Screening the yeast deletant mutant collection for hypersensitivity and hyper-resistance to sorbate, a weak organic acid food preservative

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1 Yeast Yeast 2004; 21: Published online in Wiley InterScience ( DOI: /yea.1141 Yeast Functional Analysis Report Screening the yeast deletant mutant collection for hypersensitivity and hyper-resistance to sorbate, a weak organic acid food preservative Mehdi Mollapour 1, Dahna Fong 1, Krishna Balakrishnan 1, Nicholas Harris 1, Suzanne Thompson 1, Christoph Schüller 2, Karl Kuchler 2 and Peter W. Piper 1 * 1 Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK 2 Department of Medical Biochemistry, Division of Molecular Genetics, Max F. Perutz Laboratories, University and Biocenter of Vienna, A-1030 Vienna, Austria *Correspondence to: Peter W. Piper, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK. peter.piper@sheffield.ac.uk Received: 20 February 2004 Accepted: 23 May 2004 Abstract Certain yeasts cause large-scale spoilage of preserved food materials, partly as a result of their ability to grow in the presence of the preservatives allowed in food and beverage preservation. This study used robotic methods to screen the collection of Saccharomyces cerevisiae gene deletion mutants for both increased sensitivity and increased resistance to sorbic acid, one of the most widely-used weak organic acid preservatives. In this way it sought to identify the non-essential, non-redundant activities that influence this resistance, activities that might be the potential targets of new preservation strategies. 237 mutants were identified as incapable of growth at ph 4.5 in presence of 2 mm sorbic acid, while 34 mutants exhibit even higher sorbate resistance than the wild-type parental strain. A number of oxidative stress-sensitive mutants, also mitochondrial mutants, are sorbate-sensitive. This appears to reflect the importance of sustaining a reducing intracellular environment (high reduced glutathione levels and NADH/NAD and NADPH/NADP ratios). Sorbate resistance is also very severely compromised in mutants lacking an acidified vacuole, in vacuolar protein sorting (vps) mutants, in mutants defective in ergosterol biosynthesis (erg mutants) and with several defects in actin and microtubule organization. Sorbate resistance is, however, elevated with the loss of the Yap5 transcription factor; with single losses of two B-type cyclins (Clb3p, Clb5p); and with loss of a plasma membrane calcium channel activated by endoplasmic reticulum stress (Cch1p/Mid1p). Copyright 2004 John Wiley & Sons, Ltd. Keywords: Saccharomyces cerevisiae; organic acid stress; food preservatives; weak acid adaptation Introduction Certain yeasts and fungi pose a spoilage threat for many materials preserved by low ph, low water activity; and/or high levels of preservative (Fleet, 1992). A relatively small number of yeasts are of major importance in this regard and therefore classified as spoilage yeasts. They include a number of the Zygosaccharomyces, as well as some isolates of Saccharomyces cerevisiae (see Steels et al., 2000), and references cited therein). Remarkably, these yeasts can sometimes grow in the presence of the highest weak organic acid preservative levels allowed in food preservation (Fleet, 1992; Piper et al., 2001; Steels et al., 1999, 2000). S. cerevisiae growth in the presence of moderately lipophilic carboxylate preservatives is Copyright 2004 John Wiley & Sons, Ltd.

2 928 M. Mollapour et al. dependent on the induction of a specific stress response, involving the War1p transcription factordependent induction of the Pdr12p ATP-binding cassette (ABC) transporter (Hatzixanthis et al., 2003; Holyoak et al., 1999; Kren et al., 2003; Piper et al., 1997, 1998; Schüller et al., 2004). So strong is this Pdr12p induction, that the Pdr12p levels in the plasma membrane of sorbate-stressed cells frequently approach those of the most abundant plasma membrane protein, the plasma membrane H + -ATPase (Piper et al., 1998, 2001). Pdr12p action has been shown to lower the intracellular levels of benzoate and fluorescein (Holyoak et al., 1999; Piper et al., 1998). It appears, therefore, to be conferring resistance by catalysing the active efflux of the weak acid preservative anion from the cell. Z. bailii (another spoilage yeast) does not show this strong induction of a plasma membrane transporter when subjected to sorbate stress (Piper et al., 2001). It may instead place more reliance upon limiting the initial diffusional entry of the acid to the cells (Piper et al., 2001). Z. bailii, unlike S. cerevisiae, possesses a mitochondrial monooxygenase that enables it to catalyse the oxidative degradation of both sorbate and benzoate (Mollapour and Piper, 2001a, 2001b). In aerobic cells a major benefit of Pdr12p lowering the levels of moderately lipophilic carboxylate compounds is substantial reduction in the endogenous oxidative stress that these acids generate. Sorbate and benzoate act upon the mitochondrial inner membrane, so as to greatly enhance superoxide free radical production by the mitochondrial electron transport chain (Piper, 1999). As a result, superoxide dismutases also make major contributions to sorbate resistance in aerobic cultures of S. cerevisiae (Piper, 1999). Sorbate- and benzoatestressed S. cerevisiae and Z. bailii are also experiencing a severe energy (ATP) depletion due, in part, to the high energy demands of counteracting weak acid stress (Piper et al., 1997, 2001; Warth and Nickerson, 1991). This energy crisis is exacerbated by the mitochondrial dysfunction and by the strong inhibitory effects of sorbate and benzoate on glycolysis (an inhibition exerted mainly at the phosphofructokinase (Pfk) reaction (Cole and Keenan, 1987; Krebs et al., 1983; Pearce et al., 2001). To obtain a more complete picture of the activities that contribute to yeast growth in the presence of moderately lipophilic carboxylic acids, we have used robotic procedures to screen the collection of S. cerevisiae gene deletant strains for sensitivity to sorbate. We also screened the mutant collection for an increased resistance to sorbate, in view of the finding that S. cerevisiae can be engineered for higher weak acid resistance (Mollapour and Piper, 2001b) and the possibility that certain activities might counteract this resistance. As described in this report, these screens uncovered a large number of non-essential and non-redundant functions that influence sorbate resistance. Materials and methods Yeast strains, media The screens described below utilized the EUROSCARF collection of diploid S. cerevisiae mutant strains ( mikro/euroscarf/); homozygous kanmx4 cassette gene deletions in BY4743 (MAT a/mat α; his3-1/his3-1; leu2-0/leu2-0; met15-0/met15; LYS2/lys2-0; ura3-0/ura3-0). A set of mnn mutants in the SEY6210 background (MAT α ura3-52 his3-200 leu2,3-112 trp1-901 lys2-801 suc2-9) was kindly provided by Sean Munro. The latter were all insertions of the Schizosaccharomyces pombe HIS5 sequence (Rayner and Munro, 1998), with the sole exception of the mnn8 mutant (a LEU2 disruptant of this gene). Strains were maintained on ph 6.8 YPD agar [1% Difco yeast extract, 2% peptone, 2% glucose, 2% agar (all % w/v)]. For screening purposes, liquid YPD was adjusted to ph 4.5 or ph 6.8 with either HCl or NaOH before autoclaving, 2 mm or 5mM sorbic acid being added (from a 1 M ph 7.0 stock solution in water) just prior to use. Screening for sorbic acid sensitivity and resistance All replications and inoculations were carried out using Biomek 2000 Laboratory Automation Workstation (Beckman), with movements programmed using the BioWorks Version software (Beckman). Mutant strains were transferred from standard 96-well microtitre master plates to (YPDagar) OMNI trays (128 mm 86 mm; NUNC, USA) using the 96-pin replicator of the Biomek. Mutant strains from four separate 96-well

3 S. cerevisiae mutants sensitive or resistant to sorbic acid 929 plates were combined, so as to generate collections of the strains in 384 format. These plates were then incubated for 2 days at 28 C until large colonies formed. The Biomek 384-pin replicator was then used to inoculate colonies into 50 µl liquid YPD in 384-well microtitre format. These plates were then cultured for 24 h at 30 C. After gentle vortexing in order to resuspend the cells, these 384-well microtitre plates were used to inoculate (sequentially, with intermediate cleansing and sterilization of the replicator pins): 50 µl liquid ph 6.8 YPD; 50 µl ph 4.5 YPD; 50 µl ph 4.5 YPD, 2 mm sorbate; and 50 µl ph 4.5 YPD, 5 mm sorbate. The latter plates were grown for 3 days (5 days for YPD plus 5 mm sorbate) at 30 C and the optical density (OD 600 nm ) of the wells measured using a microtitre plate reader (Multiscan Ascent, ThermoLabsystems, Finland). Sorbate inhibition was apparent by comparison of OD 600 nm values to the OD 600 nm values of control ph 6.8 cultures, reductions in OD 600 nm greater than 80 90% being scored as significant. All screens were performed in duplicate, the very few strains that did not behave identically in each of these screens being rechecked before a phenotype was assigned. Analysis of PDR12 LacZ expression and Pdr12p levels Measurements of the expression of a PDR12 LacZ gene reporter of PDR12 promoter activity; also of total cellular levels of Pdr12p and Sba1p (the latter a loading control on the Western blots), were all as previously described (Hatzixanthis et al., 2003; Kren et al., 2003; Piper et al., 1998). Results and discussion Designing a screen for both sensitivity and resistance to sorbate stress We recently identified a new stress response in S. cerevisiae, a response that is regulated by the War1p zinc finger transcription factor and induced by sorbate, benzoate and other moderately lipophilic carboxylate compounds (Piper et al., 1998; Hatzixanthis et al., 2003; Kren et al., 2003; Schüller et al., 2004). The War1p regulon is small. Remarkably, it appears that the only War1p target gene that is needed for the pronounced effects of War1p loss on sorbate resistance is the gene encoding the Pdr12p transporter (Schüller et al., 2004). This is despite several other activities in addition to Pdr12p, activities that are not War1p-regulated, being required for resistance to these moderately lipophilic carboxylate compounds (see Introduction). This study sought to identify most of these other activities, by robotic screening of the S. cerevisiae deletant mutant collection for altered sorbate resistance. Sorbic acid (pk a 4.76) exerts much stronger inhibitory effects at low ph, when it is substantially undissociated (reviewed in Piper et al., 2001). It cannot be degraded by S. cerevisiae (Mollapour and Piper, 2001b) so that, when added to cultures of this yeast, it imposes constant weak acid stress. Following the addition of mm sorbate, S. cerevisiae cultures in growth at ph 4.5 exit the cell cycle and enter a long period of stasis. Eventually, after a period of several hours to days, they resume growth. In this state total cell biomass increases only slowly, since the energy yields of a high rate of glycolytic flux are mostly used in counteracting the stress. The cells are, however, now stressadapted, in that they will not display another transient growth arrest if re-inoculated into fresh ph 4.5 medium containing the same, subinhibitory level of sorbate (Holyoak et al., 1999; Piper, et al., 1997, 1998). Genetic background can have a strong influence over the level of a carboxylate compound needed to inhibit S. cerevisiae growth. Auxotrophic requirements for aromatic amino acids exert a particularly pronounced effect, mutations such as trp1 rendering S. cerevisiae very sensitive to inhibition by both moderately lipophilic weak organic acids such as sorbate, and by high levels of (the relatively non-lipophilic) acetate. This is because these acids are strongly inhibitory to uptake of aromatic amino acids from the medium, rendering cells particularly sensitive if they have to catalyse such uptake in order to grow (Bauer et al., 2003). Thus, the minimal inhibitory concentration of sorbate in ph 4.5 cultures is 4 5 mm for strains competent in aromatic amino acid biosynthesis, but only mm for strains with auxotrophic requirements for aromatic amino acids. Our choice of the sorbate concentrations used for these screens was therefore based on our previous work showing the levels that would allow weak acid adaptation in a TRP + genetic background (Bauer et al., 2003).

4 930 M. Mollapour et al. Recent large-scale phenotypic screens of the S. cerevisiae deletant collection have included selections for sensitivity to oxidative stress, brefeldin A and monesin, caffeine, Calcofluor white and bud site selection pattern (Higgins et al., 2002; Muren et al., 2001; Ni and Snyder, 2001). Automated microtitre plate-based screening increases the number of phenotypes that can be scored (Rieger et al., 1997, 1999). Although we have already reported the preliminary results of manually screening the haploid deletant mutant collection for growth inhibition by 1 mm sorbate (Schüller et al., 2004), we perceived the need to conduct a much more thorough robotic screen: (a) using the library of homozygous diploid mutants (in view of the possibility that the phenotypes of few of the strains in the haploid mutant collection might be due to second-site kanmx4 cassette integrations); and (b) screening for both increased sensitivity and increased resistance to sorbate (see Methods). Before these sorbate effects on the arrays of deletant mutants could be screened, the effects of the moderately low ph alone had first to be examined. Only 17 deletion strains were clearly compromised in growth at ph 4.5, as compared to ph 6.8 (Table 1). Nevertheless, previous studies appear not to have identified this low ph sensitivity associated with loss of the Sfl1p transcription factor; with loss of Sti1p (a Hsp90 system co-chaperone), or with the loss of Ssn8p cyclin and its associated cyclin-dependent kinase (Ssn3p). Strains sensitive or resistant to sorbate at ph 4.5 Gene losses that render cells of BY4743 genetic background sensitive to 2 mm sorbate at ph 4.5 are listed in Table 2, while those generating a resistance to 5 mm sorbate at this ph are listed in Table 3. Of the 4794 homozygous diploid mutants tested, 237 ( 5% of the total) were sorbatesensitive (Table 2), while a further 34 (1.2% of those screened) were more sorbate-resistant than the wild-type (Table 3). Of these, 20% and 39%, respectively, represent genes of unknown function. The number of non-essential, non-redundant genes whose loss leads to sorbate sensitivity is considerably greater than the number showing an appreciable induction by sorbic acid (de Nobel et al., 2001; Schüller et al., 2004). This is not surprising, since some genes that contribute to sorbate resistance are known not to be upregulated by sorbate (e.g. WAR1 ; Kren et al., 2003). There is, in addition, relatively little correspondence between the gene losses that generate sensitivity or resistance to sorbic acid and changes in the transcriptome that occur in response to this stress (de Nobel et al., 2001; Schüller et al., 2004). It should be noted, though, that the phenotypic screens were conducted in a TRP + genetic background, whereas the strains used in the transcript profiling studies (FY c; W303) are both trp1 mutants and therefore sensitized to weak acid stress (Bauer et al., 2003). We have found that certain mutations exert very dissimilar effects on weak acid resistance in the Table 1. Gene losses resulting in sensitivity to YPD growth at ph 4.5, relative to ph 6.8 Gene ORF Product BUD23 YCR047c S-adenosylmethionine-dependent methyltransferase GAL11 YOL051w RNA polymerase II holoenzyme complex component positive and negative transcriptional regulator of genes involved in mating-type specialization GRR1 YJR090c F box protein; ubiquitin-dependent protein catabolism HIR2 YOR038c Transcription co-repressor MAP1 YLR244c Methionine aminopeptidase RTS1 YOR014w Protein phosphatase 2A (PP2A) B-type regulatory subunit SFL1 YOR140w Transcription factor; required for cell surface assembly and flocculence SSN3 YPL042c Cyclin (SSN8)-dependent serine/threonine protein kinase SSN8 YNL025c C-type cyclin; associates with the Ssn3p cyclin-dependent kinase STI1 YOR027w Co-chaperone; interacts with Hsp70 and Hsp90 VPS34 YLR240w PtdIns 3-kinase APT1 YLR118c Acyl protein thioesterase Unknown function: YOL002c (PHO36); YHL014c; YDR042c; YNR014w; YOR050c

5 S. cerevisiae mutants sensitive or resistant to sorbic acid 931 Table 2. Gene losses resulting in sensitivity to YPD plus 2 mm sorbate ph 4.5 Gene ORF Product SENSITIVE MUTANTS INDICATING THE IMPORTANCE OF MAINTAINING GLYCOLYTIC FLUX, THE PENTOSE PHOSPHATE PATHWAY, ANTIOXIDANT PROTECTION AND MITOCHONDRIAL INTEGRITY (i) Glycolysis, pentose phosphate pathway and glucose sensing PFK1 YGR240c Phosphofructokinase α-subunit PFK2 TMR205c Phosphofructokinase β-subunit TPS2 YDR074w Trehalose-6-phosphate phosphatase GND1 YHR183w 6-Phosphogluconate dehydrogenase GSF2 YMLO48w Glucose signalling factor RPE1 YJL121c D-Ribulose-5-phosphate 3-epimerase SNF7 YLRO25w Involved in derepression of SUC2 in response to glucose limitation TDH3 YGR192c Glyceraldehyde-3-phosphate dehydrogenase, isozyme 3 (ii) Antioxidant protection CYS3 YAL012w Cystathionine γ -lyase, cysteine biosynthesis CYS4 YGR155w Cystathionine β-synthase, cysteine biosynthesis GRX5 YPL059w Mitochondrial glutaredoxin GSH1 YJL101C γ -Glutamylcysteine synthetase GSH2 YOL049w Glutathione synthetase SOD1 YJR104c Cu, Zn superoxide dismutase (iii) Mitochondrial metabolism; mitochondrial organization and biogenesis COX9 YDL067c Cytochrome c oxidase subunit ETR1 YBR026c Mitochondrial 2-enoyl thioester reductase; fatty acid synthesis OAR1 YKL055c 3-Oxoacyl-[acyl-carrier-protein] reductase; fatty acid synthesis LPD1 YFL018c Dihydrolipoamide dehydrogenase component of pyruvate dehydrogenase complex PDX1 YGR193c Pyruvate dehydrogenase complex protein X component MDJ1 YFL016c Co-chaperone involved in mitochondrial biogenesis and protein folding MDM20 YOL076w Mitochondrial inheritance; cytoskeletal regulator MDM31 YHR194w Mitochondrial distribution and morphology MDM39 YGL020c Mitochondrial distribution and morphology MRF1 YGL143c Mitochondrial polypeptide chain release factor MRPL39 YML009c Mitochondrial ribosomal protein MRPL27 YBR282w Mitochondrial ribosomal protein IMG2 YCR046c Mitochondrial ribosomal protein MSW1 YDR268w Mitochondrial tryptophanyl-trna synthetase MTF1 YMR228w Mitochondrial RNA polymerase specificity factor OXA1 YER154w Mediates export of proteins from the mitochondrial matrix to the intermembrane space OCT1 YKL134c Octapeptidyl amino-peptidase; processes proteins after mitochondrial import PET8 YML003c S-Adenosylmethionine transporter of the mitochondrial inner membrane POR1 YNL055c Outer mitochondrial membrane porin (voltage-dependent anion channel) RIM1 YCR028c Single-stranded DNA-binding protein; essential for mitochondrial genome maintenance SENSITIVE MUTANTS AFFECTED IN RIBOSOME BIOGENESIS; MRNA TURNOVER; PROTEIN TRANSLATION AND PROTEIN FOLDING RPL7A YPL266w Ribosomal protein L7A RPL39 YJL189w Ribosomal protein L39 RPS4A YJR145c Ribosomal protein S4A RPS6A YPL090c Ribosomal protein S6A RPS27B YHR021c Ribosomal protein S27B RPS29B YDL061c Ribosomal protein S29B RAI1 YGL246c Processing of 27S pre-rrna DIM1 YPL266w Dimethyladenosine transferase; rrna modification FUN12 YAL035w Translation initiation; promotes Met-tRNA Met i binding to ribosomes EAP1 YKL204w Translation initiation factor eif-4e associated protein MRT4 YKL009w Ribosome large subunit biogenesis; mrna turnover DBP7 YKL024c RNA helicase; pre-rrna processing and ribosome large subunit biogenesis YDJ1 YNL064c Co-chaperone of Hsp70/Hsp90 system CPR7 YJR032w Co-chaperone; cyclophilin 40 peptidyl-prolyl cis-trans isomerase

6 932 M. Mollapour et al. Table 2. Continued Gene ORF Product GIM5 YML094w Chaperone involved in tubulin folding ZUO1 YGR285c Chaperone; putative Z-DNA binding activity SENSITIVE MUTANTS AFFECTED IN TRANSCRIPTION, CHROMATIN REMODELLING AND NUCLEAR TARGETING (i) Regulators of RNA polymerase II CAF17 YJR122w Component of CCR4 transcriptional complex CTK1 YKL139w Subunit of kinase that phosphorylates the CTD of RNA polymerase II large subunit to affect both transcription and pre-mrna 3 end processing CTK3 YML112w Subunit kinase that phosphorylates the CTD of RNA polymerase II large subunit to affect both transcription and pre-mrna 3 end processing DEF1 YKLO54w RNA polymerase II degradation factor 1; forms a complex with Rad26p to enable ubiquitination and proteolysis of RNA polymerase II ELA1 YNL230w Elongin A; elongation factor for RNA polymerase II GAL11 YOL051w Component of Gal11p Pgd1p Sin4p complex regulator of RNA polymerase II PAF1 YBR279w RNA polymerase II-associated protein that may serve as both a positive and negative gene regulator, perhaps operating in parallel with Gal11p MET18 YIL128w Required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription; possible role in assembly of multiprotein complex(es) required for NER and RNAP II transcription NOT5 YPR072w Component of CCR4 complex, a global negative regulator of transcription POP2 YNR052c Component of CCR4 complex; nuclease component of yeast mrna deadenylase RLR1 YNL139c Role in transcription elongation by RNA polymerase II RPB4 YJL140w RNA polymerase II core subunit ROX3 YBL093c Subunit of RNA polymerase II holoenzyme/mediator complex SIN4 YNL236w Component of Gal11p Pgd1p Sin4p RNA polymerase II holoenzyme/mediator complex SRB2 YHR041c Subunit of RNA polymerase II holoenzyme/mediator complex SRB5 YGR104c Subunit of RNA polymerase II holoenzyme/mediator complex PGD1 YGL025c Component of Gal11p Pgd1p Sin4p RNA polymerase II holoenzyme/mediator complex POG1 YIL122w Probable transcription factor SPT4 YGR063c With Spt5p forms a complex that mediates both activation and inhibition of transcription elongation; Spt4p-Spt5p also plays a role in pre-mrna processing STB5 YHR178c Transcription factor WAR1 YML076c Transcription factor; mediates response to sorbate stress (ii) Chromatin remodelling BRE1 YDL074c E3 ubiquitin ligase for Rad6p, required for ubiquitination of histone H2B, recruitment of Rad6p to promoter chromatin and subsequent methylation of histone H3 HFI1 YPL254w Adaptor protein required for structural integrity of the SAGA complex, a histone acetyltransferase co-activator complex that is involved in global regulation of gene expression through acetylation and transcription functions NGG1 YDR176w Component of two transcriptional adaptor/histone acetyltransferase complexes, ADA and SAGA SPT20 YOL148c Component of histone acetyltransferase SAGA complex HMO1 YDR174w One of seven S. cerevisiae high mobility group (HMG-box) proteins SNF5 YBR289w Chromatin remodelling; subunit of Swi/Snf complex RSC2 YLR357w Component of RSC complex, which remodels the structure of chromatin NBP2 YDR162c Binds Nap1p, which is involved in nucleosome assembly (iii) Nuclear import and export NUP120 YKL057c Nuclear pore complex subunit NUP133 YKR082c Nuclear pore complex subunit NUP170 YBL079w Nuclear pore complex subunit NPL6 YMR091c Nuclear protein, possible role in nuclear protein import SAC3 YDR159w Nuclear pore component; involved in nuclear export of both mrna and protein SENSITIVE MUTANTS AFFECTED IN CYTOSKELETON, BUDDING AND CELL CYCLE (i) Organization of cytoskeleton; polarized growth and bud site selection; microtubules; spindle checkpoint; chromosome cohesion/segregation, exit from mitosis ARC18 YLR370c Arp2/3p complex subunit; structural constituent of cytoskeleton BUD3 YCL014w Cell cycle regulated protein required for axial bud formation; co-assembles with Bud4p at bud sites

7 S. cerevisiae mutants sensitive or resistant to sorbic acid 933 Table 2. Continued Gene ORF Product BUD31 YCR063w Random budding BUD25 YER014c Involved in bipolar budding PEA2 YER149c Localized with Spa2p to sites of polarized growth and required for efficient mating, bipolar budding and pheromone-induced shmoo formation BUR2 YLR226w Cyclin-dependent protein kinase; chromosome segregation, transcription BIM1 YER016w Bim1p and Kar9p together make up the cortical microtubule-capture site; delays exit from mitosis when the spindle is oriented abnormally BUB1 YGR188c Checkpoint kinase involved in permitting entry into mitosis depending upon the assembly state of microtubules BUB3 YOR026c Required for cell cycle arrest in response to loss of microtubule function DBF2 YGR092w Kinase required for late nuclear division; Cdc15p promotes exit from mitosis by directly switching on the kinase activity of Dbf2p DCC1 YCL016c Sister chromatid cohesion; loss leads to benomyl sensitivity NUM1 YDR150w Tubulin binding; may function in nuclear migration during mitosis and meiosis by affecting astral microtubule functions, perhaps by being involved in polymerization and stabilization of microtubules SPC72 YAL047c Protein of spindle pole bodies. Cells lacking Spc72 generate only very short and unstable astral microtubules TPM1 YNL079c Tropomyosin I; actin-binding protein that stabilizes actin filaments VRP1 YLR337c Verprolin; involved in cytoskeletal organization and cellular growth (ii) DNA replication RAD27 YKL113c 5 to 3 Exonuclease, 5 flap endonuclease; required for Okazaki fragment processing and long-patch base-excision repair RNR1 YER070w Ribonucleotide diphosphate reductase, large subunit RNR4 YGR180c Ribonucleotide diphosphate reductase, small subunit SENSITIVE MUTANTS AFFECTED IN PTDINS METABOLISM, VESICLE TRAFFICKING, VACUOLE FUNCTION, AUTOPHAGY AND ENDOCYTOSIS (i) Vacuolar H(+)-ATPase VMA4 YOR332w Vacuolar H(+)-ATPase V1 domain subunit E VMA5 YKL080w Vacuolar H(+)-ATPase V1 domain subunit C VMA7 YGR020c Vacuolar H(+)-ATPase V1 domain subunit F VMA8 YEL051c Vacuolar H(+)-ATPase V1 domain subunit D VMA13 YPR036w Vacuolar H(+)-ATPase V1 domain subunit H VMA21 YGR105w Involved in vacuolar H(+)-ATPase assembly or function VPH2 YKL119c Required for biogenesis of a functional vacuolar H(+) ATPase (ii) PtdIns metabolism ARG82 YDR173c Transcriptional regulator; phosphorylates Ins(1,4,5)P3 to Ins(1,4,5,6)P4 and then Ins(1,3,4,5,6)P5; also phosphorylates Ins(1,3,4,5,6)P5 to generate a mixture of diphosphorylated Ins polyphosphates IPK1 YDR315c Ins(1,3,4,5,6)P5-2-kinase KCS1 YDR017c InsP6 kinase; converts InsP6 to diphosphoinositol polyphosphates VPS34 YLR240w PtdIns 3-kinase SAC1 YKL212w PtdIns phosphatase (iii) Vacuolar protein sorting COG1 YGL223c Conserved oligomeric Golgi complex DOA4 YDR069c Deubiquitinating enzyme; vacuole biogenesis function ERV14 YGL054c Vacuolar protein sorting ERV29 YGR284c ER Golgi transport vesicle protein GTR1 YML121w Small GTPase (putative); Pho84p phosphate transporter function PEP12 YOR036w Integral membrane protein; located in endosome SSO2 YMR183c Syntaxin homologue (post-golgi t-snare); acts in late stages of secretion VID21 YDR359c Vacuolar import and degradation VPS1 YKR001c GTPase; probably required for membrane-protein retention in a late Golgi compartment VPS3 YDR495c Vacuolar sorting protein VPS16 YPL045w Vacuolar sorting protein VPS22 YPL002c Vacuolar sorting protein STP22 YCL008c Vacuolar protein sorting VPS24 YKL041w Vacuolar protein sorting

8 934 M. Mollapour et al. Table 2. Continued Gene ORF Product VPS25 YJR102c Vacuolar protein sorting VPS28 YPL065w Protein involved in transport of precursors for soluble vacuolar hydrolases from the late endosome to the vacuole VPS36 YLR417w Vacuolar protein sorting VPS45 YGL095c Vacuolar protein sorting VPS53 YJL029c Vacuolar protein sorting VPS65 YLR322w Vacuolar protein sorting VPS66 YPR139c Vacuolar protein sorting (iv) Autophagy and endocytosis CHC1 YGL206c Clathrin heavy chain RVS161 YCR009c Cytoskeletal protein binding; part of a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress AKR1 YDR264c Protein palmitoylation; required for endocytosis of pheromone receptors APG17 YLR423c Required for activation of Apg1 protein kinase; autophagy pathway PLASMA MEMBRANE PROTEINS AND LIPIDS (i) Plasma membrane activities BAP2 YBR068c Amino acid permease for leucine, valine, and isoleucine (putative) FET4 YMR319c Low affinity Fe 2+ transport protein PDR12 YPL058c Multidrug resistance transporter TRK1 YJL129c 180 kda high-affinity potassium transporter (ii) Synthesis and turnover of plasma membrane lipids ERG2 YMR202w C-8 sterol isomerase ERG4 YGL012c Sterol C-24 reductase ERG6 YML008c (24)-sterol C-methyltransferase ERG3 YLR056w C-5 sterol desaturase ERG24 YNL280c Sterol C-14 reductase ARV1 YLR242c Protein involved in sterol distribution ISC1 YER019w Phospholipase C; hydrolyses inositolphosphosphingolipids as well as sphingomyelin (spingolipid catabolism) CELL WALL BGL2 YGR282c Cell wall endo-β-1,3-glucanase CDC10 YCR002c Component of septin ring of the mother-bud neck; required for cytokinesis CWH36 YCL007c Unknown function in cell wall organization and biogenesis FKS1 YLR342w 1,3-β-D-glucan synthase GAS1 YMR307w Glycophospholipid-anchored surface 1,3-β-glucanosyltransferase KRE6 YPR159w β-1,6-glucan synthase MNN8 YEL036c Mannosyltransferase complex MNN10 YDR245w Mannosyltransferase complex MNN11 YJL183w Mannosyltransferase complex AMINO ACID BIOSYNTHESIS AAT2 YLR027c Aspartate aminotransferase ARO1 YDR127w 3-dehydroquinate synthase ARO2 YGL148w Chorismate synthase ARO7 YPR060c Chorismate mutase GLY1 YEL046c Threonine aldolase TRP1 YDR007w Phosphoribosylanthranilate isomerase TRP2 YER090w Anthranilate synthase component I TRP3 YKL211c Anthranilate synthase component II indole-3-phosphate TRP4 YDR354w Anthranilate phosphoribosyl transferase TRP5 YGL026c Tryptophan synthetase THR1 YHR025w Homoserine kinase THR4 YCR053w Threonine synthase

9 S. cerevisiae mutants sensitive or resistant to sorbic acid 935 Table 2. Continued Gene ORF Product MISCELLANEOUS FYV4 YHR059w Required for yeast viability on toxin exposure FYV5 YCLO58c Required for yeast viability on toxin exposure FYV6 YNL133c Required for yeast viability on toxin exposure FYV13 YGR160w Required for yeast viability on toxin exposure GRR1 YJR090c Component of the SCF ubiquitin ligase complex. Required for Cln1p and Cln2p degradation, involved in carbon catabolite repression, glucose-dependent divalent cation transport, high-affinity glucose transport, and morphogenesis HPR1 YDR183w Suppresses intrachromosomal recombination; possibly also involved in RNA elongation from polymerase II promoters and mrna nuclear export KEM1 YGL173c 5 3 exonuclease; plays a role in cytoplasmic mrna degradation; also possibly chromosome pairing and exchange MEI5 YPL121c Meiotic protein required for synapsis and meiotic recombination PDX3 YBR035c Pyridoxine (pyridoxiamine) phosphate oxidase; pyridoxal-5-phosphate salvage pathway PHM6 YDR281c Phosphate metabolism; transcription is regulated by PHO system PHO85 YPL031c Cyclin-dependent protein kinase; involved in phosphate and glycogen metabolism as well as cell cycle progression PTC1 YDL006w Serine threonine protein phosphatase SIT4 YDL047w Type 2A-related protein phosphatase; role in G 1 S transition of cell cycle PER1 YCR044c Protein processing in the ER RMD1 YER083c Required for meiotic nuclear division; functions in DNA replication and damage response SPF1 YEL031w P-type ATPase of ER membrane TEP1 YNL128w Protein tyrosine phosphatase TPD3 YAL016w Protein phosphatase 2A regulatory subunit A Unknown function: YBL058c (SHP1), YBR077c, YCL046w, YCR063w (BUD31), YCR331c, YDR008c, YDR149c, YDR161w, YEL044w (IES6), YEL045c, YER014c (BUD25), YFL013c (IES1), YGL127c (SOH1), YGL218w, YGL262w, YGR064w, YGR163w (GTR2), YGR262c (BUD32), YGR283c, YHR067w (RMD12), YHR162w, YJL046w, YJL075c (APQ13), YJL184w, YJL188c (BUD19), YJR118c (ILM1), YKL033c, YKL050c, YKL162c, YKL118w, YKL124w (SSH4), YKR007w, YLR235c, YLR338w, YLR426w, YLR428c, YLR431c, YLR435w (TSR2), YNL217w, YNL195c, YNL296w, YMR279c, YNL081c, YNL297c (MON2), YNR036c, YOL013w, YOL072w (BUD29), YPL024w (NCE4) A gene loss also sensitizing cells to growth at ph 4.5 in the absence of sorbic acid (Table 1). trp1 and TRP + genetic backgrounds (Bauer et al., 2003). Both below and in Tables 2 and 3, genes are categorized according to their literature and their annotated functions in SGD ( stanford.edu/saccharomyces/). This categorization is, to a certain extent, arbitrary. Thus, mutants in actin organization are mostly listed under mutants affected in cytoskeleton, budding and cell cycle, even though their defects also generate a defective mitochondrial morphology and inheritance (the mdm phenotype; Ni and Snyder, 2001), endocytosis and vesicle transport. In a similar way, antioxidant activities could conceivably be providing resistance by protecting a specific critical target [e.g. acting to prevent oxidative inactivation of the vacuolar H + -ATPase (Oluwatosin and Kane, 1997) that, it transpires from this work, is an important activity for sorbate resistance]. As previously reported (Bauer et al., 2003), all the mutants in the collection defective in the biosynthesis of aromatic amino acids were sorbate-sensitive (Table 2). These are not discussed further in this report. Mutants indicating that maintaining glycolytic flux, the pentose phosphate pathway, antioxidant protection andmitochondrial integrity are important for sorbate resistance The importance of Pfk for sorbate resistance was shown in an earlier study (Cheng et al., 1999) and confirmed with the identification of the pfk1 /pfk1 and pfk2 /pfk2 mutants as sorbatesensitive in this screen (Table 2). Overcoming the inhibition of glycolysis at the Pfk reaction is probably extremely important for weak acid adaptation (for literature, see Pearce et al., 2001; Piper et al., 2001). This Pfk inhibition leads, in turn, to a trehalose accumulation in sorbate-treated S. cerevisiae (Cheng et al., 1999). The tps2 /tps2 mutant is probably sensitive because Pfk inhibition

10 936 M. Mollapour et al. Table 3. Gene losses resulting in resistance to 5 mm sorbate at ph 4.5 Gene ORF Description GLYCOLYSIS AND GLUCOSE SENSING TPK2 YPL203W camp-dependent protein kinase catalytic subunit MITOCHONDRIAL CCE1 YKL011C Mitochondrial cruciform cutting endonuclease MCX1 YBR227C Mitochondrial ATP-binding protein, similar to ClpX MDH1 YKL085W Mitochondrial malate dehydrogenase RIBOSOME BIOGENESIS; TRANSLATION RPL22A YLR061W Ribosomal protein L22A RPL37B YDR500C Ribosomal protein L37B RPL41A YDL184C Ribosomal protein L41A RPS1B YML063W Ribosomal protein S1B DTD1 YDL219W D-Tyr-tRNA(Tyr) deacylase; functions in protein translation TRANSCRIPTION; CHROMATIN REMODELLING CAF40 YNL288W CCR4 (carbon catabolite repression)-associated factor UGA3 YDL170W Zinc finger transcriptional activator; nitrogen utilization YAP5 YIR018W bzip (basic-leucine zipper) transcription factor CELL CYCLE CLB3 YDL155W B-type cyclin. Involved in DNA replication during S phase CLB5 YPR120C B-type cyclin. Involved in DNA replication during S phase VACUOLE FUNCTION; VESICLE TRAFFICKING GVP36 YIL041W Golgi-vesicle protein of 36 kda SYN8 YAL014C SNARE protein related to mammalian SYNtaxin 8, ULP1 interacting protein 2 VID28 YIL017C Vacuole import and degradation VTA1 YLR181C Class E vacuolar-protein sorting YPT52 YKR014C Rab5-like GTPase involved in vacuolar protein sorting and endocytosis ER AND THE RESPONSE TO ER STRESS CCH1 YGR217W Plasma membrane calcium channel MID1 YNL291C N-glycosylated plasma membrane protein involved in calcium influx DER1 YBR201W Endoplasmic reticulum membrane protein, required for the protein degradation process associated with the ER, involved in the retrograde transport of misfolded or unassembled proteins HRD3 YLR207W Component of HRD complex responsible for the ER-associated degradation (ERAD) of numerous ER-resident proteins CELL WALL ECM7 YLR443W Cell wall maintenance ECM29 YHL030W Cell wall maintenance MISCELLANEOUS IKS1 YJL057C Serine threonine kinase (putative). Ira1 kinase suppressor PPG1 YNR032W Serine threonine protein phosphatase involved in glycogen accumulation PRO1 YDR300C γ -Glutamyl kinase USA1 YML029W Pre-mRNA splicing factor (putative) WHI2 YOR043W Phosphatase activator involved in growth regulation; Whi2p and its binding partner, Psr1p-phosphatase, are required for a full activation of the general stress response, through dephosphorylation of Msn2/4p SLX9 YGR081c Synthetic fitness effect with loss of Sgs1p GPP1 YIL053W DL-glycerol-3-phosphatase; glycerol biosynthesis AQY2 YLL052C Aquaporin (putative) IML3 YBR107C Increased minichromosome loss Unknown function: YBR134w, YBR141c, YBR144c, YBR157c (ICS2), YDL094c, YDL157c, YDL177c, YDR018c, YGR268c (HUA1), YHR009c, YHR016c (YSC84), YIL029c, YIL077c, YIL110w, YJR107w, YLR062c (BUD28), YDL151c (BUD30), YLR072w, YLR211c, YLR444c, YNR063w, YPL071c

11 S. cerevisiae mutants sensitive or resistant to sorbic acid 937 in this strain will cause intracellular accumulation of a large, potentially cytotoxic pool of trehalose- 6-phosphate. Sorbate sensitivity was apparent in tdh3 /tdh3, a mutant that is oxidative stress-sensitive (Grant et al., 1999) and that lacks one isoform of glyceraldehyde-3-phosphate dehydrogenase. This oxidoreductase enzyme is one of the proteins most strongly induced by sorbic acid (de Nobel et al., 2001). It may be important, not just for sustaining the high glycolytic flux of weak acid-stressed cells, but also for providing reducing power (NADH) in these cells with their severely compromised mitochondrial function (Piper et al., 2001). The importance of maintaining a reducing environment in sorbate-stressed cells is highlighted by other gene losses causing sensitivity. Thus sensitivity is associated with defects in the pentose phosphate pathway (gnd1 /gnd1 ; rpe1 /rpe1 ); pyruvate dehydrogenase (pdx1 /pdx1 ; lpd1 / lpd1 ); and in glutathione (GSH; γ -L-glutamyl- L-cysteinylglycine) biosynthesis (gsh1 /gsh1 ; gsh2 /gsh2 ). GSH provides important protection against both oxidative stress (Grant, 2001) and sorbate stress (Table 2). One presumes that cys3 /cys3 and cys4 /cys4 (defective in the synthesis of cysteine, needed for GSH production from homocysteine) are sorbate-sensitive because of their low GSH levels (Oluwatosin and Kane, 1997). Curiously, gpp1 /gpp1, lacking glycerol-3- phosphatase, exhibited an elevated sorbate resistance (Table 3). This possibly reflects the reactions of glycerol synthesis (involving the consumption of NADH) acting to the detriment of sustaining a reducing environment in sorbate-stressed cells. Similarly, it is interesting to note an increased resistance in mdh1 /mdh1 (Table 3) lacking the mitochondrial malate dehydrogenase (important for the malate aspartate shuttle that is responsible for introducing reducing equivalents from cytosolic NADH to the electron transport chain of mitochondria). Is this gene loss leading to a shutting down of respiratory activity, therefore less oxidative stress in sorbate-stressed cells? Another sensitive mutant was grx5 /grx5, lacking the Grx5p mitochondrial glutaredoxin (Table 2). Grx5p is important for antioxidant protection, its loss severely impairing the synthesis and assembly of iron sulphur (Fe S) proteins (Rodriguez-Manzaneque et al., 1999). In aerobic S. cerevisiae a major cause of sorbateand benzoate-sensitivity is an elevated superoxide production by the mitochondrial respiratory chain (see Introduction). Even though the loss of an assembled respiratory chain will eliminate the major source of this free radical production, respiratory-deficient petites are in general very sensitive to oxidative stress, since they cannot mount a proper response to oxidants (Jamieson, 1998; Moradas-Ferreira and Costa, 2000). It is noteworthy that nuclear genes for mitochondrial components whose loss generated sorbate sensitivity were (with the exception of cox9 /cox9 ) not those encoding respiratory chain components, but genes associated with the mitochondrial transcription/translation machinery, mitochondrial integrity and/or mitochondrial inheritance (Table 2). Thus, our screen identified an increased sensitivity in three mdm mutants; in mdj1 /mdj1 lacking a mitochondrial co-chaperonin (protein folding); as well as with losses of components of the mitochondrial transcription and translation machinery (msw1 /msw1 ; mtf1 /mtf1 ; rim1 /rim1 and ypl183w-a /ypl183w-a ). Most of the latter mutants are respiration-defectives, although mdm20 /mdm20 (defective in actin organization; Ni and Snyder, 2001) and mdm31 /mdm31 ; mdm39 /mdm39 are respiration-competent, but exhibit defects in mitochondrial morphology (Dimmer et al., 2002). Mutants indicating a role for ribosome biogenesis, translation and protein folding in sorbate resistance The small and large subunits of the cytosolic ribosomes of S. cerevisiae have 32 and 46 proteins, respectively. Many of these ribosomal proteins are encoded by duplicated genes, resulting in a total genome complement of 137 ribosomal protein (rp) genes (Planta and Mager, 1998). Remarkably, some of these duplicated rp genes influence sorbic acid resistance, since losses of individual rp genes can cause either a decreased (rps4a /rps4a ; rps6a /rps6a ; rps27b /rps27b ; rps29b /rps29b ; rpl7a / rpl7a ) oranincreased resistance (rps1b /rps1b; rpl22a / rpl22a ; rpl37b /rpl37b ; rpl41a, rpl41a ) (Tables 2 and 3). It is noteworthy that none of the strains lacking any of the homologues of these genes were identified as having altered sorbate resistance.

12 938 M. Mollapour et al. Other phenotypes have been identified associated with losses of individual members of duplicated rp gene pairs in S. cerevisiae (e.g. reduced growth rates and random budding patterns; Ni and Snyder, 2001). It appears, therefore, that certain paralogous rp gene copies have evolved separately following the ancestral duplication of the S. cerevisiae genome (Wolfe and Shields, 1997), since in several instances the two respective gene copies (although encoding ribosomal proteins that are generally >90% identical in sequence) are not equivalent in function. In certain cases the phenotype may result from inefficient synthesis of the relevant ribosomal protein (e.g. rps27b /rps27b ; rpl7a /rpl7a (sorbate-sensitive) and rpl22a /rpl22a (sorbateresistant) all display reduced growth rates; Ni and Snyder, 2001). Nevertheless, the observation that single gene losses within at least 9 rp gene pairs can be associated with either decreased or increased sorbate resistance merits further study. It suggests the intriguing possibility that subtle alterations to the protein composition of the ribosome might be important for adaptation to sorbic acid (even though transcript analysis has so far failed to reveal any major change in the expression of the above rp genes with sorbate stress; de Nobel et al., 2001). Certain of the sorbate-sensitive mutants in Table 2 correspond to defects in ribosome maturation (rai1 /rai1 ; dim1 /dim1 ; dbp7 /dbp7 ), translation initiation (eap1 /eap1 ; fun12 /fun 12 ) or non-essential co-chaperones (ydj1 /ydj1 ; cpr7 /cpr7 ; gim5 /gim5 ; zuo1 /zuo1 ). The latter co-chaperone defects all generate a general stress sensitivity and slow growth (Caplan and Douglas, 1991; Duina et al., 1996; Yan et al., 1998). Mutants indicating the importance of transcriptional components, chromatin remodelling and nuclear targeting in sorbate resistance RNA polymerase II regulators The screen identified a number of mutants that lack components of the RNA polymerase II transcription machinery as being sorbate-sensitive (Table 2), as well as three that exhibited an increased sorbate resistance (caf40 /caf40 ; uga3 /uga3 and yap5 /yap5 ; Table 3). Of the former, only war1 /war1 was defective in Pdr12p induction (Kren et al., 2003, and data not shown). Sensitivity was apparent with the loss of Gal11p, Sin4p and Pgd1p, auxiliary transcription factors that (as the Gal11p Sin4p Pgd1p subcomplex component of the RNA polymerase II holoenzyme) regulate the expression of several genes (see Nishizawa, 2001, for literature). Gal11p is generally a positive regulator and Sin4p a negative regulator, with Pgd1p mediating the interaction between Gal11p and Sin4p. Sensitivity was also apparent with the loss of components of CCR4, another transcriptional complex exerting both positive and negative effects on transcription (Liu et al., 1998). CCR4 is required for the transcription of several genes involved in non-fermentative growth and cell wall structure (see Chang et al., 1999; Kaeberlein and Guarente, 2002; Liu et al., 1998, for literature). Although increased sensitivity was apparent with the loss of certain CCR4 components [Not5p, Pop2p, Dbf2p (a protein kinase) and Caf17p], an increased resistance was found with loss of the CCR4-associated factor Caf40p. Several mutants lacking RNA polymerase II subunits, or altered in the modification of the RNA polymerase II large subunit C-terminal domain (CTD), were sorbate-sensitive (Table 2). Ctk1p and Ctk3p are subunits of a protein kinase that phosphorylates the CTD (Lee and Greenleaf, 1997). Their loss causes defects in both transcriptional repression and activation (Kuchin and Carlson, 1998; Ni and Snyder, 2001). PDR12 LacZ induction by sorbate was reduced in both ctk1 /ctk1 and ctk3 /ctk3, but Western blot analysis showed that these CTD-kinase-deficient mutants were still displaying sorbate induction of Pdr12p (Figure 1). YAP5 encodes the Yap5p transcription factor, one of eight bzip proteins encoded by the S. cerevisiae genome (Fernandes et al., 1997). The screen identified yap5 /yap5 as being more resistant to sorbate (Table 3). As shown in Figure 2a,b, this enhanced weak acid resistance of yap5 cells was also evidence during agar growth and appeared to be more apparent on benzoate-containing, rather than sorbate-containing, ph 4.5 plates (certain other yap mutants also displaying a slightlyincreased benzoate resistance; Figure 2b). Unexpected was the finding that the Pdr12p induction by sorbate, and especially benzoate, was reduced in yap5 /yap5 cells (Figure 2c). We had previously found that a strain engineered for GAL1 promoterdirected PDR12 expression exhibits higher Pdr12p levels when grown on galactose, but slightly lower

13 S. cerevisiae mutants sensitive or resistant to sorbic acid 939 nup mutations affect the nuclear localization of the War1p transcription factor (Kren et al., 2003). Figure 1. Analyses of basal and sorbate-induced PDR12 LacZ reporter gene expression (open and shaded bars, respectively; upper diagram); also total Pdr12p level (Western blot analysis; lower diagram) in various protein kinase mutants. Measurements were on 30 C, ph 4.5 cultures of the indicated mutants, before ( ) and1hafter(+) addition of 2 mm sorbate resistances to sorbate (Schüller et al., 2004) and to propionate (Hatzixanthis et al., 2003), as compared to the wild-type cells, where the PDR12 gene is under the normal regulation of War1p. This is an indication that only a moderate Pdr12p induction is needed for an optimal resistance to these moderately lipophilic acids and that still higher levels of Pdr12p induction are actually slightly detrimental for this resistance. YAP5 is a target of the SBF (Swi4 Swi6 cell cycle box binding) transcription factor (Horak et al., 2002). Its actions as a modulator of weak acid resistance and a possible co-regulator of Pdr12p expression (Figure 2) are currently the subject of further study. Nuclear import and export Macromolecular transport between the nucleus and cytoplasm occurs through the nuclear pore complexes (NPCs) of the nuclear envelope, the latter composed of 30 different nucleoporins or nups. Our screen identified nup120 /nup120 ; nup133 /nup133 and nup170 /nup170 as sorbate-sensitive (Table 2). Since nup mutations are frequently associated with defects in nuclear import or export (Elion, 2002; Komeili and O Shea, 2001), we are currently investigating whether these three Mutants indicating sensitivity is associated with defects in the cytoskeleton, polarized growth and bud site selection and the cell cycle As shown in Table 2, sorbate sensitivity is associated with several defects in actin organization (arc18 /arc18 ; pea2 /pea2 ; trm1/trm1 ; vrp1 /vrp1 ), as well as with defects in polarized growth and bud site selection [the latter the bud (random budding) mutants]. The actin cytoskeleton is essential for polarized growth and bud site selection (for literature, see Goode et al., 2000; Ni and Snyder, 2001). Sorbate sensitivity is also associated with several defects in microtubule organization and biogenesis (bim1 /bim1 ; num1 /num1 ; spc72 /spc72 ); as well as with defects in the spindle checkpoint, chromosome cohesion/segregation, and the exit from mitosis. The sorbate sensitivity that characterizes a number of spindle checkpoint defects (bim1 /bim1, bub1 /bub1 and bub3 /bub3 ; Table 2) is an indication that a large influx of the weak acid into dividing cells with sudden exposure to sorbate might cause severe disruption of the events of spindle assembly, chromosome segregation and cytokinesis. Survival of the acid stress may therefore require such cells to undergo a cell cycle arrest at the spindle checkpoint. A few other cell cycle mutants (dbf2 /dbf2, pho85 /pho85 ) showed sorbate sensitivity (Table 2). In contrast, single losses of two of the B-type cyclins present during mitosis in budding yeast, Clb3p or Clb5p, generated an increased resistance to sorbate stress (Table 3). The weak acid sensitivity associated with defects in the ribonucleotide diphosphate reductase (RNR) complex is consistent with the general stress sensitivity of rnr mutants (Wang et al., 1997). Mutants indicating sorbate sensitivity results from defects in vacuole function, PI metabolism, endocytosis and autophagy Several sorbate-sensitive mutants indicated the importance of both an acidified vacuole and an intact endosomal sorting pathway in weak acid resistance. Thus sensitivity was found to result from:

14 940 M. Mollapour et al. Figure 2. (a) Growth of BY4741 (wt) and different yap mutants of BY4741 genetic background in the presence of the indicated level of sorbate or benzoate. An undiluted overnight culture, also 1 : 10 and 1 : 100 serial dilutions, were pinned onto solid ph4.5 YPD and the plates photographed after 3 days at 30 C. (b) Western blot analysis of the sorbate and benzoate induction of Pdr12p in wild-type (BY4741; wt) and yap5 cells 1. Defects in vacuolar H + -ATPase. Mutants defective in the vacuolar membrane H + -ATPase (vma) are markedly sorbate-sensitive, indicating that this enzyme is critically important for weak acid resistance. Vacuolar H + -ATPase maintains the acidity of the vacuole (ph 6.2) and generates the electrogenic potential that is used to drive secondary transport of many small molecules

15 S. cerevisiae mutants sensitive or resistant to sorbic acid 941 into this organelle (cations such as Ca ++, Mg ++, Zn ++, Fe ++, Na +, phosphate, amino acids, etc.). The vacuole contains the major pool of intracellular Ca ++ (Ohya et al., 1991) and acts as a major site of GSH storage/degradation and of bulk turnover of many cytosolic components via autophagy (Abeliovich and Klionsky, 2001). vma mutants also exhibit a lowered level of plasma membrane H + -ATPase (Perzov et al., 2000), another activity important for acid resistance (Holyoak et al., 1996; Piper et al., 2001). 2. Defects in phosphoinositide metabolism: (arg82 /arg82, ipk1 /ipk1, kcs1 /kcs1, sac1 /sac1 ). Inositol pyrophosphates regulate both endocytic trafficking (Saiardi et al., 2000, 2002) and mrna export from the nucleus (York et al., 1999). Arg82p phosphorylates Ins(1,4,5)P3 to Ins(1,4,5,6)P4 and then Ins(1,3,4,5,6)P5. It also phosphorylates Ins(1,3,4,5,6)P5 so as to generate a mixture of diphosphoinositol polyphosphates (Zhang et al., 2001). Ipk1p, in turn, phosphorylates Ins(1,3,4,5,6)P5 to InsP6 and is nuclear in location (see Wera et al., 2001, for literature). The sorbate-sensitive kcs1 /kcs1 mutant exhibits slow growth, has diphosphoinositol polyphosphate levels 60 80% lower than wild-type cells, and possesses abnormally small and fragmented vacuoles (Saiardi et al., 2000). InsP6 accumulates under stress conditions in Sz. pombe (Ongusaha et al., 1998) and a precedent exists for the direct modulation of vesicle movement through InsP6 binding (InsP6 binding to mammalian synaptotagmin may alter its interaction with the AP2 clathrin assembly protein and thereby inhibit synaptic vesicle trafficking (Voglmaier et al., 1992). Sorbate sensitivity was also associated with the loss of Sac1p, a phosphoinositol phosphatase located at the endoplasmic reticulum and at the Golgi (see Wera et al., 2001, for literature). 3. Defects in vacuolar protein sorting. Several vacuolar protein sorting (vps) mutants were sorbate-sensitive. This is probably just one manifestation of the general stress sensitivity that characterizes vps mutants (Shimoni and Schekman, 2002). vps3, 34, 45 and 53 are class D vps mutants, characterized by an inability to generate a ph gradient across the vacuolar membrane (Raymond et al., 1992)(a defect also apparent in vma mutants; see above). vps24, 25, and 28 (class E vps mutants) accumulate an exaggerated form of a pre-vacuolar compartment, such that their 60 kda vacuolar H + -ATPase subunit is sequestered in a separate compartment from the vacuole; Raymond et al., 1992). Certain class E VPS gene products are proposed to form three distinct endosomal sorting complexes (ESCRT-I,II,III), required for the sorting of ubiquitinated cargo into the multivesicular bodies (MVB) prior to transport to the vacuolar lumen (Babst et al., 2002a, 2002b). ESCRT-I (Vps23/28/37p) acts in concert with Vps27 to initiate the sorting of ubiquitinated cargo at the late endosome/mvb (Katzmann et al., 2001). ESCRT-II (Vps22/25/26p) and ESCRT-III (Vps32/20p and Vps2/24p) direct the sorting of ubiquitinated cargo into inwardbudding vesicles, the ubiquitin being released by the actions of Doa4p, a deubiquitinating enzyme associated with the removal of ubiquitin from proteins of the late endosome/mvb. Although Doa4p is not specifically required for vesicle formation, it does help to maintain cellular levels of free ubiquitin by reducing the vacuolar degradation of ubiquitin (several class E VPS genes being extragenic suppressors of doa4-1 ; Amerik et al., 2000). It is noteworthy that losses of components of all three ESCRT complexes can generate sorbate sensitivity (Table 2); also that doa4 /doa4 is the only mutant of the ubiquitination system for intracellular protein degradation (with the possible exception of mutants lacking the F-box proteins Def1p and Grr1p) that we found to be sorbatesensitive. The importance of endocytosis for sorbate resistance is also indicated from the sensitivity of akr1 /akr1 and chc1 /chc1 (the latter lacking the clathrin required for coated pit formation); also cog1 /cog1, erv14 /erv14 and erv29 /erv29 (defective in sorting of vacuolar proteins from the cell surface or from a late Golgi compartment; Conboy and Cyert, 2000). A requirement for another vacuole-associated function, the ability to catalyse autophagy, is indicated by the sensitivity of apg17 /apg17. Finally, consideration must also be given to the possibility that the sorbate sensitivity that is associated with several defects in the actin cytoskeleton (see above) might be due to the influences that such defects will exert on the events of vesicle transport.

16 942 M. Mollapour et al. Many of the more severe vps mutants, such as vps1 /vps1 and pep12 /pep12 (both sorbatesensitive; Table 2), missort proteins both in the endosomal system and at other locations (e.g. the Golgi; Shimoni and Schekman, 2002). The endocytic and secretory systems are intricately connected, one branch of the exocytic pathway appearing to transit through endosomes before reaching the cell surface (Harsay and Schekman, 2002). Thus, Kex2p, the protease required for mating factor processing, is no longer in the Golgi in vps1 and class E vps mutants, but instead degraded in vacuoles or a vacuole-like compartment (Spelbrink and Nothwehr, 1999). Finally, some plasma membrane proteins are actually recycling (even though their steady state is on the plasma membrane). Mutations such as vps1 /vps1, vps45 /vps45 and vps53 /vps53 will interfere with such recycling, with the result that several of these vps mutants might display an altered plasma membrane level of Pdr12p and Pma1p (both of which are important activities for sorbate resistance; Piper et al., 2001). Mutants indicating sensitivity associated with defects in plasma membrane protein and lipid composition; also the response to ER stress In addition to Pdr12p, a number of plasma membrane activities contribute to sorbate resistance. Mutants with a reduced plasma membrane H + - ATPase activity are sorbate-sensitive (Holyoak et al., 1996), weak acid stress stimulating the plasma membrane H + -ATPase catalysed efflux of protons from the cell (Holyoak et al., 1996; Piper et al., 1997; Viegas and Sa Correia, 1991). An important charge-balancing mechanism for this H + efflux may be K + influx, since sorbate sensitivity results from the loss of the Trk1p highaffinity K + uptake transporter (Bertl et al., 2003) in trk1 /trk1. Together Mid1p and Cch1p (the latter a homologue of the voltage-gated Ca 2+ channels found in the plasma membrane of electrically-excitable animal cells; Locke et al., 2000) form a plasma membrane channel that triggers Ca 2+ influx in the Ire1-independent response to endoplasmic reticulum (ER) stress (Bonilla and Cunningham, 2003). This Ca 2+ influx in turn activates calcineurin, which helps to prevent cell death during the multiple forms of ER stress (Bonilla and Cunningham, 2003). It appears that this process may be detrimental for sorbate resistance, since both cch1 /cch1 and mid1 /mid1 were found to display elevated resistance to this stress (Table 3). The increased resistance of the der1 /der1 and hrd3 /hrd3 mutants (Table 3) provides yet further evidence of a connection between ER stress and weak acid resistance. Sorbate sensitivity was apparent in mutants defective in the biosynthesis of membrane sterols (erg2 /erg2 ; erg3 /erg3 ; erg4 /erg4 ; erg6 /erg6 and erg24 /erg24 ; Table 2), mutants that exhibit a pleiotropic drug-sensitivity (Kaur and Bachhawat, 1999). Sorbate induction of Pdr12p in the haploid erg4 and erg5 mutants was unaltered relative to that of the BY4741 wildtype (data not shown), showing that the altered plasma membrane sterol composition of these erg mutants is not affecting the capacity of these cells to induce Pdr12p. Instead, this altered sterol composition may facilitate passive diffusion of the weak acid across the plasma membrane, thus seriously compromising any ability of the Pdr12pcatalysed acid anion efflux to lower intracellular levels of the acid (Piper et al., 1998). Studies on the erg6 mutant have revealed that loss of the Erg6p sterol methylase increases passive diffusion of several drugs across the plasma membrane, without compromising the ability of another ABC transporter (Pdr5p) to catalyse active drug export (Emter et al., 2002). It is probable, therefore, that the same defect facilitates sorbate entry, without affecting the ability of Pdr12p to catalyse the active extrusion of the sorbate anion. Mutants indicating sensitivity associated with cell wall defects The cell wall of S. cerevisiae functions to preserve the osmotic integrity of the cell and to sustain morphology during budding, post-mating development, sporulation and the formation of pseudohyphae (Horie and Isono, 2001). The major components of the cell wall are highly mannosylated proteins, ß-1,3- and ß-1,6-glucans that constitute the inner layer, and a small amount of chitin that is localized at the septum (Kapteyn et al., 1997). The large mannan structure on the cell wall proteins consists of a long backbone of up to 200 α-1,6-linked mannoses, to which are attached branches up to three mannoses in length, the

17 S. cerevisiae mutants sensitive or resistant to sorbic acid 943 Figure 3. The effects of 0.5 mm and 2 mm sorbate on the growth of various mannosyltransferase (mnn) mutant strains of SEY6210 genetic background (ph 4.5 YPD; 30 C, 3 days). The plates were supplemented with 50 mm tryptophan, a strategy shown to overcome the effects of the trp1 mutation sensitizing cells to weak acid stress (Bauer et al., 2003) first two of these mannoses being α-1,2-linked and the final one α-1,3-linked. Addition of the first and second of these branching mannoses is catalysed by Mnn2p and Mnn5p, respectively (Rayner and Munro, 1998). The α-1,3-linked mannose is, in turn, added by Mnn1p. Finally, phosphomannose is added to many of these termini by Mnn6p. Deficiencies in these different Golgi mannosyltransferases produces strains with different cell wall mannose morphologies, including truncated side-chains or cells with reduced phosphomannan content. The screen revealed sorbate sensitivity associated with a number of these mutants (mnn8 /mnn8, mnn10 /mnn10, mnn11 /mnn11 ); also with mutants of glucan synthesis (fks1 /fks1 and kre6 /kre6 ) and remodelling (bgl2 /bgl2 and gas1 /gas1 (the latter lacking a glycosidase that is thought to direct ß-1,6-glucans to cross-link to ß-1,3-glucans rather than to chitin; Vai et al., 1991). To further clarify the involvement of MNN genes in sorbate resistance we plated on solid medium an isogenic series of mnn mutants in the SEY6210 genetic background (Rayner and Munro, 1998). Of these, mnn1 and mnn6, lacking the enzymes that terminate the mannose chains in the outer branches of the mannan structure on cell wall proteins, were the most sorbate-sensitive (Figure 3). In contrast, mnn2 and mnn5, strains lacking the enzymes for α-1,2-linked mannose addition were slightly more sorbate resistant than the wildtype (Figure 3). It is clear, therefore, that different mnn mutants display either an increased or a decreased sorbate resistance, depending on their altered mannan content. Mannosylphosphorylation is induced under certain conditions of stress (Jigami and Odani, 1999), but whether it is induced during weak acid adaptation is unknown. It is a modification that may make the cell wall less permeable to the acid, since mnn6, a mutant lacking this modification, is sorbate-sensitive (Figure 3). Acknowledgements We thank Chris Grant and Sean Munro for providing mutant strains; also Ewald Hettema, Bettina Bauer, Yasmine Mamnun and Peter Coote for fruitful discussions and for sharing reagents. This work was supported by a BBSRC grant (31/D17849) to P.W.P. and a CASE Studentship supported