transcriptomes from anaerobic glucose-limited cultures of the rim101Δ strain (run in duplicate) were compared with those of the reference strain CEN.PK113-7D (run in triplicate) obtained under identical conditions (of growth and sample preparation) (De Nicola, 2007; Knijnenburg, 2009). The average deviation from the mean among microarrays from replicate cultures was not >9%. Transcript levels of two commonly used loading standards for Northern analysis, ACT1 and PDA1, varied by <15% over the arrays. Two hundred and nine genes were differentially expressed in anaerobic, glucose-limited chemostat cultures of the snf7Δ strain and the CEN.PK113-7D reference strain (Table S2). Of these 209 genes, 95 showed a higher transcript level in the snf7Δ strain (GG3201). In anaerobic chemostat cultures of an isogenic rim101Δ strain (IMK229), 401 genes showed a different transcript level from that in cultures of the reference strain. Of these genes, 270 showed a higher transcript level than in the reference cultures (Table S2). Role of Snf7p/Rim101p in transcriptional repression To investigate whether Snf7p and Rim101p were involved in the repression of genes expression, transcriptomes of anaerobic chemostat cultures of the mutant strains GG3201 (snf7Δ) and IMK229 (rim101Δ) were compared with that of the reference strain CEN.PK113-7D. This analysis identified sets of 95 and 270 genes upregulated in the snf7Δ and the rim101Δ strains, respectively (Table S2). GO analysis revealed enrichment in six GO categories in the gene set upregulated in the rim101Δ strain (Table 3). However, a similar GO category enrichment analysis in the set of upregulated genes in the snf7Δ strain identified stress response as the only enriched category. Table 3 Overrepresented biological processes in genes differently expressed in snf7Δ and rim101Δ strains relative to the reference strain CEN.PK113-7D   K out of N  P-value  Genes  GO leaf categories significantly enriched in rim101Δ upregulated genes relative to the reference CEN.PK113-7D strain  Telomere maintenance via recombination  12 out of 33  1.0E−11  YJL225C, YIL177C, YHL050C, YNL339C, YLR462W, YLR464W, YLR466W, YLR467W, YLR467W, YLL067C, YHR218W, YML133C  Regulation of transcription, DNA-dependent  40 out of 501  1.3E−04  AFT1, AHC1, ASF1, ARP7, CCR4, CYC8, DOT6, ESC1, FKH1, GIS1, HHO1, HMO1, HMS2, HSF1, IXR1, MCM3, MSN2, NRG1, PHO4, REB1, RIS1, RSC2, RSC8, RRN5, SIN3, SMP1, SNF2, SOH1, SOK2, SPP41, SPT5, SPT8, SSL2, STB5, SUM1, TOS8, TUP1, UME6, WHI5, YFL052W  Iron ion transport  7 out of 29  2.0E−04  ARN1, FET3, FIT2, FIT3, FRE5, FTR1, SIT1  Response to stress  16 out of 153  3.7E−04  DDR2, DDR48, HAL1, HSF1, HSP26, HSP31, HSP32, HSP42, HSP150, WSC4, MDJ1 MNN4, MSN2, TIR2, TPS2, TSL1  Cell wall organization and biogenesis  23 out of 158  5.7E−05  BMH1, CRH1, DON1, DSE1, ECM4, FMP45, GIS1, HPF1, HSP150, INP52, KRE6, MYO3, MYO5, PIR3, PSK2, SCW10, SCW11, SED1, SRL1, SUN4, TAX4, WSC4, YPS3  Pentose-phosphate shunt, oxidative branch  3 out of 5  7.9E−04  GND2, SOL4, ZWF1  GO leaf category significantly enriched in snf7Δ upregulated genes relative to the reference CEN.PK113-7D strain  Response to stress  25 out of 153  3.68E−10  AHP1, ATG13, CRG1, DAN2, DAN3, DDR2, DDR48, GTT1, HAC1, HAL1, IMP2, KTR2, MNN4, MGR1, PAU2, PAU5, PAU7, PAU17, PAU15, PRB1, PRM5, RAP1, RRD1, TIR2, YIL108W  GO leaf category significantly enriched in rim101Δ downregulated genes relative to the reference CEN.PK113-7D strain  Protein oligomerization  3 out of 6  1.6E−04  STE2, TIM11, ATP20  GO leaf categories significantly enriched in snf7Δ downregulated genes relative to the reference CEN.PK113-7D strain  Transport  27 out of 797  7.3 E−04  AAC3, AQR1, ATR1, BAP3, CTR3, CYC1, DIP5, ENB1, FET4, FIT2, GNP1, MCH5, OPT1, OPT2, PDR12, PHO84, SGE1, SMF1, SNF7, SUL1, TAT1, YHC3, VPS63, YLR364W, YMR279C, ZRT1, ZRT2  Amino acid transport  5 out of 36  4.2 E−04  BAP3, DIP5, GNP1, TAT1, YHC3  Amino acid biosynthetic process  9 out of 97  5.54E−05  ADI1, ARG1, ARG3, ARO4, ASN1, BAT2, ILV5, LYS20, TRP4    K out of N  P-value  Genes  GO leaf categories significantly enriched in rim101Δ upregulated genes relative to the reference CEN.PK113-7D strain  Telomere maintenance via recombination  12 out of 33  1.0E−11  YJL225C, YIL177C, YHL050C, YNL339C, YLR462W, YLR464W, YLR466W, YLR467W, YLR467W, YLL067C, YHR218W, YML133C  Regulation of transcription, DNA-dependent  40 out of 501  1.3E−04  AFT1, AHC1, ASF1, ARP7, CCR4, CYC8, DOT6, ESC1, FKH1, GIS1, HHO1, HMO1, HMS2, HSF1, IXR1, MCM3, MSN2, NRG1, PHO4, REB1, RIS1, RSC2, RSC8, RRN5, SIN3, SMP1, SNF2, SOH1, SOK2, SPP41, SPT5, SPT8, SSL2, STB5, SUM1, TOS8, TUP1, UME6, WHI5, YFL052W  Iron ion transport  7 out of 29  2.0E−04  ARN1, FET3, FIT2, FIT3, FRE5, FTR1, SIT1  Response to stress  16 out of 153  3.7E−04  DDR2, DDR48, HAL1, HSF1, HSP26, HSP31, HSP32, HSP42, HSP150, WSC4, MDJ1 MNN4, MSN2, TIR2, TPS2, TSL1  Cell wall organization and biogenesis  23 out of 158  5.7E−05  BMH1, CRH1, DON1, DSE1, ECM4, FMP45, GIS1, HPF1, HSP150, INP52, KRE6, MYO3, MYO5, PIR3, PSK2, SCW10, SCW11, SED1, SRL1, SUN4, TAX4, WSC4, YPS3  Pentose-phosphate shunt, oxidative branch  3 out of 5  7.9E−04  GND2, SOL4, ZWF1  GO leaf category significantly enriched in snf7Δ upregulated genes relative to the reference CEN.PK113-7D strain  Response to stress  25 out of 153  3.68E−10  AHP1, ATG13, CRG1, DAN2, DAN3, DDR2, DDR48, GTT1, HAC1, HAL1, IMP2, KTR2, MNN4, MGR1, PAU2, PAU5, PAU7, PAU17, PAU15, PRB1, PRM5, RAP1, RRD1, TIR2, YIL108W  GO leaf category significantly enriched in rim101Δ downregulated genes relative to the reference CEN.PK113-7D strain  Protein oligomerization  3 out of 6  1.6E−04  STE2, TIM11, ATP20  GO leaf categories significantly enriched in snf7Δ downregulated genes relative to the reference CEN.PK113-7D strain  Transport  27 out of 797  7.3 E−04  AAC3, AQR1, ATR1, BAP3, CTR3, CYC1, DIP5, ENB1, FET4, FIT2, GNP1, MCH5, OPT1, OPT2, PDR12, PHO84, SGE1, SMF1, SNF7, SUL1, TAT1, YHC3, VPS63, YLR364W, YMR279C, ZRT1, ZRT2  Amino acid transport  5 out of 36  4.2 E−04  BAP3, DIP5, GNP1, TAT1, YHC3  Amino acid biosynthetic process  9 out of 97  5.54E−05  ADI1, ARG1, ARG3, ARO4, ASN1, BAT2, ILV5, LYS20, TRP4  K is the number of differentially expressed genes that belong to a functional category and N represents the number of genes that belongs to the same category genomewide. The P-value is the result of a Fisher's exact test that compares the two values. View Large Table 3 Overrepresented biological processes in genes differently expressed in snf7Δ and rim101Δ strains relative to the reference strain CEN.PK113-7D   K out of N  P-value  Genes  GO leaf categories significantly enriched in rim101Δ upregulated genes relative to the reference CEN.PK113-7D strain  Telomere maintenance via recombination  12 out of 33  1.0E−11  YJL225C, YIL177C, YHL050C, YNL339C, YLR462W, YLR464W, YLR466W, YLR467W, YLR467W, YLL067C, YHR218W, YML133C  Regulation of transcription, DNA-dependent  40 out of 501  1.3E−04  AFT1, AHC1, ASF1, ARP7, CCR4, CYC8, DOT6, ESC1, FKH1, GIS1, HHO1, HMO1, HMS2, HSF1, IXR1, MCM3, MSN2, NRG1, PHO4, REB1, RIS1, RSC2, RSC8, RRN5, SIN3, SMP1, SNF2, SOH1, SOK2, SPP41, SPT5, SPT8, SSL2, STB5, SUM1, TOS8, TUP1, UME6, WHI5, YFL052W  Iron ion transport  7 out of 29  2.0E−04  ARN1, FET3, FIT2, FIT3, FRE5, FTR1, SIT1  Response to stress  16 out of 153  3.7E−04  DDR2, DDR48, HAL1, HSF1, HSP26, HSP31, HSP32, HSP42, HSP150, WSC4, MDJ1 MNN4, MSN2, TIR2, TPS2, TSL1  Cell wall organization and biogenesis  23 out of 158  5.7E−05  BMH1, CRH1, DON1, DSE1, ECM4, FMP45, GIS1, HPF1, HSP150, INP52, KRE6, MYO3, MYO5, PIR3, PSK2, SCW10, SCW11, SED1, SRL1, SUN4, TAX4, WSC4, YPS3  Pentose-phosphate shunt, oxidative branch  3 out of 5  7.9E−04  GND2, SOL4, ZWF1  GO leaf category significantly enriched in snf7Δ upregulated genes relative to the reference CEN.PK113-7D strain  Response to stress  25 out of 153  3.68E−10  AHP1, ATG13, CRG1, DAN2, DAN3, DDR2, DDR48, GTT1, HAC1, HAL1, IMP2, KTR2, MNN4, MGR1, PAU2, PAU5, PAU7, PAU17, PAU15, PRB1, PRM5, RAP1, RRD1, TIR2, YIL108W  GO leaf category significantly enriched in rim101Δ downregulated genes relative to the reference CEN.PK113-7D strain  Protein oligomerization  3 out of 6  1.6E−04  STE2, TIM11, ATP20  GO leaf categories significantly enriched in snf7Δ downregulated genes relative to the reference CEN.PK113-7D strain  Transport  27 out of 797  7.3 E−04  AAC3, AQR1, ATR1, BAP3, CTR3, CYC1, DIP5, ENB1, FET4, FIT2, GNP1, MCH5, OPT1, OPT2, PDR12, PHO84, SGE1, SMF1, SNF7, SUL1, TAT1, YHC3, VPS63, YLR364W, YMR279C, ZRT1, ZRT2  Amino acid transport  5 out of 36  4.2 E−04  BAP3, DIP5, GNP1, TAT1, YHC3  Amino acid biosynthetic process  9 out of 97  5.54E−05  ADI1, ARG1, ARG3, ARO4, ASN1, BAT2, ILV5, LYS20, TRP4    K out of N  P-value  Genes  GO leaf categories significantly enriched in rim101Δ upregulated genes relative to the reference CEN.PK113-7D strain  Telomere maintenance via recombination  12 out of 33  1.0E−11  YJL225C, YIL177C, YHL050C, YNL339C, YLR462W, YLR464W, YLR466W, YLR467W, YLR467W, YLL067C, YHR218W, YML133C  Regulation of transcription, DNA-dependent  40 out of 501  1.3E−04  AFT1, AHC1, ASF1, ARP7, CCR4, CYC8, DOT6, ESC1, FKH1, GIS1, HHO1, HMO1, HMS2, HSF1, IXR1, MCM3, MSN2, NRG1, PHO4, REB1, RIS1, RSC2, RSC8, RRN5, SIN3, SMP1, SNF2, SOH1, SOK2, SPP41, SPT5, SPT8, SSL2, STB5, SUM1, TOS8, TUP1, UME6, WHI5, YFL052W  Iron ion transport  7 out of 29  2.0E−04  ARN1, FET3, FIT2, FIT3, FRE5, FTR1, SIT1  Response to stress  16 out of 153  3.7E−04  DDR2, DDR48, HAL1, HSF1, HSP26, HSP31, HSP32, HSP42, HSP150, WSC4, MDJ1 MNN4, MSN2, TIR2, TPS2, TSL1  Cell wall organization and biogenesis  23 out of 158  5.7E−05  BMH1, CRH1, DON1, DSE1, ECM4, FMP45, GIS1, HPF1, HSP150, INP52, KRE6, MYO3, MYO5, PIR3, PSK2, SCW10, SCW11, SED1, SRL1, SUN4, TAX4, WSC4, YPS3  Pentose-phosphate shunt, oxidative branch  3 out of 5  7.9E−04  GND2, SOL4, ZWF1  GO leaf category significantly enriched in snf7Δ upregulated genes relative to the reference CEN.PK113-7D strain  Response to stress  25 out of 153  3.68E−10  AHP1, ATG13, CRG1, DAN2, DAN3, DDR2, DDR48, GTT1, HAC1, HAL1, IMP2, KTR2, MNN4, MGR1, PAU2, PAU5, PAU7, PAU17, PAU15, PRB1, PRM5, RAP1, RRD1, TIR2, YIL108W  GO leaf category significantly enriched in rim101Δ downregulated genes relative to the reference CEN.PK113-7D strain  Protein oligomerization  3 out of 6  1.6E−04  STE2, TIM11, ATP20  GO leaf categories significantly enriched in snf7Δ downregulated genes relative to the reference CEN.PK113-7D strain  Transport  27 out of 797  7.3 E−04  AAC3, AQR1, ATR1, BAP3, CTR3, CYC1, DIP5, ENB1, FET4, FIT2, GNP1, MCH5, OPT1, OPT2, PDR12, PHO84, SGE1, SMF1, SNF7, SUL1, TAT1, YHC3, VPS63, YLR364W, YMR279C, ZRT1, ZRT2  Amino acid transport  5 out of 36  4.2 E−04  BAP3, DIP5, GNP1, TAT1, YHC3  Amino acid biosynthetic process  9 out of 97  5.54E−05  ADI1, ARG1, ARG3, ARO4, ASN1, BAT2, ILV5, LYS20, TRP4  K is the number of differentially expressed genes that belong to a functional category and N represents the number of genes that belongs to the same category genomewide. The P-value is the result of a Fisher's exact test that compares the two values. View Large A total of 37 genes showed a significant co-upregulation in both strains (Fig. 2). The comparison of these genes with the published transcriptome data obtained from shake-flask cultures of the rim101Δ mutant (Lamb & Mitchell, 2003; Mira, 2009) confirmed the large similarity of the results. We found a total of 12 genes that were common in at least two studies (Fig. 2). The high correlation between the three Rim101p responsive gene sets would suggest that the observed regulation is largely independent of oxygen availability. Fig. 2 View largeDownload slide Identification of the genes co-upregulated in strains GG3201 (snf7Δ) and IMK229 (rim101Δ). (a) The 95 genes upregulated in GG3201 relative to CEN.PK113-7D were compared with the 270 genes upregulated in IMK229 relative to CEN.PK113-7D. (b) The heat map represents the expression in CEN.PK113-7D, IMK229 and GG3201 of the 37 co-upregulated genes in GG3201 and IMK229 relative to CEN.PK113-7D. The blue box identifies the presence of a putative Rim101p cis-regulatory element in the gene promoter sequence. (c) Venn diagram of the results from three transcriptome studies on rim101Δ mutants of Saccharomyces cerevisiae. The rim101Δ upregulated genes obtained from aerobic shake-flask cultures from Lamb & Mitchell (2003) and Mira (2009) were compared with the rim101Δ chemostat-based transcriptome data generated in the present study. The asterisk identifies the Rim101p targets specifically regulated in the presence of propionic acid. (d) Overrepresented motif found in the 37 genes co-upregulated in GG3201 and in IMK229 relative to CEN.PK113-7D. Fig. 2 View largeDownload slide Identification of the genes co-upregulated in strains GG3201 (snf7Δ) and IMK229 (rim101Δ). (a) The 95 genes upregulated in GG3201 relative to CEN.PK113-7D were compared with the 270 genes upregulated in IMK229 relative to CEN.PK113-7D. (b) The heat map represents the expression in CEN.PK113-7D, IMK229 and GG3201 of the 37 co-upregulated genes in GG3201 and IMK229 relative to CEN.PK113-7D. The blue box identifies the presence of a putative Rim101p cis-regulatory element in the gene promoter sequence. (c) Venn diagram of the results from three transcriptome studies on rim101Δ mutants of Saccharomyces cerevisiae. The rim101Δ upregulated genes obtained from aerobic shake-flask cultures from Lamb & Mitchell (2003) and Mira (2009) were compared with the rim101Δ chemostat-based transcriptome data generated in the present study. The asterisk identifies the Rim101p targets specifically regulated in the presence of propionic acid. (d) Overrepresented motif found in the 37 genes co-upregulated in GG3201 and in IMK229 relative to CEN.PK113-7D. A systematic search for the cis-regulatory element in the promoter sequences of the 37 coinduced genes in the snf7Δ and rim101Δ revealed the presence of a conserved motif (g/t CTT g/a G c/a) in 24 of the promoter sequences (Fig. 2c). This motif resembles the Rim101p-binding motif (Macisaac, 2006). At least three mechanisms might explain the upregulated transcriptional profile in the absence of Snf7p/Rim101p under anaerobic conditions: (1) Rim101p is directly, or in coordination with (an) additional repressor(s), involved in the gene repression, (2) Rim101p does not directly act in cis, but is an upstream element of a regulatory cascade or (3) transcriptional upregulation of genes is an indirect consequence of rim101 knockout (e.g. via altered metabolite levels). The observed overrepresentation of the Rim101p cis-regulatory element in promoter sequences of genes upregulated in Snf7p and Rim101p strain would support the first assumption. Expression of anaerobically upregulated yeast genes in snf7Δ and rim101Δ mutants To investigate whether deletion of SNF7 and RIM101 affected the anaerobic upregulation of genes other than TIR1, the set of 114 genes whose transcript levels in anaerobic cultures were reduced in the snf7Δ strain and the set of 131 genes whose transcript levels in anaerobic cultures were reduced in the rim101Δ strain were compared with each other. A significant (P-value<1.0E−05) 27 genes were co-downregulated in both deletion strains relative to their respective reference (Fig. 3). Out of these 27 genes, seven (TIR1, MUC1, SML1, AAC3, FET4, YBR300C, YDL241W) belonged to a set of 65 genes that were previously found to have a consistently higher transcript level in the absence of oxygen under four different nutrient limitation regimes (Tai, 2005) (Fig. 3). Fig. 3 View largeDownload slide Identification of the genes co-downregulated in strains GG3201 (snf7Δ) and IMK229 (rim101Δ). (a) The 114 genes downregulated in GG3201 relative to CEN.PK113-7D were compared with the 131 genes downregulated in IMK229 relative to CEN.PK113-7D. These data sets were further matched up with a set of 65 genes that were previously found to have a consistently higher transcript level in the absence of oxygen under four different nutrient limitation regimes (Tai, 2005). (b) The heat map represents the expression in CEN.PK113-7D, IMK229 and GG3201 of the 27 co-downregulated genes in GG3201 and IMK229 relative to CEN.PK113-7D. The underlined genes correspond to the seven genes also found in common with the data described in Tai (2005). (c) Overrepresented motif found in the 27 genes co-downregulated in GG3201 and in IMK229 relative to CEN.PK113-7D. Fig. 3 View largeDownload slide Identification of the genes co-downregulated in strains GG3201 (snf7Δ) and IMK229 (rim101Δ). (a) The 114 genes downregulated in GG3201 relative to CEN.PK113-7D were compared with the 131 genes downregulated in IMK229 relative to CEN.PK113-7D. These data sets were further matched up with a set of 65 genes that were previously found to have a consistently higher transcript level in the absence of oxygen under four different nutrient limitation regimes (Tai, 2005). (b) The heat map represents the expression in CEN.PK113-7D, IMK229 and GG3201 of the 27 co-downregulated genes in GG3201 and IMK229 relative to CEN.PK113-7D. The underlined genes correspond to the seven genes also found in common with the data described in Tai (2005). (c) Overrepresented motif found in the 27 genes co-downregulated in GG3201 and in IMK229 relative to CEN.PK113-7D. A search for cis-regulatory element in the promoter sequences of 27 genes that showed reduced transcript levels in both the snf7Δ and the rim101Δ strains revealed the presence of a conserved motif (A/g G/a ACCCT) in 17 of the promoter sequences (Fig. 3c). This motif resembles the Nrg1p-binding motif (Macisaac, 2006). This suggested that induction of TIR1 transcription in anaerobic cultures could involve the relief of the Nrg1p repression role on TIR1 through Rim101p repression of NRG1 expression. It also indicated that TIR1 is not the only anaerobically upregulated gene influenced by the Snf7p/Rim101p signalling. When, similarly, genes with a reduced transcript level in the snf7Δ and rim101Δ strains were compared with the set of genes whose transcript level is increased under anaerobic conditions in glucose-limited cultures only (Tai, 2005), sets of 19 and 36 genes were identified, respectively (Table 4). Table 4 Saccharomyces cerevisiae genes whose transcript levels are reduced in anaerobic, glucose-limited chemostat cultures of GG3201 (snf7Δ) and/or IMK229 (rim101Δ) strain relative to cultures of an isogenic reference strain and whose transcript levels are higher in anaerobic chemostat cultures of the reference strain CEN.PK113-7D relative to aerobic cultures of the reference strain Gene  Functional description  snf7Δ anaerobic  Ref1 anaerobic  Ref1 aerobic  rim101Δ anaerobic  Ref2 anaerobic  Ref2 aerobic  Genes downregulated in GG3201 (snf7Δ) and IMK229 (rim101Δ) and upregulated under anaerobiosis in glucose-limited chemostat  FET4  Low-affinity Fe(II) transport protein  51.8 ± 21  171.9 ± 33  12.4 ± 0  89.8 ± 68  236.2 ± 27  115.7 ± 13  AAC3  Mitochondrial ADPVATP translocator  75.5 ± 50  372.5 ± 18  12.5 ± 0  66.5 ± 78  173.0 ± 17  71.5 ± 103  TIR1  Protein of the Srp1pVTip1p family of serine–alanine-rich proteins  171.7 ± 9  4248.8 ± 292  100.3 ± 21  303.6 ± 71  7726.2 ± 625  252.5 ± 46  MUC1  Cell surface flocculin with structure/GPI-anchored cell wall proteins  88.8 ± 19  1106.8 ± 102  34.4 ± 5  96.9 ± 30  1149.4 ± 104  410.3 ± 61  SML1  Identified by SAGE  82.8 ± 33  439.6 ± 46  17.8 ± 1  396.8 ± 157  939.0 ± 141  266.7 ± 32  YBR300C  Strong similarity to hypothetical protein YGR293c  17.9 ± 5  49.5 ± 10  12.0 ± 0  13.1 ± 1  29.0 ± 5  15.8 ± 6  YDL241W  Hypothetical protein  12.0 ± 0  64.0 ± 7  12.0 ± 0  13.1 ± 0  43.3 ± 7  21.9 ± 14  YJL213W  similarity to Methanobacterium aryldialkylphosphatase  50.6 ± 22  216.9 ± 33  25.0 ± 4  45.7 ± 16  104.3 ± 24  68.2 ± 38  YLR460C  Similarity to C. carbonum toxD protein  61.1 ± 16  144.5 ± 17  17.7 ± 5  52.7 ± 11  178.7 ± 31  62.9 ± 72  SKT5  Protoplast regeneration and killer toxin resistance gene,  76.4 ± 7  171.2 ± 4  65.0 ± 20  118.1 ± 5  282.2 ± 32  125.8 ± 99  YCL049C  Hypothetical protein  178.9 ± 31  478.3 ± 35  220.2 ± 24  179.0 ± 46  522.0 ± 59  257.3 ± 15  OPT2  Similarity to S. pombe isp4 protein  12.0 ± 0  167.0 ± 39  12.0 ± 0  14.5 ± 3  205.2 ± 32  83.9 ± 124  Genes downregulated in GG3201 (snf7Δ) and upregulated under anaerobiosis in glucose-limited chemostat  YOL014W  Hypothetical protein  12.2 ± 0  26.7 ± 4  12.0 ± 0  12.1 ± 0  15.9 ± 0  12.0 ± 0  ASN1  Asparagine synthetase  449.6 ± 89  1208.1 ± 131  305.1 ± 98  579.6 ± 23  936.1 ± 118  376.0 ± 373  BAP3  Valine transporter  35.4 ± 8  153.1 ± 12  12.0 ± 0  141.8 ± 55  84.5 ± 12  43.5 ± 54  DIP5  Dicarboxylic amino acid permease  83.8 ± 86  1054.0 ± 88  32.4 ± 7  408.7 ± 97  789.5 ± 118  304.3 ± 50  IMD1  Strong similarity to IMP dehydrogenases  211.1 ± 75  494.2 ± 23  105.7 ± 7  327.4 ± 47  236.9 ± 34  131.6 ± 86  SSK22  Protein kinase  31.1 ± 4  73.0 ± 3  28.9 ± 4  65.8 ± 3  89.9 ± 17  54.0 ± 20  TIR4  Similarity to Tir1p and Tir2p  1143.5 ± 265  2668.0 ± 130  125.9 ± 32  2104.9 ± 727  4859.3 ± 310  164.5 ± 24  Genes downregulated in IMK229 (rim101Δ) and upregulated under anaerobiosis in glucose-limited chemostat  YDL085C  Identified by SAGE  193.0 ± 91  347.1 ± 35  204.3 ± 47  202.6 ± 16  408.3 ± 7  1897 ± 91  YBR004C  Similarity to S. pombe hypothetical protein SPAC18B11.05  61.8 ± 3  114.7 ± 13  111.5 ± 7  49.0 ± 11  158.0 ± 8  39.8 ± 19  YMR325W  Strong similarity to members of the Srp1p/Tip1p family  136.6 ± 12  87.9 ± 5  83.7 ± 60  65.7 ± 10  241.7 ± 57  73.7 ± 68  YJL218W  Strong similarity to E. coli galactoside O-acetyltransferase  73.5 ± 58  115.5 ± 8  29.6 ± 9  22.3 ± 0  57.8 ± 11  12.0 ± 0  YGR131W  Strong similarity to Nce2p  92.3 ± 12  133.4 ± 17  46.4 ± 1  72.9 ± 17  153.1 ± 33  44.9 ± 9  YHR210C  UDP-glucose-4-epimerase (GAL10, galE)  366.8 ± 250  267.0 ± 30  31.3 ± 7  100.8 ± 26  311.5 ± 99  127.8 ± 88  ADA2  Transcription factor, member of ADA and SAGA  31.9 ± 12  29.9 ± 10  24.6 ± 5  12.2 ± 0  54.5 ± 1  27.2 ± 26  DAN2  Strong similarity to members of the Srp1p/Tip1p family  1366.8 ± 400  229.8 ± 32  154.5 ± 16  221.4 ± 31  827.2 ± 155  162.6 ± 89  DAN4  Similarity to mucin proteins, YKL224c, Sta1p  218.1 ± 43  243.7 ± 33  144.9 ± 36  183.2 ± 18  422.2 ± 167  128.4 ± 38  HMRA1  Homeobox-domain containing protein  493.2 ± 84  549.5 ± 20  304.8 ± 29  18.8 ± 4  645.8 ± 116  248.7193  LYS14  Transcriptional activator of lysine pathway genes  17.4 ± 4  21.9 ± 10  12.0 ± 0  28.7 ± 1  59.7 ± 43  23.3 ± 9  MEP3  Ammonia permease of high capacity and low affinity  75.3 ± 24  133.7 ± 12  26.6 ± 1  45.6 ± 33  217.3 ± 23  82.1 ± 93  MFA2  Mating a-factor pheromone precursor  2893.5 ± 118  2036.3 ± 251  2239.5 ± 938  1940.0 ± 13  4016.6 ± 332  1960.3 ± 147  NIP7  Nip7p is required for 60S ribosome subunit biogenesis  111.4 ± 53  158.1 ± 34  135.8 ± 9  79.2 ± 8  169.9 ± 16  49.0 ± 15  PAU3  Member of the seripauperin protein\gene family  143.4 ± 22  74.1 ± 18  58.4 ± 38  83.9 ± 12  294.3 ± 51  121.3 ± 81  Q0280  Ubiquinol–cytochrome c reductase subunit (cytochrome b) F.  12.0 ± 0  12.0 ± 0  12.0 ± 0  35.8 ± 15  72.1 ± 6  31.8 ± 33  RPR1  RNAse P RNA  21.8 ± 17  12.1 ± 0  12.0 ± 0  13.8 ± 2  164.8 ± 35  72.1 ± 94  RPS26B  Ribosomal protein S26B  464.7 ± 172  649.5 ± 60  806.2 ± 312  426.0 ± 30  887.2 ± 90  325.3 ± 98  SNR17B  Encodes snRNA U3, SNR17A also encodes snRNA U3  322.8 ± 136  439.0 ± 12  531.3 ± 147  207.6 ± 46  503.7 ± 36  176.4 ± 122  SST2  Regulator of G protein-signaling family member  64.7 ± 10  93.0 ± 12  28.9 ± 5  42.4 ± 11  87.8 ± 10  31.7 ± 25  STE2  Pheromone α-factor G protein-coupled receptor (GPCR)  394.3 ± 64  346.2 ± 6  183.1 ± 21  143.3 ± 24  415.2 ± 37  73.0 ± 52  SUC2  Invertase (sucrose-hydrolyzing enzyme)  1114.4 ± 263  1281.9 ± 41  487.9 ± 57  410.2 ± 3  2097.1 ± 268  978.8 ± 230  SUC4  Invertase (sucrose-hydrolyzing enzyme)  865.3 ± 292  1015.3 ± 62  316.0 ± 29  335.6 ± 10  1252.2 ± 220  325.4 ± 212  TRS33  Involved in the targeting and fusion of ER to golgi transport vesicles  212.7 ± 22  188.9 ± 17  120.8 ± 33  96.8 ± 6  196.1 ± 45  49.4 ± 51  Gene  Functional description  snf7Δ anaerobic  Ref1 anaerobic  Ref1 aerobic  rim101Δ anaerobic  Ref2 anaerobic  Ref2 aerobic  Genes downregulated in GG3201 (snf7Δ) and IMK229 (rim101Δ) and upregulated under anaerobiosis in glucose-limited chemostat  FET4  Low-affinity Fe(II) transport protein  51.8 ± 21  171.9 ± 33  12.4 ± 0  89.8 ± 68  236.2 ± 27  115.7 ± 13  AAC3  Mitochondrial ADPVATP translocator  75.5 ± 50  372.5 ± 18  12.5 ± 0  66.5 ± 78  173.0 ± 17  71.5 ± 103  TIR1  Protein of the Srp1pVTip1p family of serine–alanine-rich proteins  171.7 ± 9  4248.8 ± 292  100.3 ± 21  303.6 ± 71  7726.2 ± 625  252.5 ± 46  MUC1  Cell surface flocculin with structure/GPI-anchored cell wall proteins  88.8 ± 19  1106.8 ± 102  34.4 ± 5  96.9 ± 30  1149.4 ± 104  410.3 ± 61  SML1  Identified by SAGE  82.8 ± 33  439.6 ± 46  17.8 ± 1  396.8 ± 157  939.0 ± 141  266.7 ± 32  YBR300C  Strong similarity to hypothetical protein YGR293c  17.9 ± 5  49.5 ± 10  12.0 ± 0  13.1 ± 1  29.0 ± 5  15.8 ± 6  YDL241W  Hypothetical protein  12.0 ± 0  64.0 ± 7  12.0 ± 0  13.1 ± 0  43.3 ± 7  21.9 ± 14  YJL213W  similarity to Methanobacterium aryldialkylphosphatase  50.6 ± 22  216.9 ± 33  25.0 ± 4  45.7 ± 16  104.3 ± 24  68.2 ± 38  YLR460C  Similarity to C. carbonum toxD protein  61.1 ± 16  144.5 ± 17  17.7 ± 5  52.7 ± 11  178.7 ± 31  62.9 ± 72  SKT5  Protoplast regeneration and killer toxin resistance gene,  76.4 ± 7  171.2 ± 4  65.0 ± 20  118.1 ± 5  282.2 ± 32  125.8 ± 99  YCL049C  Hypothetical protein  178.9 ± 31  478.3 ± 35  220.2 ± 24  179.0 ± 46  522.0 ± 59  257.3 ± 15  OPT2  Similarity to S. pombe isp4 protein  12.0 ± 0  167.0 ± 39  12.0 ± 0  14.5 ± 3  205.2 ± 32  83.9 ± 124  Genes downregulated in GG3201 (snf7Δ) and upregulated under anaerobiosis in glucose-limited chemostat  YOL014W  Hypothetical protein  12.2 ± 0  26.7 ± 4  12.0 ± 0  12.1 ± 0  15.9 ± 0  12.0 ± 0  ASN1  Asparagine synthetase  449.6 ± 89  1208.1 ± 131  305.1 ± 98  579.6 ± 23  936.1 ± 118  376.0 ± 373  BAP3  Valine transporter  35.4 ± 8  153.1 ± 12  12.0 ± 0  141.8 ± 55  84.5 ± 12  43.5 ± 54  DIP5  Dicarboxylic amino acid permease  83.8 ± 86  1054.0 ± 88  32.4 ± 7  408.7 ± 97  789.5 ± 118  304.3 ± 50  IMD1  Strong similarity to IMP dehydrogenases  211.1 ± 75  494.2 ± 23  105.7 ± 7  327.4 ± 47  236.9 ± 34  131.6 ± 86  SSK22  Protein kinase  31.1 ± 4  73.0 ± 3  28.9 ± 4  65.8 ± 3  89.9 ± 17  54.0 ± 20  TIR4  Similarity to Tir1p and Tir2p  1143.5 ± 265  2668.0 ± 130  125.9 ± 32  2104.9 ± 727  4859.3 ± 310  164.5 ± 24  Genes downregulated in IMK229 (rim101Δ) and upregulated under anaerobiosis in glucose-limited chemostat  YDL085C  Identified by SAGE  193.0 ± 91  347.1 ± 35  204.3 ± 47  202.6 ± 16  408.3 ± 7  1897 ± 91  YBR004C  Similarity to S. pombe hypothetical protein SPAC18B11.05  61.8 ± 3  114.7 ± 13  111.5 ± 7  49.0 ± 11  158.0 ± 8  39.8 ± 19  YMR325W  Strong similarity to members of the Srp1p/Tip1p family  136.6 ± 12  87.9 ± 5  83.7 ± 60  65.7 ± 10  241.7 ± 57  73.7 ± 68  YJL218W  Strong similarity to E. coli galactoside O-acetyltransferase  73.5 ± 58  115.5 ± 8  29.6 ± 9  22.3 ± 0  57.8 ± 11  12.0 ± 0  YGR131W  Strong similarity to Nce2p  92.3 ± 12  133.4 ± 17  46.4 ± 1  72.9 ± 17  153.1 ± 33  44.9 ± 9  YHR210C  UDP-glucose-4-epimerase (GAL10, galE)  366.8 ± 250  267.0 ± 30  31.3 ± 7  100.8 ± 26  311.5 ± 99  127.8 ± 88  ADA2  Transcription factor, member of ADA and SAGA  31.9 ± 12  29.9 ± 10  24.6 ± 5  12.2 ± 0  54.5 ± 1  27.2 ± 26  DAN2  Strong similarity to members of the Srp1p/Tip1p family  1366.8 ± 400  229.8 ± 32  154.5 ± 16  221.4 ± 31  827.2 ± 155  162.6 ± 89  DAN4  Similarity to mucin proteins, YKL224c, Sta1p  218.1 ± 43  243.7 ± 33  144.9 ± 36  183.2 ± 18  422.2 ± 167  128.4 ± 38  HMRA1  Homeobox-domain containing protein  493.2 ± 84  549.5 ± 20  304.8 ± 29  18.8 ± 4  645.8 ± 116  248.7193  LYS14  Transcriptional activator of lysine pathway genes  17.4 ± 4  21.9 ± 10  12.0 ± 0  28.7 ± 1  59.7 ± 43  23.3 ± 9  MEP3  Ammonia permease of high capacity and low affinity  75.3 ± 24  133.7 ± 12  26.6 ± 1  45.6 ± 33  217.3 ± 23  82.1 ± 93  MFA2  Mating a-factor pheromone precursor  2893.5 ± 118  2036.3 ± 251  2239.5 ± 938  1940.0 ± 13  4016.6 ± 332  1960.3 ± 147  NIP7  Nip7p is required for 60S ribosome subunit biogenesis  111.4 ± 53  158.1 ± 34  135.8 ± 9  79.2 ± 8  169.9 ± 16  49.0 ± 15  PAU3  Member of the seripauperin protein\gene family  143.4 ± 22  74.1 ± 18  58.4 ± 38  83.9 ± 12  294.3 ± 51  121.3 ± 81  Q0280  Ubiquinol–cytochrome c reductase subunit (cytochrome b) F.  12.0 ± 0  12.0 ± 0  12.0 ± 0  35.8 ± 15  72.1 ± 6  31.8 ± 33  RPR1  RNAse P RNA  21.8 ± 17  12.1 ± 0  12.0 ± 0  13.8 ± 2  164.8 ± 35  72.1 ± 94  RPS26B  Ribosomal protein S26B  464.7 ± 172  649.5 ± 60  806.2 ± 312  426.0 ± 30  887.2 ± 90  325.3 ± 98  SNR17B  Encodes snRNA U3, SNR17A also encodes snRNA U3  322.8 ± 136  439.0 ± 12  531.3 ± 147  207.6 ± 46  503.7 ± 36  176.4 ± 122  SST2  Regulator of G protein-signaling family member  64.7 ± 10  93.0 ± 12  28.9 ± 5  42.4 ± 11  87.8 ± 10  31.7 ± 25  STE2  Pheromone α-factor G protein-coupled receptor (GPCR)  394.3 ± 64  346.2 ± 6  183.1 ± 21  143.3 ± 24  415.2 ± 37  73.0 ± 52  SUC2  Invertase (sucrose-hydrolyzing enzyme)  1114.4 ± 263  1281.9 ± 41  487.9 ± 57  410.2 ± 3  2097.1 ± 268  978.8 ± 230  SUC4  Invertase (sucrose-hydrolyzing enzyme)  865.3 ± 292  1015.3 ± 62  316.0 ± 29  335.6 ± 10  1252.2 ± 220  325.4 ± 212  TRS33  Involved in the targeting and fusion of ER to golgi transport vesicles  212.7 ± 22  188.9 ± 17  120.8 ± 33  96.8 ± 6  196.1 ± 45  49.4 ± 51  The CEN.PK113-7D Ref1 corresponds to the reference of GG3201 (snf7Δ) [the array samples have been processed using the protocol described in Daran-Lapujade (2004)]. The CEN.PK113-7D Ref2 corresponds to the reference of IMK229 (rim101Δ) [the array samples have been processed using the protocol described in De Nicola (2007)]. Genes underlined showed a consistent anaerobic transcriptional upregulation under four different nutrient limitation regimes as described previously in Tai (2005). The mean expression value and mean deviation were calculated from triplicate cultures, except for IMK229, whose values were derived from duplicate cultures. View Large Table 4 Saccharomyces cerevisiae genes whose transcript levels are reduced in anaerobic, glucose-limited chemostat cultures of GG3201 (snf7Δ) and/or IMK229 (rim101Δ) strain relative to cultures of an isogenic reference strain and whose transcript levels are higher in anaerobic chemostat cultures of the reference strain CEN.PK113-7D relative to aerobic cultures of the reference strain Gene  Functional description  snf7Δ anaerobic  Ref1 anaerobic  Ref1 aerobic  rim101Δ anaerobic  Ref2 anaerobic  Ref2 aerobic  Genes downregulated in GG3201 (snf7Δ) and IMK229 (rim101Δ) and upregulated under anaerobiosis in glucose-limited chemostat  FET4  Low-affinity Fe(II) transport protein  51.8 ± 21  171.9 ± 33  12.4 ± 0  89.8 ± 68  236.2 ± 27  115.7 ± 13  AAC3  Mitochondrial ADPVATP translocator  75.5 ± 50  372.5 ± 18  12.5 ± 0  66.5 ± 78  173.0 ± 17  71.5 ± 103  TIR1  Protein of the Srp1pVTip1p family of serine–alanine-rich proteins  171.7 ± 9  4248.8 ± 292  100.3 ± 21  303.6 ± 71  7726.2 ± 625  252.5 ± 46  MUC1  Cell surface flocculin with structure/GPI-anchored cell wall proteins  88.8 ± 19  1106.8 ± 102  34.4 ± 5  96.9 ± 30  1149.4 ± 104  410.3 ± 61  SML1  Identified by SAGE  82.8 ± 33  439.6 ± 46  17.8 ± 1  396.8 ± 157  939.0 ± 141  266.7 ± 32  YBR300C  Strong similarity to hypothetical protein YGR293c  17.9 ± 5  49.5 ± 10  12.0 ± 0  13.1 ± 1  29.0 ± 5  15.8 ± 6  YDL241W  Hypothetical protein  12.0 ± 0  64.0 ± 7  12.0 ± 0  13.1 ± 0  43.3 ± 7  21.9 ± 14  YJL213W  similarity to Methanobacterium aryldialkylphosphatase  50.6 ± 22  216.9 ± 33  25.0 ± 4  45.7 ± 16  104.3 ± 24  68.2 ± 38  YLR460C  Similarity to C. carbonum toxD protein  61.1 ± 16  144.5 ± 17  17.7 ± 5  52.7 ± 11  178.7 ± 31  62.9 ± 72  SKT5  Protoplast regeneration and killer toxin resistance gene,  76.4 ± 7  171.2 ± 4  65.0 ± 20  118.1 ± 5  282.2 ± 32  125.8 ± 99  YCL049C  Hypothetical protein  178.9 ± 31  478.3 ± 35  220.2 ± 24  179.0 ± 46  522.0 ± 59  257.3 ± 15  OPT2  Similarity to S. pombe isp4 protein  12.0 ± 0  167.0 ± 39  12.0 ± 0  14.5 ± 3  205.2 ± 32  83.9 ± 124  Genes downregulated in GG3201 (snf7Δ) and upregulated under anaerobiosis in glucose-limited chemostat  YOL014W  Hypothetical protein  12.2 ± 0  26.7 ± 4  12.0 ± 0  12.1 ± 0  15.9 ± 0  12.0 ± 0  ASN1  Asparagine synthetase  449.6 ± 89  1208.1 ± 131  305.1 ± 98  579.6 ± 23  936.1 ± 118  376.0 ± 373  BAP3  Valine transporter  35.4 ± 8  153.1 ± 12  12.0 ± 0  141.8 ± 55  84.5 ± 12  43.5 ± 54  DIP5  Dicarboxylic amino acid permease  83.8 ± 86  1054.0 ± 88  32.4 ± 7  408.7 ± 97  789.5 ± 118  304.3 ± 50  IMD1  Strong similarity to IMP dehydrogenases  211.1 ± 75  494.2 ± 23  105.7 ± 7  327.4 ± 47  236.9 ± 34  131.6 ± 86  SSK22  Protein kinase  31.1 ± 4  73.0 ± 3  28.9 ± 4  65.8 ± 3  89.9 ± 17  54.0 ± 20  TIR4  Similarity to Tir1p and Tir2p  1143.5 ± 265  2668.0 ± 130  125.9 ± 32  2104.9 ± 727  4859.3 ± 310  164.5 ± 24  Genes downregulated in IMK229 (rim101Δ) and upregulated under anaerobiosis in glucose-limited chemostat  YDL085C  Identified by SAGE  193.0 ± 91  347.1 ± 35  204.3 ± 47  202.6 ± 16  408.3 ± 7  1897 ± 91  YBR004C  Similarity to S. pombe hypothetical protein SPAC18B11.05  61.8 ± 3  114.7 ± 13  111.5 ± 7  49.0 ± 11  158.0 ± 8  39.8 ± 19  YMR325W  Strong similarity to members of the Srp1p/Tip1p family  136.6 ± 12  87.9 ± 5  83.7 ± 60  65.7 ± 10  241.7 ± 57  73.7 ± 68  YJL218W  Strong similarity to E. coli galactoside O-acetyltransferase  73.5 ± 58  115.5 ± 8  29.6 ± 9  22.3 ± 0  57.8 ± 11  12.0 ± 0  YGR131W  Strong similarity to Nce2p  92.3 ± 12  133.4 ± 17  46.4 ± 1  72.9 ± 17  153.1 ± 33  44.9 ± 9  YHR210C  UDP-glucose-4-epimerase (GAL10, galE)  366.8 ± 250  267.0 ± 30  31.3 ± 7  100.8 ± 26  311.5 ± 99  127.8 ± 88  ADA2  Transcription factor, member of ADA and SAGA  31.9 ± 12  29.9 ± 10  24.6 ± 5  12.2 ± 0  54.5 ± 1  27.2 ± 26  DAN2  Strong similarity to members of the Srp1p/Tip1p family  1366.8 ± 400  229.8 ± 32  154.5 ± 16  221.4 ± 31  827.2 ± 155  162.6 ± 89  DAN4  Similarity to mucin proteins, YKL224c, Sta1p  218.1 ± 43  243.7 ± 33  144.9 ± 36  183.2 ± 18  422.2 ± 167  128.4 ± 38  HMRA1  Homeobox-domain containing protein  493.2 ± 84  549.5 ± 20  304.8 ± 29  18.8 ± 4  645.8 ± 116  248.7193  LYS14  Transcriptional activator of lysine pathway genes  17.4 ± 4  21.9 ± 10  12.0 ± 0  28.7 ± 1  59.7 ± 43  23.3 ± 9  MEP3  Ammonia permease of high capacity and low affinity  75.3 ± 24  133.7 ± 12  26.6 ± 1  45.6 ± 33  217.3 ± 23  82.1 ± 93  MFA2  Mating a-factor pheromone precursor  2893.5 ± 118  2036.3 ± 251  2239.5 ± 938  1940.0 ± 13  4016.6 ± 332  1960.3 ± 147  NIP7  Nip7p is required for 60S ribosome subunit biogenesis  111.4 ± 53  158.1 ± 34  135.8 ± 9  79.2 ± 8  169.9 ± 16  49.0 ± 15  PAU3  Member of the seripauperin protein\gene family  143.4 ± 22  74.1 ± 18  58.4 ± 38  83.9 ± 12  294.3 ± 51  121.3 ± 81  Q0280  Ubiquinol–cytochrome c reductase subunit (cytochrome b) F.  12.0 ± 0  12.0 ± 0  12.0 ± 0  35.8 ± 15  72.1 ± 6  31.8 ± 33  RPR1  RNAse P RNA  21.8 ± 17  12.1 ± 0  12.0 ± 0  13.8 ± 2  164.8 ± 35  72.1 ± 94  RPS26B  Ribosomal protein S26B  464.7 ± 172  649.5 ± 60  806.2 ± 312  426.0 ± 30  887.2 ± 90  325.3 ± 98  SNR17B  Encodes snRNA U3, SNR17A also encodes snRNA U3  322.8 ± 136  439.0 ± 12  531.3 ± 147  207.6 ± 46  503.7 ± 36  176.4 ± 122  SST2  Regulator of G protein-signaling family member  64.7 ± 10  93.0 ± 12  28.9 ± 5  42.4 ± 11  87.8 ± 10  31.7 ± 25  STE2  Pheromone α-factor G protein-coupled receptor (GPCR)  394.3 ± 64  346.2 ± 6  183.1 ± 21  143.3 ± 24  415.2 ± 37  73.0 ± 52  SUC2  Invertase (sucrose-hydrolyzing enzyme)  1114.4 ± 263  1281.9 ± 41  487.9 ± 57  410.2 ± 3  2097.1 ± 268  978.8 ± 230  SUC4  Invertase (sucrose-hydrolyzing enzyme)  865.3 ± 292  1015.3 ± 62  316.0 ± 29  335.6 ± 10  1252.2 ± 220  325.4 ± 212  TRS33  Involved in the targeting and fusion of ER to golgi transport vesicles  212.7 ± 22  188.9 ± 17  120.8 ± 33  96.8 ± 6  196.1 ± 45  49.4 ± 51  Gene  Functional description  snf7Δ anaerobic  Ref1 anaerobic  Ref1 aerobic  rim101Δ anaerobic  Ref2 anaerobic  Ref2 aerobic  Genes downregulated in GG3201 (snf7Δ) and IMK229 (rim101Δ) and upregulated under anaerobiosis in glucose-limited chemostat  FET4  Low-affinity Fe(II) transport protein  51.8 ± 21  171.9 ± 33  12.4 ± 0  89.8 ± 68  236.2 ± 27  115.7 ± 13  AAC3  Mitochondrial ADPVATP translocator  75.5 ± 50  372.5 ± 18  12.5 ± 0  66.5 ± 78  173.0 ± 17  71.5 ± 103  TIR1  Protein of the Srp1pVTip1p family of serine–alanine-rich proteins  171.7 ± 9  4248.8 ± 292  100.3 ± 21  303.6 ± 71  7726.2 ± 625  252.5 ± 46  MUC1  Cell surface flocculin with structure/GPI-anchored cell wall proteins  88.8 ± 19  1106.8 ± 102  34.4 ± 5  96.9 ± 30  1149.4 ± 104  410.3 ± 61  SML1  Identified by SAGE  82.8 ± 33  439.6 ± 46  17.8 ± 1  396.8 ± 157  939.0 ± 141  266.7 ± 32  YBR300C  Strong similarity to hypothetical protein YGR293c  17.9 ± 5  49.5 ± 10  12.0 ± 0  13.1 ± 1  29.0 ± 5  15.8 ± 6  YDL241W  Hypothetical protein  12.0 ± 0  64.0 ± 7  12.0 ± 0  13.1 ± 0  43.3 ± 7  21.9 ± 14  YJL213W  similarity to Methanobacterium aryldialkylphosphatase  50.6 ± 22  216.9 ± 33  25.0 ± 4  45.7 ± 16  104.3 ± 24  68.2 ± 38  YLR460C  Similarity to C. carbonum toxD protein  61.1 ± 16  144.5 ± 17  17.7 ± 5  52.7 ± 11  178.7 ± 31  62.9 ± 72  SKT5  Protoplast regeneration and killer toxin resistance gene,  76.4 ± 7  171.2 ± 4  65.0 ± 20  118.1 ± 5  282.2 ± 32  125.8 ± 99  YCL049C  Hypothetical protein  178.9 ± 31  478.3 ± 35  220.2 ± 24  179.0 ± 46  522.0 ± 59  257.3 ± 15  OPT2  Similarity to S. pombe isp4 protein  12.0 ± 0  167.0 ± 39  12.0 ± 0  14.5 ± 3  205.2 ± 32  83.9 ± 124  Genes downregulated in GG3201 (snf7Δ) and upregulated under anaerobiosis in glucose-limited chemostat  YOL014W  Hypothetical protein  12.2 ± 0  26.7 ± 4  12.0 ± 0  12.1 ± 0  15.9 ± 0  12.0 ± 0  ASN1  Asparagine synthetase  449.6 ± 89  1208.1 ± 131  305.1 ± 98  579.6 ± 23  936.1 ± 118  376.0 ± 373  BAP3  Valine transporter  35.4 ± 8  153.1 ± 12  12.0 ± 0  141.8 ± 55  84.5 ± 12  43.5 ± 54  DIP5  Dicarboxylic amino acid permease  83.8 ± 86  1054.0 ± 88  32.4 ± 7  408.7 ± 97  789.5 ± 118  304.3 ± 50  IMD1  Strong similarity to IMP dehydrogenases  211.1 ± 75  494.2 ± 23  105.7 ± 7  327.4 ± 47  236.9 ± 34  131.6 ± 86  SSK22  Protein kinase  31.1 ± 4  73.0 ± 3  28.9 ± 4  65.8 ± 3  89.9 ± 17  54.0 ± 20  TIR4  Similarity to Tir1p and Tir2p  1143.5 ± 265  2668.0 ± 130  125.9 ± 32  2104.9 ± 727  4859.3 ± 310  164.5 ± 24  Genes downregulated in IMK229 (rim101Δ) and upregulated under anaerobiosis in glucose-limited chemostat  YDL085C  Identified by SAGE  193.0 ± 91  347.1 ± 35  204.3 ± 47  202.6 ± 16  408.3 ± 7  1897 ± 91  YBR004C  Similarity to S. pombe hypothetical protein SPAC18B11.05  61.8 ± 3  114.7 ± 13  111.5 ± 7  49.0 ± 11  158.0 ± 8  39.8 ± 19  YMR325W  Strong similarity to members of the Srp1p/Tip1p family  136.6 ± 12  87.9 ± 5  83.7 ± 60  65.7 ± 10  241.7 ± 57  73.7 ± 68  YJL218W  Strong similarity to E. coli galactoside O-acetyltransferase  73.5 ± 58  115.5 ± 8  29.6 ± 9  22.3 ± 0  57.8 ± 11  12.0 ± 0  YGR131W  Strong similarity to Nce2p  92.3 ± 12  133.4 ± 17  46.4 ± 1  72.9 ± 17  153.1 ± 33  44.9 ± 9  YHR210C  UDP-glucose-4-epimerase (GAL10, galE)  366.8 ± 250  267.0 ± 30  31.3 ± 7  100.8 ± 26  311.5 ± 99  127.8 ± 88  ADA2  Transcription factor, member of ADA and SAGA  31.9 ± 12  29.9 ± 10  24.6 ± 5  12.2 ± 0  54.5 ± 1  27.2 ± 26  DAN2  Strong similarity to members of the Srp1p/Tip1p family  1366.8 ± 400  229.8 ± 32  154.5 ± 16  221.4 ± 31  827.2 ± 155  162.6 ± 89  DAN4  Similarity to mucin proteins, YKL224c, Sta1p  218.1 ± 43  243.7 ± 33  144.9 ± 36  183.2 ± 18  422.2 ± 167  128.4 ± 38  HMRA1  Homeobox-domain containing protein  493.2 ± 84  549.5 ± 20  304.8 ± 29  18.8 ± 4  645.8 ± 116  248.7193  LYS14  Transcriptional activator of lysine pathway genes  17.4 ± 4  21.9 ± 10  12.0 ± 0  28.7 ± 1  59.7 ± 43  23.3 ± 9  MEP3  Ammonia permease of high capacity and low affinity  75.3 ± 24  133.7 ± 12  26.6 ± 1  45.6 ± 33  217.3 ± 23  82.1 ± 93  MFA2  Mating a-factor pheromone precursor  2893.5 ± 118  2036.3 ± 251  2239.5 ± 938  1940.0 ± 13  4016.6 ± 332  1960.3 ± 147  NIP7  Nip7p is required for 60S ribosome subunit biogenesis  111.4 ± 53  158.1 ± 34  135.8 ± 9  79.2 ± 8  169.9 ± 16  49.0 ± 15  PAU3  Member of the seripauperin protein\gene family  143.4 ± 22  74.1 ± 18  58.4 ± 38  83.9 ± 12  294.3 ± 51  121.3 ± 81  Q0280  Ubiquinol–cytochrome c reductase subunit (cytochrome b) F.  12.0 ± 0  12.0 ± 0  12.0 ± 0  35.8 ± 15  72.1 ± 6  31.8 ± 33  RPR1  RNAse P RNA  21.8 ± 17  12.1 ± 0  12.0 ± 0  13.8 ± 2  164.8 ± 35  72.1 ± 94  RPS26B  Ribosomal protein S26B  464.7 ± 172  649.5 ± 60  806.2 ± 312  426.0 ± 30  887.2 ± 90  325.3 ± 98  SNR17B  Encodes snRNA U3, SNR17A also encodes snRNA U3  322.8 ± 136  439.0 ± 12  531.3 ± 147  207.6 ± 46  503.7 ± 36  176.4 ± 122  SST2  Regulator of G protein-signaling family member  64.7 ± 10  93.0 ± 12  28.9 ± 5  42.4 ± 11  87.8 ± 10  31.7 ± 25  STE2  Pheromone α-factor G protein-coupled receptor (GPCR)  394.3 ± 64  346.2 ± 6  183.1 ± 21  143.3 ± 24  415.2 ± 37  73.0 ± 52  SUC2  Invertase (sucrose-hydrolyzing enzyme)  1114.4 ± 263  1281.9 ± 41  487.9 ± 57  410.2 ± 3  2097.1 ± 268  978.8 ± 230  SUC4  Invertase (sucrose-hydrolyzing enzyme)  865.3 ± 292  1015.3 ± 62  316.0 ± 29  335.6 ± 10  1252.2 ± 220  325.4 ± 212  TRS33  Involved in the targeting and fusion of ER to golgi transport vesicles  212.7 ± 22  188.9 ± 17  120.8 ± 33  96.8 ± 6  196.1 ± 45  49.4 ± 51  The CEN.PK113-7D Ref1 corresponds to the reference of GG3201 (snf7Δ) [the array samples have been processed using the protocol described in Daran-Lapujade (2004)]. The CEN.PK113-7D Ref2 corresponds to the reference of IMK229 (rim101Δ) [the array samples have been processed using the protocol described in De Nicola (2007)]. Genes underlined showed a consistent anaerobic transcriptional upregulation under four different nutrient limitation regimes as described previously in Tai (2005). The mean expression value and mean deviation were calculated from triplicate cultures, except for IMK229, whose values were derived from duplicate cultures. View Large In order to dissect even further the oxygen effect from the Rim101p response, we have compared the rim101Δ downregulated genes obtained in this study with the rim101Δ downregulated set from previous studies obtained under different environmental conditions (Lamb, 2001; Mira, 2009) (Fig. 4). This comparison identified common and specific responses. From the seven anaerobic genes earlier mentioned, five (TIR1, MUC1, SML1, AAC3, YBR300C) exhibited a rim101Δ-dependent downregulation specifically under anaerobic conditions (Fig. 4). Fig. 4 View largeDownload slide Venn diagram of results from three transcriptome studies on rim101Δ mutants of Saccharomyces cerevisiae. The rim101Δ downregulated genes obtained from aerobic shake-flask cultures from Lamb & Mitchell (2003) and Mira (2009) were compared with the rim101Δ chemostat-based transcriptome data generated in the present study. Fig. 4 View largeDownload slide Venn diagram of results from three transcriptome studies on rim101Δ mutants of Saccharomyces cerevisiae. The rim101Δ downregulated genes obtained from aerobic shake-flask cultures from Lamb & Mitchell (2003) and Mira (2009) were compared with the rim101Δ chemostat-based transcriptome data generated in the present study. Nrg1p, Nrg2p and Smp1p are not involved in Rim101-dependent regulation of TIR1 Nrg1p acts as a corepressor with Rim101p in the expression of DIT1 (Rothfels, 2005) and ENA1 (Lamb & Mitchell, 2003; Platara, 2006), and the NRG1 gene itself is repressed by active Rim101p (Lamb & Mitchell, 2003). Indeed, NRG1 showed a 3.2- and 2.0-fold higher transcript level in the rim101Δ and in the snf7Δ strain than in the reference strain, respectively. While the difference was deemed significant in the IMK229 (rim101Δ) mutant, in the strain GG3201 (snf7Δ), the gene expression difference did not pass our statistical evaluation. Similarly, Rim101p has been reported to influence ion tolerance and cell differentiation via Smp1p (Lamb & Mitchell, 2003), SMP1 showed a significant 5.5-fold increase in the expression level in the snf7Δ strain compared with the reference strain under anaerobic conditions and a 7.3-fold increase in the rim101Δ strain (Fig. 2). This would fit with the model in which, under anaerobic conditions, activated Rim101p would repress the NRG1 and/or the SMP1 expression allowing the derepression of the TIR1 and all co-upregulated genes. To test the possible involvement of Nrg1/2p and Smp1p in the anaerobic upregulation of TIR1, the pTIR construct was introduced into nrg1Δ, nrg2Δ and nrg1Δnrg2Δ mutants [the zinc-finger protein Nrg2p is a homologue of Nrg1p (Vyas, 2001; Berkey, 2004)] as well as in an smp1Δ strain (yielding strains IMZ180, IMZ182, IMZ166 and IMZ184). β-Galactosidase activity under aerobic and anaerobic conditions did not reveal significantly increased activities under either aerobic or anaerobic conditions (Fig. 5). However, the LacZ activity measured from the TIR1 promoter in the double ngr1 nrg2Δ strains was at least threefold lower than that in the control and the single mutants. While these data would preclude a unique and direct role of NRG1 and NRG2 in the TIR1 repression, they do not rule an indirect effect out that might explain the phenotype observed. Similarly, placing NRG1 behind the control of a strong constitutive promoter (TPI1pro) did not result in an anaerobic repression of the TIR1 promoter-driven expression of β-galactosidase (Fig. 5). This indicated that Snf7p/Rim101p-dependent upregulation of the TIR1 gene cannot be solely explained via repression mediated by Nrg1p, Nrg2p or Smp1p. Thus, the cis-regulatory elements identified in the promoter region of anaerobic genes whose expressions depended on Snf7p/Rim101p signalling did not play a decisive role in oxygen-dependent transcriptional regulation. However, these data should not totally exclude an involvement of NRG1 and NRG2 in TIR1 regulation. Fig. 5 View largeDownload slide Induction of TIR1 promoter-driven β-galactosidase activity. β-Galactosidase activity was measured in reference strain IMZ162 and in strains IMZ180 (nrg1Δ), IMZ182 (nrg2Δ), IMZ184 (nrg1Δ nrg2Δ), IMZ185 (nrg1Δ nrg2ΔTPI1-NRG1), IMZ166 (smp1Δ), IMZ174 (ssn6Δ), IMZ175 (tup1Δ), IMZ176 (rox1Δ), IMZ173 (upc2Δ) and IMZ181 (slt2Δ). Exponential-phase cells growing on glucose under aerobic (white bars) and anaerobic (gray bars) conditions were harvested and β-galactosidase activity was measured. β-Galactosidase activities are expressed as μM of o-nitrophenol (ONP) formed per min and per mg of protein. The activity displayed is the average of two independent biological samples and its mean deviation. Fig. 5 View largeDownload slide Induction of TIR1 promoter-driven β-galactosidase activity. β-Galactosidase activity was measured in reference strain IMZ162 and in strains IMZ180 (nrg1Δ), IMZ182 (nrg2Δ), IMZ184 (nrg1Δ nrg2Δ), IMZ185 (nrg1Δ nrg2ΔTPI1-NRG1), IMZ166 (smp1Δ), IMZ174 (ssn6Δ), IMZ175 (tup1Δ), IMZ176 (rox1Δ), IMZ173 (upc2Δ) and IMZ181 (slt2Δ). Exponential-phase cells growing on glucose under aerobic (white bars) and anaerobic (gray bars) conditions were harvested and β-galactosidase activity was measured. β-Galactosidase activities are expressed as μM of o-nitrophenol (ONP) formed per min and per mg of protein. The activity displayed is the average of two independent biological samples and its mean deviation. Aerobic/anaerobic regulation of TIR1 is Ssn6p–Tup1p dependent Several regulatory circuits have been involved in anaerobiosis-related regulation in S. cerevisiae. The transcriptional activator UPC2 has been linked to the regulation of the ABC transporters AUS1 and PDR11, which are required for sterol uptake in yeast under anaerobic conditions (Wilcox, 2002). ROX1, in cooperation with MOT3, MOX1 and MOX2, participates in the repression of several hypoxic genes (Abramova, 2001; ter Linde & Steensma, 2002). The repressor function of Rox1p is dependent on the Ssn6p–Tup1p complex. Recently, the Rim101p pathway has been shown to be actively involved in cell wall assembly and in the maintenance of neck integrity during cell division. This process required both RIM101 and SLT2, which encodes a mitogen-activated kinase (Castrejon, 2006). To verify whether any of these circuits were involved in TIR1 regulation, upc2Δ, rox1Δ, mot3Δ, ssn6Δ, tup1Δ and slt2Δ mutants were transformed with pTIR (yielding strains IMZ172, IMZ176, IMZ183, IMZ174, IMZ175 and IMZ181) and grown under aerobic and anaerobic conditions. While upc2Δ, rox1Δ, mot3Δ and slt2Δ strains did not show an obvious phenotype, the ssn6Δ and tup1Δ strains exhibited aerobic deregulation of the TIR1pr-driven β-galactosidase activity (Fig. 5). The Tup1p–Ssn6p corepressor complex is important for the repression of many genes involved in a wide variety of physiological processes (Smith & Johnson, 2000; Malave & Dent, 2006). Tup1p–Ssn6p is recruited to target genes by interaction with DNA-bound transcriptional repressors that recognize specific sequences within the target gene promoters. Such repressors include Matα2p, which regulates mating-type-specific genes (Komachi & Johnson, 1997; Malave & Dent, 2006), Mig1p, which regulates glucose-repressed genes (Treitel & Carlson, 1995; Komachi & Johnson, 1997), Rfx1p, which is involved in DNA repair (Huang, 1998), Sko1p, which is involved in stress responses (Proft, 2001), and, as mentioned previously, Nrg1p (Park, 1999). Impaired anaerobic growth of an snf7Δ strain in the presence of SDS Many phenotypical characteristics have been assigned previously to snf7Δ strains, including deficiencies in sporulation and raffinose utilization, high-temperature sensitivity (Tu, 1993), decreased tolerance to salt stress and nystatin (Giaever, 2002), inability to grow on nonfermentable carbon sources (Steinmetz, 2002), decreased glycogen accumulation (Wilson, 2002) and sensitivity to CaCl2 and tunicamycin (Unno, 2005). On plates, under anaerobic, but not aerobic conditions, a slight growth reduction was observed on complex medium, relative to the isogenic reference strain. The growth rate determination of the snf7Δ and CEN.PK113-7D reference strain from off-gas data of the anaerobic batch phase of the chemostat cultures confirmed the slower growth of the snf7Δ and rim101Δ strains relative to its isogenic reference (0.28 ± 0.01 and 0.27 ± 0.02 h−1, respectively, vs. 0.33 ± 0.02 h−1). However, the difference remained limited and did not exceed 15% of the CEN.PK113-7D maximal specific growth rate. Furthermore, the strain showed increased sensitivity to SDS (data not shown). Discussion The results presented above demonstrate that Snf7p and Rim101p are required for the strong anaerobic transcriptional upregulation of the cell wall mannoprotein-encoding TIR1 gene. Furthermore, the involvement of Snf7p and Rim101p in the anaerobic transcriptional upregulation is not unique for TIR1, because six additional genes that are transcriptionally upregulated in anaerobic chemostat cultures [irrespective of the nutrient limitation (Tai, 2005)] of an isogenic reference strain also showed strongly reduced anaerobic transcript levels in snf7Δ and rim101Δ mutants (Fig. 2). Despite these similarities, the overall genome-wide responses to snf7Δ and rim101Δ mutations were noticeably different, with only approximately 10% of the transcriptional responses being shared (Table 4). This may be explained by the fact that, although the two proteins function in the same signalling pathway, they still have distinct roles. In particular, SNF7 is involved in MVB trafficking. An snf7 mutant exhibits morphological vacuolar changes (Raymond, 1992) accompanied by large accumulation of transmembrane ubiquitinated proteins (Amerik, 2000) probably caused by deficient endosomal sorting (Babst, 1998). Many phenotypic characteristics previously attributed to snf7Δ mutants can be related to the cell surface [e.g. sensitivity to nystatin and alkaline conditions (Giaever, 2002; Boysen & Mitchell, 2006; Castrejon, 2006; Gomez, 2009)]. The increased SDS sensitivity of yeast mutants is generally considered to reflect cell wall defects that result in increased accessibility of SDS to the plasma membrane (de Groot, 2001). The increased SDS sensitivity of an snf7Δ strain [which has been demonstrated previously for rim101Δ mutants (Castrejon, 2006)] is therefore consistent with a role of Snf7p in regulating cell surface composition and/or organization. At the transcriptional level, 18 of 95 genes with a higher transcript level in the snf7Δ strain and nine of 114 genes with a lower transcript level than the reference strain encode cell wall-related proteins (Saccharomyces Genome database, http://www.yeastgenome.org/). Transcription of 11 of these genes was previously shown to be oxygen regulated (Tai, 2005) (Table S2). Among the genes that showed a transcriptional upregulation under anaerobic conditions, the cell wall protein-encoding genes MUC1 and TIR1 showed highly similar transcript profiles (Fig. 3). MUC1 (FLO11) encodes a cell wall flocculin that plays a key role in biofilm formation and pseudohyphal growth (Lambrechts, 1996; Reynolds & Fink, 2001). Both genes showed a high-level transcriptional upregulation in anaerobic chemostat cultures [under four different nutrient limitation regimes (Tai, 2005)] that was completely abolished in snf7Δ and rim101Δ mutants. Oxygen availability has been shown previously to affect the expression of cell wall protein-encoding genes in S. cerevisiae. These changes are slow, taking several generations for completion (Lai, 2005). Transcript levels of CWP1 and CWP2 decrease, while those of the seripauperin family genes, such as the DAN, TIR and PAU genes, increase (Klis, 2002), suggesting a switch from one set of cell wall proteins to another. Transcriptome analysis did not provide indications that changes in cell wall composition in an snf7Δ strain resulted in seriously compromised cell wall integrity. Rlm1p-controlled cell wall protein-encoding genes and STRE-controlled genes were not transcriptionally induced in the Δsnf7 strain (Table S2), nor were ribosomal genes and rRNA genes downregulated, as observed during exposure to calcofluor white (CFW) and zymolyase treatments (Boorsma, 2004; Garcia, 2004). Consistent with these observations, an snf7Δ strain did not exhibit increased sensitivity to CFW and electron microscopy of thin sections did not reveal morphological changes (data not shown). Interestingly, the involvement of Nrg1p and Nrg2p in the regulation of MUC1 has been demonstrated previously (Kuchin, 2002), thus providing a plausible signal transduction pathway for its Snf7-dependent anaerobic transcriptional upregulation. For TIR1, we attempted to identify a possible transcriptional repressor acting downstream of Rim101p and whose transcriptional repression by Rim101p might explain anaerobic upregulation of TIR1. However, we cannot exclude that TIR1 might be dually controlled in the absence of oxygen. Besides a relief of transcriptional repression via the Snf7p/Rim101p pathway, TIR1 might be positively controlled under anaerobiosis by an as yet unknown mechanism, which would explain the absence of aerobic β-galactosidase transcriptional activation in vsp2Δ, vps24Δ, RIM101-531 and perhaps nrg1Δ mutants (Figs 1 and 5). Furthermore, increased TIR1 transcript levels in aerobic cultures of ssn6Δ and tup1Δ mutants confirmed that the mechanism involved in aerobic TIR1 repression depends on the Ssn6p–Tup1 complex. This analysis strongly indicated a specific regulatory path downstream of Rim101p for the five ‘anaerobic’ genes TIR1, AAC3, MUC1, SML1 and YBR300C. Further research is required to investigate whether anaerobic upregulation of TIR1 involves an alternative Rim101p-controlled transcriptional repressor or whether it results from indirect mechanisms. In this respect, it is relevant to note that deletion of SNF7 affected the expression of a number of homologous cell wall protein-encoding genes in opposite directions (e.g. reduced transcript levels of TIR1 and TIR4 were accompanied by increased transcript levels of TIR2 and DAN2). This raises the possibility that some of the transcriptional responses may involve autoregulation [as shown within other gene families, e.g. autoregulation of the pyruvate decarboxylase genes PDC1 and PDC5 (Hohmann & Cederberg, 1990)] or indirect mechanisms that involve sensing of cell wall structure or composition. The impact of Snf7p/Rim101p on anaerobic upregulation of yeast genes was not confined to cell wall protein-encoding genes. A particularly interesting example of this is AAC3, which encodes the hypoxic isoenzyme of mitochondrial ADP–ATP translocase (Kolarov, 1990), and that shared the transcriptional profile of MUC1 and TIR1. Transcriptional regulation of AAC3 is known to involve both a heme-dependent pathway (involving Rox1p) and a carbon-source-related, heme-independent pathway involving Rap1p (Sokolikova, 2000). Further research should identify whether regulation by Snf7p/Rim101p occurs through these previously identified pathways or whether it represents an alternative pathway. The present study provides the first evidence for the involvement of Snf7p/Rim101p in the transcriptional reprogramming that occurs during anaerobic growth of S. cerevisiae and emphasizes the general role of Rim101p in the cellular adaptation to a broad range of environmental conditions. The exact contribution of these changes to cellular fitness under aerobic and anaerobic growth conditions remains to be identified, for example by competitive cultivation of deletion mutants under aerobic and anaerobic conditions (Tai, 2007). 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TI - Involvement of Snf7p and Rim101p in the transcriptional regulation of TIR1 and other anaerobically upregulated genes in Saccharomyces cerevisiae
JF - FEMS Yeast Research
DO - 10.1111/j.1567-1364.2010.00622.x
DA - 2010-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/involvement-of-snf7p-and-rim101p-in-the-transcriptional-regulation-of-7BGwlQ0U8A
SP - 367
EP - 384
VL - 10
IS - 4
DP - DeepDyve
ER -