global screening of genes essential for growth in high ...tubes were subjected to high pressure of...

Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.083063

Global Screening of Genes Essential for Growth in High-Pressure andCold Environments: Searching for Basic Adaptive Strategies Using

a Yeast Deletion Library

Fumiyoshi Abe*,1 and Hiroaki Minegishi†

*Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology, Yokosuka 237-0061, Japan and†Department of Applied Chemistry, Faculty of Engineering, Toyo University, Kawagoe 350-0815, Japan

Manuscript received October 7, 2007Accepted for publication December 17, 2007

ABSTRACT

Microorganisms display an optimal temperature and hydrostatic pressure for growth. To establish themolecular basis of piezo- and psychroadaptation, we elucidated global genetic defects that give rise tosusceptibility to high pressure and low temperature in Saccharomyces cerevisiae. Here we present 80 genesincluding 71 genes responsible for high-pressure growth and 56 responsible for low-temperature growthwith a significant overlap of 47 genes. Numerous previously known cold-sensitive mutants exhibit markedhigh-pressure sensitivity. We identified critically important cellular functions: (i) amino acid biosynthesis,(ii) microautophagy and sorting of amino acid permease established by the exit from rapamycin-inducedgrowth arrest/Gap1 sorting in the endosome (EGO/GSE) complex, (iii) mitochondrial functions, (iv)membrane trafficking, (v) actin organization mediated by Drs2-Cdc50, and (vi) transcription regulated bythe Ccr4-Not complex. The loss of EGO/GSE complex resulted in a marked defect in amino acid uptakefollowing high-pressure and low-temperature incubation, suggesting its role in surface delivery of aminoacid permeases. Microautophagy and mitochondrial functions converge on glutamine homeostasis in thetarget of rapamycin (TOR) signaling pathway. The localization of actin requires numerous associatedproteins to be properly delivered by membrane trafficking. In this study, we offer a novel route to gaininginsights into cellular functions and the genetic network from growth properties of deletion mutants underhigh pressure and low temperature.

MICROORGANISMS display an optimal growthtemperature and an optimal growth hydrostaticpressure. However, the basis of these profiles and thelimiting factor on cell growth are poorly understood. Ithas been a conventional technique in yeast cell biologyto analyze gene functions using high temperature- andlow temperature (cold)-sensitive mutants, i.e., ts and cs,respectively. Nevertheless, little is known about why themutation or deletion of genes limits the ability to growunder adverse conditions except for certain types ofmutations. It is well-known that bacterial and yeastSaccharomyces cerevisiae mutants defective in ribosomeassembly are cold sensitive, probably because ribosomeassembly has high activation energy in the absence ofcertain subunits (Friedman et al. 1969; Bryant andSypherd 1974; Singh et al. 1978; Ursic and Davies1979). Some yeast mutants that have altered sensitivitiesto the antibiotics trichodermin and cycloheximide arealso cold sensitive (Moritz et al. 1991; Dresios et al.2000, 2001, 2003). A substitution of aspartic acid forleucine in actin at position 266 confers cold sensitivity at

temperatures between 9� and 15� due to polymerizationdefects of actin at low temperatures (Chen et al. 1993).Deletion mutants for DRS2 and CDC50 that encodeproteins localize to the late Golgi and endosomes aredefective in the endocytic pathway and in organization ofthe actin cytoskeleton at 15� or 18�, resulting in cold-sensitive growth (Moir et al. 1982; Chen et al. 1999; Misuet al. 2003; Natarajan et al. 2004; Saito et al. 2004; Chenet al. 2006), but it is unclear which step is blocked by lowtemperature. Deletion of LTE1 that encodes the Cdc25family guanine-nucleotide exchanging factor causes cellsto arrest at telophase at low temperature (Shirayamaet al. 1994a,b). The loss of Ccr4 and Pop2 that constitutesthe Ccr4-Not transcriptional regulator (Tucker et al.2001; Collart 2003; Denis and Chen 2003) causesmarked cold sensitivity, but the reason is unknown(Hata et al. 1998). Mutants defective in tryptophanbiosynthesis are cold sensitive due to decreased trypto-phan uptake at low temperature (Singh and Manney1974; Gaber et al. 1989; Chen et al. 1994). Although thecold sensitivity of these mutants is evident, their growthhas not been examined at high hydrostatic pressureexcept in our study on tryptophan uptake.

Increasing hydrostatic pressure has an effect analo-gous to decreasing temperature in terms of increasing

1Corresponding author: Extremobiosphere Research Center, JapanAgency for Marine-Earth Science and Technology (JAMSTEC), 2-15Natsushima-cho, Yokosuka 237-0061, Japan. E-mail: [email protected]

Genetics 178: 851–872 (February 2008)

order and decreasing the fluidity of biological mem-branes. We demonstrated that the uptake of tryptophanis a limiting factor for yeast cell growth at high pressureas well as at low temperature (Abe and Horikoshi2000). Wild-type strains having trp1 as a nutrient auxo-trophic marker (e.g., YPH499 or W303-1A) exhibit di-minished cell growth at high pressure of 25 MPa at 24�or at low temperature of 10�–15� at atmospheric pres-sure (�0.1 MPa ¼ 1 bar ¼ 0.9869 atm ¼ 1.0197 kg offorce/cm2; to avoid confusion, MPa is used throughout)although tryptophan–prototrophic strains can effi-ciently grow under the same conditions. The introduc-tion of the TRP1 gene or tryptophan permease genesTAT1 and TAT2 (Heitman et al. 1993; Schmidt et al.1994; Beck et al. 1999) in a multicopy vector enabledcells to grow at high pressure and low temperature (Abeand Horikoshi 2000; Abe and Iida 2003). Mutations inthe catalytic domain of Rsp5 ubiquitin ligase (Abe andIida 2003), the cytoplasmic tails of Tat2 (Nagayamaet al. 2004) or ubiquitin-specific protease genes DOA4,UBP6, or UBP14 (Miura and Abe 2004) result in markedstabilization of Tat2 and/or Tat1, and thereby themutants grow at high pressure and low temperature.The effect of high pressure on tryptophan auxotrophicstrains is also similar to the effect of an immunosup-pressive drug, rapamycin (Beck et al. 1999), from theaspect of downregulation of Tat2 and the arrest of cellcycle in the G1 phase (Abe and Horikoshi 2000).However, high pressure and rapamycin treatment differin terms of the regulation on Npr1 and the generalamino acid permease Gap1. In contrast to a rapid de-phosphorylation of Npr1 and induction of Gap1 ex-pression upon rapamycin treatment, high pressure didnot affect the phosphorylation state of Npr1, and itdecreased the level of Gap1 protein, suggesting that thepressure-sensing pathway is likely to be independent ofthe Npr1 function (Abe and Horikoshi 2000).

To achieve a molecular understanding of cellularresponses to changes in pressure and temperature, wescreened the yeast deletion library (Giaever et al. 2002)to isolate mutants that were defective in growth underhigh pressure and low temperature. It would provide aunique clue to linking the disrupted gene and its func-tion through thermal energy and work, by varying tem-perature and hydrostatic pressure, respectively. Then wewould analyze the rate-limiting step on the growth of amutant strain from the two mutually dependent ener-gies (supplemental Figure S1 at http://www.genetics.org/supplemental/).

Our results highlight the marked similarity of theeffects of high pressure and low temperature at thegenomewide level. The approach and results describedhere offer a novel route to gaining insights into aminoacid availability, nutrient sensing, membrane trafficking,or organization of macromolecules arising from pre-sumed changes in the activation volume, and the acti-vation energy upon the loss of genes. We focus on the

role of the exit from rapamycin-induced growth arrest(EGO) complex to distinguish between responses to highpressure/low temperature and known responses to rapa-mycin treatment and describe that the EGO complex isinvolved in the regulation of amino acid uptake.

MATERIALS AND METHODS

Yeast strains, culture media, and plasmids: The EUROSCARFyeast deletion library (cat. no. 95400.H3, Invitrogen, Carlsbad,CA) containing 4828 haploid gene deletion mutants and theparental strain BY4742 (MATa his3D1 leu2D0 lys2D0 ura3D0;wild type) were used in this study (Giaever et al. 2002). Thefollowing strains were kindly provided by C. De Virgilio of theUniversity of Geneva Medical School, Switzerland (Duboulozet al. 2005): Y2922 (wild type), CDV207 (ego1D), CDV208 (ego3D),and CDV209 (gtr2D); double-deletion mutants ego1Dnpr1D,ego1Darg3D, ego1Dcpa2D, and ego1Dcbp6D; CDV213 (wild type,EGO1-GFP), CDV214 (wild type, GTR2-GFP), and CDV215(wild type, EGO3-GFP). Cells were grown in YPD (1% bactoyeast extract, 2% bacto peptone, 2% d-glucose), syntheticdextrose (SD, 0.67% yeast extract nitrogen base w/o aminoacids, adenine 20 mg/liter, uracil 20 mg/liter, methionine 20mg/liter, tryptophan 40 mg/liter, histidine-HCl 20 mg/liter,leucine 90 mg/liter, lysine-HCl 30 mg/liter, 2% d-glucose)medium and synthetic complete (SC, 0.67% yeast extractnitrogen base w/o amino acids, adenine sulfate 20 mg/liter,uracil 20 mg/liter, tryptophan 40 mg/liter, histidine-HCl 20mg/liter, leucine 90 mg/liter, lysine-HCl 30 mg/liter, arginine-HCl 20 mg/liter, methionine 20 mg/liter, tyrosine 30 mg/liter,isoleucine 30 mg/liter, phenyalanine 50 mg/liter, glutamicacid 100 mg/liter, aspartic acid 100 mg/liter, threonine 200mg/liter, serine 400 mg/liter, 2% d-glucose) medium. YPDmedium was used for the first qualitative screening. SC me-dium was used for the subsequent quantitative analysis. The53 SC medium contains five-fold concentration of SD withthe regular concentration of nonessential amino acids. SDmedium was used for examining amino acid auxotrophy ofsome mutant strains.

Plasmids: The entire coding region and its upstream anddownstream noncoding region for the EGO1 allele were am-plified using oligonucleotides, XbaI-EGO1-F1 (GCTCTAGAGC-AGCCTCGTTAGTGCCTTCTTCAATATCC) and XbaI-EGO1-R1(GCTCTAGAGC-CTCTTGGTTTTTAGGATGTTTTCCCGGC).Likewise, those of the EGO3 allele were amplified using oli-gonucleotides, XbaI-EGO3-F1 (GCTCTAGAGC-ATGGTTGTTTACTGCACGTTGCCTTTGT) and XbaI-EGO3-R1 (GCTCTAGAGC-AAAGCTGTCATGTAGGGCCCTCTGAGCA). The re-sultant DNA fragments were digested with XbaI (underlined)and were inserted into YCplac33 (URA3 CEN4) to give pEGO1cand pEGO3c. pL137 (CEN4 URA3) containing GTR1 regulatedby its own promoter was kindly provided by T. Sekiguchi ofKyusyu University, Japan (Nakashima et al. 1999). pCDV987(CEN4 URA3) containing GTR2 regulated by its own promoterwas kindly provided by C. De Virgilio (Dubouloz et al. 2005).YCplac33, YCplac111 (LEU2 CEN4), pRS313 (HIS3 CEN4), andpKI (LYS2 2m) were used to confer prototrophy for uracil, leu-cine, histidine, and lysine together on strains of the BY4742genetic background.

Screening of mutants defective in growth under highpressure and low temperature: To determine screening con-ditions, we first compared the growth of the wild-type strain andthe trp1D mutant in YPD medium in 96-well culture plates atpressures from 0.1 MPa to 50 MPa and temperatures from 4� to37�. Judging from the gross yield of cell mass, a clear differencewas observed in culture at 35 MPa and 24� (high-pressure

852 F. Abe and H. Minegishi

condition) for 4 days and 0.1 MPa and 6� (low-temperaturecondition) for 4 days. Small aliquots from 4828 mutant cellcultures were transferred to fresh YPD medium in 96-well plates,followed by incubation at 24� overnight. Then, 3 ml of eachpreculture was transferred to fresh YPD medium in 96-wellplates. After sealing the plates with sterilized plastic film, thecells were subjected to pressure of 35 MPa at 24� in hydrostaticchambers (Rigo-sha, Saitama, Japan) using a hand pump(TP200L, Teramecs, Kyoto, Japan) or to low temperature at0.1 MPa and 6�. After 4 days, the growth-cell yields were checkedvisually. For more quantitative analysis, the candidate mutantcells were grown in SC medium at 0.1 MPa and 24� with vigorousshaking (150 rpm) in the exponential phase of growth (OD600 ,1.5). Then the culture was diluted with SC medium to anOD600 value of 0.15. The diluted cultures were placed in ster-ilized tubes and the tubes were sealed with parafilm. The culturetubes were subjected to high pressure of 25 MPa at 24� inhydrostatic chambers (PV100-360 and PV100-500, Teramecs) orto low temperature of 0.1 MPa at 15� for 20 hr. At the end of theculture period, the pressure was released and apparent opticaldensity was measured at 600 nm (OD600ap) using a spectro-photometer. The OD600 value, which was proportional to celldensity, was calculated using a conversion formula obtained by apolynomial approximation in a separate experiment using aspectrophotometer,

OD600 ¼ �0:415 3 A5 1 2:311 3 A4 � 3:522 3 A3

1 2:772 3 A2 1 0:292 3 A 1 0:047;

where A is OD600ap. For example, the OD600ap values of 0.5, 1.0,and 1.5 are comparable to the OD600 values of 0.58, 1.48, and3.38, respectively, and are comparable to 6.96 3 106, 1.78 3107, and 4.06 3 107 cells/ml in our experiment. The celldensity of the culture was determined using hemocytometers.

Amino acid uptake assay: The following radiolabeled aminoacids were purchased from Moravek Biochemicals (Brea, CA):l-½4,5-3H� leucine (MT-672E, 2.85 TBq/mmol), l-½4,5-3H�lysine (MT-909, 1.67 TBq/mmol), and l-½2,5-3H� histidine(MT905, 1.63 TBq/mmol). Cells of the wild type, ego1D and

gtr1D were incubated in SC medium at 0.1 MPa and 24�, 25MPa and 24�, and 0.1 MPa and 15� for 5 hr. After the releasefrom high-pressure and low-temperature incubation, theuptake of amino acid was analyzed at 0.1 MPa and 24� asdescribed previously (Abe and Iida 2003). Data are expressedas mean values of amino acid incorporated (DPM 107 cells�1

min�1) with standard deviations obtained from three to fiveindependent experiments.

RESULTS AND DISCUSSION

Screening of gene deletion mutants defective ingrowth under high pressure and low temperature: Toidentify genes responsible for growth under high pres-sure at low temperature, we screened the yeast deletionlibrary consisting of 4828 haploid deletion mutants. All ofthe mutants were cultured in 96-well plates at 35 MPa and24� or 0.1 MPa and 6� for 4 days. The first gross screeninganticipated 1022 candidate strains including 779 high-pressure-sensitive mutant and 560 low-temperature-sensitive mutant strains with a significant overlap of 317strains. To evaluate the growth of the candidates morequantitatively, it was feasible to standardize conditionsby measuring the OD600 values. Figure 1 indicates thegrowth properties of the wild-type strain in SC mediumin comparison with the trp1D mutant, a previously knownmutant showing growth defects (Abe and Horikoshi2000). The difference in the ability to grow between thetwo strains was clear at pressures between 20 and 30 MPaat 24�, and at temperatures between 13� and 16� at 0.1MPa. Therefore, further analysis was performed underthree culture conditions, 0.1 MPa and 24� (normal con-dition), 25 MPa and 24� (high-pressure condition), and

Figure 1.—Growth properties of the wild-typestrain BY4742 and the trp1D mutant. Cells werecultured under the pressures shown at 24� (A)or at the temperatures shown at 0.1 MPa (B).The OD600ap value was measured immediately af-ter decompression. The OD600ap values were con-verted into OD600 values using the formuladescribed in materials and methods.

High-Pressure and Low-Temperature Growth Genes 853

0.1 MPa and 15� (low-temperature condition). At 0.1MPa and 24�, the wild-type strain grew to the OD600 valueof 5.0 6 0.14 (n ¼ 10) in 20 hr when the culture startedwith an OD600 value of 0.15. We chose a cutoff value ofOD600 1.0 for the normal growth condition and excluded33 slow-growth strains from the 1022 mutants. At 25 MPaand 24�, the wild-type strain grew to an OD600 value of2.24 6 0.18 (n¼ 10) in 20 hr (45% with respect to normalgrowth). At 0.1 MPa and 15�, the wild-type strain grew toan OD600 value of 1.41 6 0.14 (n ¼ 10) in 20 hr (28.3%with respect to normal growth). The mutants exhibitedvarious degrees of diminished growth at high pressureand low temperature. To assign high-pressure sensitivity,we set a threshold value of 22.5% with respect to theOD600 value of normal growth of individual mutants(50% with respect to the wild-type strain). To assign low-temperature sensitivity, we set a threshold value of 14.2%with respect to the OD600 value of normal growth (50%with respect to the wild-type strain). We then obtained 309candidate strains including 152 high-pressure-sensitivemutants and 239 low-temperature-sensitive mutants.During the course of the experiments, however, webecame aware that the cell density of the preculturelargely affected the ability to grow at high pressure andlow temperature. Once the preculture OD600 valueexceeded �3.0, the cells resumed growth with somedelay after exposure to high pressure or low tempera-ture. Accordingly, this underestimates the ability to growand overestimates the number of growth-deficientmutants. We refined the analysis by maintaining thepreculture OD600 value at ,1.5. This allowed us toidentify 80 strains including 71 high-pressure-sensitivemutants and 56 low-temperature-sensitive mutants withan overlap of 47 strains (Table 1). It should be notedthat most of the 80 mutants exhibited normal cellgrowth at high temperature of 37� except for the mot2D,vps45D, and mdj1D mutants (data not shown). Thus, thereasons for growth defects at high pressure and lowtemperature could generally differ from those at hightemperature. Genes identified in the screening areinvolved in diverse cellular functions. In particular,genes involved in amino acid biosynthesis, microau-tophagy, a subset of genes involved in mitochondrialfunctions, and membrane trafficking are ranked in thetop 20 of which deletion causes marked growth defectsat high pressure and/or low temperature (Table 1, foot-notes c and d). The remainders comprise genes involvedin actin organization and bud formation, inositolphosphate metabolism, transcription and mRNA deg-radation, ribosomal functions, chromatin maintenance,stress response, and unknown open reading frames(ORFs). Next we describe their possible roles in growthat high pressure and low temperature.

Amino acid biosynthesis: The amino acid biosyn-thetic genes we obtained were ARO1, HOM3, THR4,ARO2, TRP4, TRP2, TRP1, TRP5, and SER1 (Table 1 andFigure 2B). On the basis of our previous findings, the

sensitivity of aro1D, aro2D, trp4D, trp2D, trp1D, and trp5Dmutants to high pressure and low temperature can beattributable to auxotrophy for tryptophan, and proba-bly for phenylalanine and tyrosine. Aro1 is a pentafunc-tional protein that catalyzes five steps in the biosynthesisof chorismate. Chorismate is a precursor of tryptophan,tyrosine, and phenylalanine (Duncan et al. 1988). Aro2is a bifunctional chorismate synthase and flavin reductase,which catalyzes the conversion of 5-enolpyruvylshikimate3-phosphate to form chorismate (Jones et al. 1991). Todetermine amino acid auxotrophy, these mutants werecultured on SD agar plates supplemented with appro-priate amino acids at 0.1 MPa and 24�. As expected,aro1D and aro2D strains required not only tryptophanbut also tyrosine and phenylalanine for cell growth(data not shown). The result suggests that high pressureand low temperature impair the uptake of these aro-matic amino acids. One of the unknown strains thatdisplayed substantial high-pressure/low-temperaturesensitivity was ydr008cD. This is a hypothetical ORF thatis located on the coding strand opposite TRP1. Thus,deletion of YDR008C also results in disruption of TRP1and hence confers high-pressure and low-temperaturesensitivity. The ydr008cD mutant indeed displayed tryp-tophan auxotrophy (data not shown). For an unknownreason, deletion of TRP2 resulted in moderate sensi-tivity to low temperature compared with other trypto-phan biosynthetic mutants.

HOM3 encodes l-aspartate 4-phosphate-transferase thatcatalyzes the first step in the common pathway for methi-onine and threonine biosynthesis (Rafalski and Falco1988). We confirmed that the hom3D mutant requiredboth methionine and threonine for growth (data notshown). THR4 encodes threonine synthase that catalyzesthe formation of threonine from o-phosphohomoserine(Aas and Rognes 1990; Ramos and Calderon 1994). Weconfirmed that the thr4D mutant required threonine forgrowth (data not shown). SER1 encodes 3-phosphoserineaminotransferase that catalyzes the formation of phos-phoserine from 3-phosphohydroxypyruvate (Melcheret al. 1995). We confirmed that the ser1D mutant re-quired serine for growth (data not shown). These find-ings suggest that, in a manner analogous to tryptophanuptake, high pressure and low temperature impair theability to take up threonine and serine, potentially inac-tivating their permeases. The ser1D mutant exhibitedmore moderate sensitivity than others (Table 1 andFigure 2B). SC medium contains a higher concentra-tion of serine (400 mg/liter) than other amino acids.Therefore, a high concentration of serine is likely tocompensate for the presumed defect in serine uptake asobserved in the case of tryptophan (Abe and Horikoshi2000).

The leu3D mutant was defective in growth at highpressure but grew normally at low temperature reflect-ing a fundamental rule that pressure and temperatureseparately affect any reactions (Table 1 and Figure 2B).


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16

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614

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56

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312

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76

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320

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LEU3 encodes a zinc-finger transcription factor thatregulates genes involved in the biosynthesis and uptakeof branched-chain amino acids (Friden and Schimmel1988; Nielsen et al. 2001). Strain BY4742 is a leucineauxotroph, and its growth depends on external leucine.Because Leu3 activates the transcription of BAP2 that

encodes a leucine permease, deletion of LEU3 is likely todecrease the Bap2 protein level. Hence, it should bemore difficult for leu3D mutant cells to take up sufficientamounts of leucine from the medium.

Microautophagy and regulation of amino aciduptake: We found that genes encoding components of

Figure 2.—Representation of growth defects of deletion mutants on a pressure–temperature diagram. The OD600 values arenormalized to those of the wild-type strain BY4742 (A) in Table 1. Eighty mutants were classified into 11 categories: (B) amino acidbiosynthesis, (C) microautophagy, (D) mitochondrial functions, (E) actin organization and bud formation, (F) membrane traf-ficking, (G) transcription and mRNA degradation, (H) ribosome function, (I) inositol phosphate (Ins-P) metabolism, (J) chro-matin maintenance, (K) stress response, and (L) unknown function. All results are merged in M. Data are represented as meanvalues with standard deviations obtained from three independent experiments.


the EGO complex were substantially responsible forgrowth under high pressure and low temperature (Table1 and Figure 2C). The EGO complex is a vacuolarmembrane-associated protein complex that consists ofEgo1 (also known as Meh1 and Gse2), Ego3 (also knownas Slm4, Nir1, and Gse1), and Gtr2 (Dubouloz et al.2005). It is known that cells lacking one of EGO1, EGO3,and GTR2 normally show growth arrest after the additionof rapamycin to culture medium, but no longer recoverto the growth phase upon release of the rapamycin block.Deletion of EGO1, EGO3, or GTR2 resulted in markedgrowth defects at high pressure and low temperature.Introduction of the plasmid containing EGO1, EGO3, orGTR2 into the ego1D, ego3D, or gtr2D mutant, respectively,

restored growth at high pressure and low temperature tothe wild-type level, confirming the role of these genes(Figure 3). Gtr2 and Gtr1 are homologous GTP-bindingproteins under the control of Ran/Gsp1-GTPase, whichplays an essential role in nuclear macromolecular traf-ficking (Nakashima et al. 1999). The gtr1D mutant alsoexhibited similar growth defects (Table 1 and Figure 2C).Introduction of the plasmid containing GTR1 into thegtr1D mutant also restored growth at high pressure andlow temperature, confirming the role of GTR1 (Figure3). The localization of Ego1-GFP (CDV213), Ego3-GFP(CDV215), and Gtr2-GFP (CDV214) was unaffectedupon shifts to high-pressure and low-temperature in-cubation for 10 hr (supplemental Figure S2 at http://

Figure 3.—The EGO complex is essential forgrowth at high pressure and low temperature.Cells containing the specified plasmids were cul-tured at 0.1 MPa and 24�, 25 MPa and 24�, or 0.1MPa and 15�. The OD600 values were measuredafter 20 hr of culture. pEGO1, low-copy plasmidcontaining EGO1 driven by its own promoter;pEGO3, low-copy plasmid containing EGO3 drivenby its own promoter; pL137, low-copy plasmidcontaining GTR1 driven by its own promoter;pCDV987, containing GTR2 driven by its ownpromoter. Data are represented as mean valueswith standard deviations obtained from three in-dependent experiments.


www.genetics.org/supplemental/). Our finding suggestsa novel aspect of microautophagy in terms of the reg-ulation of growth under stressful conditions.

To extend our understandings of the EGO complexfunction, we attempted to find the difference andsimilarity between effects of rapamycin treatment andhigh pressure/low temperature with respect to growthrecovery and the levels of intracellular glutamine/glutamate, key regulators in the target of rapamycin(TOR) signaling pathway. Upon the addition of rapa-

mycin, ego1D, ego3D, and gtr2D mutants are known toenter into growth arrest, but fail to resume growthfollowing rapamycin release (Dubouloz et al. 2005).While cell growth was immediately resumed followingrelease from low-temperature incubation (0.1 MPa and15� for 10 hr) in all strains tested, ego1D, gtr1D, and gtr2Dmutants failed to resume growth following release fromhigh-pressure incubation (25 MPa and 24� for 10 hr)during at least the first 12 hr (Figure 4A). Until 30 hrfollowing the pressure release, these mutants restored

Figure 4.—Cell growth of the EGO complex mutants following release from high-pressure and low-temperature incubation.(A) Cells were cultured in SC medium at 0.1 MPa and 24� following release from a 10-hr incubation at 0.1 MPa and 24�, 25 MPaand 24�, or 0.1 MPa and 15�. (B) Cells were cultured in glutamine- and glutamate-enriched SC medium at 0.1 MPa and 24� fol-lowing release from a 10-hr incubation at 0.1 MPa and 24�, 25 MPa and 24� or 0.1 MPa and 15�. (C) Cells were cultured in 53 SCmedium at 0.1 MPa and 24� following release from a 10-hr incubation at 0.1 MPa and 24�, 25 MPa and 24�, or 0.1 MPa and 15�.Data are confirmed by duplicate experiments.


the ability of growth. A similar result was obtained withthe cells after being cultured for 20 hr under high pres-sure or low temperature (data not shown). The resultsuggests that the EGO complex is not essential for re-covery of growth but is required for quick resumptionfrom high-pressure-induced growth arrest as well as forgrowth under high pressure and low temperature.Unlike rapamycin treatment, high pressure and low tem-perature did not induce the formation of autophagicbodies in the vacuole when the wild-type cells werecultured in SC medium containing phenylmethylsulfo-nylfluoride (data not shown). It is reported that rapa-mycin exerts transcriptional regulations in a mannerdependent on the function of Tor or Ego3/Nir1 or theshared function of Tor and Ego3/Nir1 (Huang et al.2004). While GAT1, GAP1, GDH1, DAL2, DAL3, andDUR1 and -2 are upregulated by rapamycin treatment by2- to 57-fold (Huang et al. 2004), these genes remainedunchanged upon the shifts of growth condition to highpressure or low temperature except for downregulationof GDH1 by 2.5-fold under low temperature (the micro-array data is available at Gene Expression Omnibus,accession no. GSE9136). These results suggest that cellsrespond to high pressure and low temperature in adistinct program from that in response to rapamycintreatment.

It is reported that npr1D, arg3D, cpa2D, and cbp6Drender the ability of growth following release fromrapamycin treatment in the ego mutants arising fromthe increase in intracellular glutamine/glutamate levels(Dubouloz et al. 2005). To elucidate the role of intra-cellular glutamine/glutamate, we analyzed the effect ofnpr1D, arg3D, cpa2D, and cbp6D mutations on growth ofthe ego1D mutant (CDV207). The double mutants usedin this experiment are isogenic with Y2922. The egoD andgtrD mutations also caused growth defects in strainY2922 under high pressure and low temperature asobserved in strain BY4742 (supplemental Figure 3A athttp://www.genetics.org/supplemental/). In contrast torestoration of growth observed in the double mutantsfollowing release from rapamycin treatment (Duboulozet al. 2005), the ego1Dnpr1D, ego1Darg3D, ego1Dcpa2D, andego1Dcbp6D mutants were unable to grow during high-pressure and low-temperature incubation, indicatingthat the increase in intracellular glutamine/glutamatedoes not compensate for the growth defects in the ego1Dmutant (supplemental Figure 3A). Regarding growth fol-lowing release from high-pressure incubation, there wasa difference in property of the ego1D mutant between theBY4742 and Y2922 genetic background. The ego1D mu-tant in the Y2922 background (CDV207) resumed growthwithout delay following release from high-pressure in-cubation (supplemental Figure 3B) while that in theBY4742 background exhibited a significant delay (Figure4A). This means that strain Y2922 is not applicable tovalidate the role of intracellular glutamine/glutamatein terms of restoration of growth following high-pressure

release. Indeed, none of npr1D, arg3D, cpa2D, and cbp6Dmutations facilitated growth of the ego1D mutant follow-ing high-pressure release (supplemental Figure 3B).Instead, we examined supply of excess glutamine andglutamate on growth of the egoD and gtrD mutants in theBY4742 genetic background. We found that glutamineand glutamate at concentrations of 2 g/liter in SC me-dium did not compensate for growth defects in ego1D,ego3D, gtr1D, and gtr2D mutants during high-pressureand low-temperature incubation (data not shown) anddid not restore quick recovery from high-pressure re-lease (Figure 4B). These results suggest that the EGOcomplex has a role in a manner independent of intra-cellular glutamine and glutamate levels to regulategrowth under high pressure and low temperature. Weassumed that the uptake of other nutritional substratessuch as glucose, vitamins, or ions might be diminishedin the egoD and gtrD mutants under high pressure andlow temperature, and thereby the growth may be im-paired. However, it is unlikely because the egoD and gtrDmutants still exhibited the growth defects in a 53 SCmedium under high pressure and low temperature (datanot shown) and also showed retardation of growthresumption after release from high-pressure incubation(Figure 4C).

Recently, the EGO complex has been revealed to beequivalent to the GTPase-containing complex for thegeneral amino acid permease Gap1 sorting in the endo-some (GSE) complex, containing Gtr1, Gtr2, Ego3/Gse1, Ego1/Gse2, and Ltv1 (Gao and Kaiser 2006).The EGO/GSE complex plays a specific role in traffick-ing Gap1 out of the endosome to the plasma membraneupon a shift of growth on glutamate to urea. Deletion ofindividual EGO/GSE components was shown to cause adecrease in other EGO/GSE components, suggesting ahierarchical order of assembly (Gao and Kaiser 2006).This supports our finding that deletion mutants forEGO1, EGO3, GTR1, and GTR2 exhibit equivalentgrowth defects at high pressure and low temperature(Table 1 and Figure 2C). Gap1 is not necessary forgrowth at high pressure and low temperature under ournitrogen-rich culture conditions. Because strain BY4742has amino acid auxotrophy for histidine, lysine, andleucine, there was a possibility that amino acid uptakewas impaired under high pressure and low temperaturewhen the EGO/GSE complex was lost. This couldpotentially be due to the lack of surface delivery ofpermease proteins, and thereby the mutant cells ex-hibited growth defects. To examine that possibility, weanalyzed the uptake of histidine, lysine, and leucine at0.1 MPa and 24� after the wild-type, ego1D, and gtr1Dstrains had been incubated in SC medium for 5 hr at 0.1MPa and 24�, 25 MPa and 24�, and 0.1 MPa and 15�. Forcomparison, we also examined mutants of which high-pressure and low-temperature sensitivity was caused by adefect possibly unrelated to trafficking of amino acidperemeases. CDC50 encodes a protein required for


actin cytoskeleton organization. The cdc50D mutantshowed growth defects under high pressure and lowtemperature (Table 1 and Figure 2E, see below). Underthe normal condition, there was no marked differencein the uptake of histidine between the wild-type and themutant strains (Figure 5). While the uptake of histidinewas not considerably affected by high pressure and lowtemperature in the wild-type strain, it was significantlydiminished in the ego1D and gtr1D mutants. The effectof high pressure and low temperature in the cdc50Dmutant was the comparable level with the wild-typestrain (Figure 5). Similar results were obtained for theuptake of lysine and leucine (Figure 5). In our pre-liminary observation, deletions for other genes such asPLC1, CAF17, and SEC22 (see below) resulted in a slightdecrease in amino acid uptake following high-pressureand low-temperature incubation, but the effect was not

remarkable compared with the losses of EGO1 and GTR1(data not shown). These findings support the hypoth-esis that the EGO/GSE complex is required to maintainthe activity of amino acid uptake under high pressureand low temperature, probably due to the appropriatecell-surface delivery of permease proteins. Apparently,high pressure caused a more significant defect in aminoacid uptake in the ego1D and gtr1D mutants than lowtemperature did. This would explain the differentialsusceptibility to high pressure and low temperature inthe ego1D and gtr1D mutants in which relative growthwas �9% (high pressure) and 37% (low temperature)compared with that of the wild-type strain (Table 1 andFigure 2C) and delay in resumption of growth followingrelease from high-pressure incubation (Figure 4A, 25MPa, 24�). If our hypothesis is valid, prototrophy for thenutrients should restore the ability of ego1D and gtr1D

Figure 5.—Amino acid uptake following release from high-pressure and low-temperature incubation. Cells of the wild type,ego1D, gtr1D, and cdc50D were cultured at 0.1 MPa and 24�, 25 MPa and 24�, or 0.1 MPa and 15� for 5 hr followed by the mea-surement of uptake of histidine, lysine, and leucine at 0.1 MPa and 24� using 3H-labeled substrates (see materials and methods).Data are represented as mean values with standard deviations obtained from three to seven independent experiments.


mutants to grow under high pressure and low temper-ature. Four plasmids containing LEU2, LYS2, HIS3, andURA3 together were introduced into individual mutantsto confer nutrient prototrophy. Then the cells were cul-tured under high pressure and low temperature in thepresence (Figure 6A) or absence (Figure 6B) of leucine,lysine, histidine, and uracil in SC medium. We found thatnutrient prototrophy restored low-temperature growthto egoD and gtrD mutants, suggesting that the suscep-tibility to low temperature of the mutants is attribut-able to defects in the uptake of nutrients (Figure 6C).However, nutrient prototrophy did not restore high-pressure growth to the mutants. Accordingly, the EGO/GSE complex is likely to play a primary role in the cell-surface delivery of amino acid permeases at low tem-perature, but under high pressure it is likely to playmultiple roles in cellular functions in addition to per-mease delivery. It has been shown in Gap delivery thatthe tyrosine-containing sequence KPRWYR within thecytosolic C-terminal domain of Gap1 is required forbinding of Gap1 to Gtr2 and for the efficient sortingfrom the endosome to the plasma membrane (Gao andKaiser 2006). Bap2/Bap3, Lyp1, and Hip1, which arepossible amino acid permeases for leucine, lysine, and

histidine, respectively, do not have KPRWYR sequence.Thus, it should be elucidated whether these permeasesphysically bind to any of the EGO/GSE components, orthere is a protein that mediates binding of the per-meases to the complex.

Using a DNA microarray on strain BY4742 (Abe2007a), we found that numbers of the genes encoding24-amino-acid permeases and their homologs were down-regulated upon shifts to growth under high pressure andlow temperature (Table 2; the DNA microarray data isavailable at Gene Expression Omnibus, accession no.GSE9136). In particular, genes classified into the aminoacid permease cluster I and cluster II were markedlydecreased. With reduced levels of transcripts, and therebysynthesis of permease proteins could be decreased, itbecomes more important to deliver the permease pro-teins to the cell surface to receive sufficient amounts ofessential amino acids for growth. In addition, some of thegenes encoding glucose/hexose transporters were alsodownregulated under high pressure and low temperature(Table 2). Accordingly, the level of glucose/hexose trans-porters in the plasma membrane might be decreasedupon the loss of the EGO/GSE complex when the cellsare exposed to high pressure. This might provide an

Figure 6.—Cell growth of the EGO/GSE complex mutants with or without nutriment auxotrophy. The wild-type and the EGO/GSE complex mutants with (A) auxotrophy for leucine, lysine, histidine, and uracil or (B) prototrophy were cultured at 0.1 MPaand 24�, 25 MPa and 24�, or 0.1 MPa and 15�. The prototrophic strains were cultured in SC medium in the absence of leucine,lysine, histidine, and uracil for maintenance of the plasmids. The OD600 values were measured after 20 hr of culture. (C) TheOD600 values are normalized to those of the wild-type strain BY4742 as shown in Figure 2. Data are represented as mean values withstandard deviations obtained from three independent experiments.


account for the fact that the egoD and gtrD mutants werenot rescued by conferring amino acid prototrophy(Figure 6C), but it is controversial because the 53 SCmedium (containing 10% glucose) did not compensate

for the growth defects of the egoD and gtrD mutants.Further biochemical analysis is necessary to validate thehypothesis that the EGO/GSE complex plays a role in thedelivery of various membrane proteins, but our findingwill be a good starting point to elucidate its role.

Mitochondrial function: We obtained 11 deletionmutants for mitochondrial proteins in our functionalscreening (Table 1 and Figure 2D). In particular, dele-tion of MRF1, MRPL22, and CAF17 resulted in markedgrowth defects. The lack of MRF1 causes mitochondrialgenome instability (Pel et al. 1992; Askarian-Amiri et al.2000). In accordance with mitochondrial functions, glu-tamine is a key factor that may act in the TOR signalingpathway (Dubouloz et al. 2005). To test the possibilitythat the mitochondrial defects could result in a decreasein intracellular glutamate/glutamine levels, possibly dueto the low production of 2-oxoglutarate in the tricarbox-ylic acid (TCA) cycle, we determined the requirementfor glutamine and glutamate in the 11 mitochondrialmutants. Among them, the aco1D and caf17D mutantsrequired either glutamate or glutamine for growth butothers did not (data not shown). The result suggests thatmitochondrial functions other than glutamine/gluta-mate synthesis also impinge on the high-pressure andlow-temperature responsive pathway. ACO1 encodes aco-nitase that catalyzes cis-aconitate to isocitrate. In additionto glutamate auxotrophy upon its deletion (Gangloffet al. 1990), Aco1 is required for mitochondrial DNA main-tenance (Chen et al. 2005). CAF17 encodes a mitochon-drial protein that interacts in the two-hybrid system withCcr4, a component of the Ccr4-Not transcriptional com-plex (Clark et al. 2004). Deletion of CCR4 also causesgrowth defects at high pressure and low temperature (seebelow).

Actin organization, cell polarity, and membranetrafficking: CDC50 encodes a protein required for actincytoskeleton organization and trafficking of proteins be-tween the Golgi complex and the endosome/vacuole.The lack of CDC50 is known to confer low-temperaturesensitivity (Misu et al. 2003). This is attributable to adefect in the establishment of actin networks resultingfrom improper localization of regulator proteins forpolarized growth such as Bni1 and Gic1 at low temper-ature (Saito et al. 2004). We found that the cdc50Dmutant also exhibited growth defects at high pressure,suggesting that high pressure perturbs the localizationof actin and/or associated proteins in the absence ofCdc50 (Table 1 and Figure 2E). Cdc50 is known toassociate with Drs2, a P-type ATPase of the amino-phospholipid translocase that functions in phospholi-pids asymmetry (Chen et al. 1999; Natarajan et al. 2004;Saito et al. 2004; Chen et al. 2006). The lack of DRS2 isalso known to cause low-temperature sensitivity (Chenet al. 1999). We found that the drs2D mutant exhibitedhigh-pressure sensitivity although it is not included inTable 1. This is because the OD600 value of the drs2D cellculture at 0.1 MPa and 24� reached only 0.8 in 20 hr

TABLE 2

Transcription of genes encoding amino acid permeases,hexose transporters, and their homologs in response

to high pressure and low temperaturea

Ratio

Gene HP/normal LT/normal

AAP cluster Ib TAT1 0.13 0.22TAT2 0.38 0.46GNP1 0.32 0.44AGP1 0.53 0.74BAP2 0.15 0.27BAP3 0.27 0.67

AAP cluster IIb HIP1 0.48 0.46GAP1 0.25 0.59MMP1 0.21 0.35SAM1 0.58 0.63

AAP cluster IIIb CAN1 0.60 0.80ALP1 0.87 0.94LYP1 0.78 0.61

AAP unclusteredb DIP5 1.06 0.84PUT4 0.38 0.92AGP2 0.76 1.05AGP3 1.31 1.33SSY1 1.25 1.22MUP1 0.20 0.36MUP3 2.69 1.58TPO5 0.94 0.62HNM1 0.86 1.03BIO5 0.85 1.08UGA4 1.04 1.28

Glucose/hexosetransporter

HXT1 0.30 0.17HXT2 0.09 0.20HXT3 0.88 0.74HXT4 0.54 2.95HXT5 0.20 0.85HXT6 0.61 3.32HXT7 0.57 2.96HXT8 0.69 0.78HXT9 0.99 1.04HXT10 0.69 0.82HXT11 1.22 1.27HXT13 1.19 1.25HXT14 1.01 0.87HXT15 0.88 0.72HXT16 0.94 0.94HXT17 0.99 0.86

Normal, 0.1 MPa and 24�; HP (high pressure), 25 MPa and24�; LT (low temperature), 0.1 MPa and 15�.

a Data from the Gene Expression Omnibus, accession no.GSE9136.

b Classification of amino acid permeases (AAPs) accordingto Nelissen et al. (1997).


when the culture started with an OD600 value of 0.15,and hence the drs2D mutant was assumed to be a slowgrowth strain, which was excluded from Table 1. In-terestingly, deletion of YBR255W, which encodes anunknown protein but was implicated in the syntheticsick/lethal interaction with Drs2 (Schuldiner et al.2005), caused marked growth defects at high pressureand low temperature (Table 1 and Figure 2E). Ourfinding is consistent with a previous report indicatingthat ybr255wD cells exhibited a slower growth rate at 15�although the growth was not examined at high pressure(Sanjuan et al. 1999). These results suggest that theDrs2-Cdc50 complex and Ybr255w redundantly have arole in establishing appropriate actin networks at highpressure and low temperature. In our unpublished ob-servation, actin cytoskeleton visualized with rhodamine-labeled phalloidin was disorganized in the drs2D, cdc50D,and ybr255wD mutants with dotted distribution in thecortical region and the cytoplasm when the cells werecultured under high pressure or low temperature (ourunpublished results). Therefore, the Drs2-Cdc50 com-plex and Ybr255w are likely to play an essential role inactin network formation at high pressure and low temper-ature by modulating protein trafficking appropriately. Itshould be elucidated whether disorganization of actincytoskeleton is the primary cause of the growth defects inthe drs2D, cdc50D, and ybr255wD mutants, or the conse-quence of diminished ability of membrane traffickingresulting from the phospholipid asymmetry defect.

MSS4 is an essential gene encoding a single phos-phoinositide-4-phosphate (PI4P) 5-kinase that gener-ates PI4,5 P2, which is required for actin cytoskeletonorganization at the plasma membrane (Desriviereset al. 1998; Homma et al. 1998). Audhya et al. (2004)identified numbers of genes encoding PI4,5P2 effectorsusing a genomewide lethality screen including six SLMgenes. SLM4 is identical to EGO3/GSE1 (Audhya et al.2004; Dubouloz et al. 2005; Gao and Kaiser 2006). Inaddition to the slm4D mutant, the slm3D mutant ex-hibited high-pressure and low-temperature sensitivitybut the slm6D mutant exhibited only low-temperaturesensitivity (Table 1 and Figure 2E). Slm1 and its homo-log Slm2 are downstream effectors of TORC2, the Tor2kinase-containing complex, which regulates the actincytoskeleton organization (Audhya et al. 2004). Theslm1Dslm2D mutant is inviable. We found that deletionmutants for SLM1, SLM2, and SLM5 grew well underhigh pressure and low temperature like the wild-typestrain, suggesting that TORC2 may not be required forhigh-pressure and low-temperature growth or that thereare other proteins redundant in the functions of SLM1,SLM2, and SLM5 (data not shown). The slm3D mutationis known to be synthetically lethal with the cdc73Dmutation (Tong et al. 2004). CDC73 encodes a constit-uent of the Paf1 complex with RNA polymerase II (Shiet al. 1997; Krogan et al. 2003). Interestingly, the cdc73Dmutant was also unable to grow under high pressure and

low temperature (see below). In our first screening on96-well plates, deletion of BOI2, ROM2, CAP2, or GIM4,which are known to display synthetic lethality with themss4ts mutation, caused growth defects at high pressureand low temperature (data not shown).

Lte1 is a putative guanine nucleotide exchange factorrequired for mitotic exit at low temperature (Wickneret al. 1987; Shirayama et al. 1994a,b). Lte1 is localized tothe bud cortex and bud cytoplasm from the S phase to Mphase and is uniformly distributed in the G1 phase(Yoshida et al. 2003). We found that the lte1D mutantwas defective in growth not only at low temperature butalso at high pressure (Table 1 and Figure 2E). Therestriction of Lte1 to the bud cortex depends on septinsCdc42, Kel1, and Cla4 but is independent of the actincytoskeleton and microtubule formation (Seshan et al.2002). The PAK-like protein kinase Cla4 is required forLte1 phosphorylation and accordingly for localization(Seshan et al. 2002). Interestingly, the cla4D mutant wasalso defective in growth at high pressure and low tem-perature even though the effect was moderate (Table 1and Figure 2E). HOF1 encodes a bud neck-localizedSrc homology 3-domain-containing protein that is re-quired for cytokinesis and regulation of actomyosin ringdynamics and septin localization (Kamei et al. 1998;Lippincott and Li 1998). The hof1D mutant exhibitedgrowth defects at high pressure (Table 1 and Figure 2E).The proteins encoded by the genes shown here haveroles in the organization of macromolecules associatedwith a serial event in the bud neck to exit from mitosis.We speculate that these proteins mediate the cellularprocesses by decreasing the system volume change andenergy barriers associated with the reactions.

Membrane trafficking: Numerous genes involved inmembrane trafficking appear to be responsible forgrowth at high pressure and low temperature (Table 1and Figure 2F). We assume that delivery of newlysynthesized proteins to appropriate locations, e.g., thebud neck, cell surface, or cell wall, is diminished at highpressure and low temperature when one of the geneslisted in Table 1 is lost. ERG24 encodes the C-14 sterolreductase that catalyzes a step in ergosterol biosynthesis(Lorenz and Parks 1992). The erg24 mutants are viablebut accumulate the abnormal sterol ignosterol (ergosta-8,14 dienol) (Parks et al. 1995). Although the erg24 muta-tion has not been examined for growth at high pressureand low temperature, the erg6 mutation is known toconfer cold sensitivity when combined with tryptophanauxotrophy (Gaber et al. 1989). In our unpublished ob-servation, tryptophan prototrophic strains carrying theerg6D, erg2D, erg3D, erg4D, or erg5D mutation fail to growthunder high pressure and low temperature, suggestingthat the structural motif of ergosterol is required for func-tion and/or trafficking of membrane proteins underhigh pressure and low temperature, and hence cellgrowth. Kishimoto et al. (2005) demonstrated that thecdc50 mutation is synthetically lethal with the five erg


mutations. In the cdc50D erg3D mutant, sterol was pre-dominantly detected either diffusely within the cytosol oras punctate dots with a concomitant decrease in theplasma membrane resulting from a possible defect inrecycling sterols to the plasma membrane. Therefore,high pressure and low temperature could lead to thesituation analogous to ergosterol deficiency in which theCdc50-Drs2 complex is unable to function in membranetrafficking and the organization of actin cytoskeleton.

In mammalian HeLa cells, low temperature of 15�blocks protein transport at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) (Martinez-Alonso et al. 2005). At this temperature, the Golgicomplex shows long tubules and newly synthesizedproteins are accumulated at the ERGIC (Martinez-Alonso et al. 2005). In this sense, vesicle-mediated pro-tein transport steps in HeLa cells are expected to havehigh activation energies. High pressure as well as lowtemperature act to increase lipid order, and hence bothpotentially have adverse effects on biological membranesthat are adapted to atmospheric pressure and moderatetemperature. Therefore, the proteins encoded by mem-brane trafficking genes revealed in this study are likelyto contribute to decreasing the activation volume andactivation energy associated with steps in vesicle buddingand/or vesicle fusion in the endocytic pathway.

Transcriptional regulation and mRNA degradation:Genes involved in transcription and mRNA degradationcomprise a major class of essential genes for growth athigh pressure and low temperature. The Ccr4-Notcomplex is a global regulator of transcription consistingof five Not proteins (Not1–Not5), Pop2 (also known asCaf1), Caf40, Caf130, and Ccr4 (Liu et al. 1998; Tuckeret al. 2001; Collart 2003). Among them, Not1 is thescaffold of the complex and is essential for viability(Maillet et al. 2000). We found that deletion of CCR4,POP2, or NOT4 (also known as MOT2) resulted insubstantial growth defects at high pressure and lowtemperature (Table 1 and Figure 2G). Low-temperaturesensitivity of the ccr4D and pop2D mutants was reportedpreviously (Hata et al. 1998). Ccr4 and Pop2 associatewith a central portion of the N-terminal domain ofNot1 and function as the major yeast deadenylases thatplay a role in mRNA degradation (Tucker et al. 2001).Meanwhile, Not4 associates with the C-terminal domainof Not1 and functions as an E3 ubiquitin ligase onubiquitination (Albert et al. 2002). Our result suggeststhat the loss of one of the components destabilizes theCcr4-Not1 complex at high pressure and low tempera-ture, or alternatively the assembly of the complex isimpaired by high pressure and low temperature.

In addition to the Ccr4-Not complex, we found thattwo complex forms of RNA polymerase II, one includingSrb proteins (Liu et al. 1997) and the second includingPaf1 and Cdc73 (Chang et al. 1999), were indispensablefor growth at high pressure and low temperature (Table1 and Figure 2G). Because double mutants paf1Dsrb5D,

paf1Dccr4D, cdc73Dccr4D, and ccr4Dsrb5D are lethal, thefunctions of these transcriptional regulators are re-dundant (Chang et al. 1999). The paf1D and ccr4Dmutants are sensitive to cell wall-damaging agents suchas caffeine or sodium dodecyl sulfate and sensitive tohigh temperature of 35.5� or 38� (Chang et al. 1999).The high-temperature sensitivity of the two mutants issuppressed by the presence of 1 m sorbitol. Thus, Paf1and Ccr4 are thought to be required for the integrity ofthe cell wall. In our recent study of global transcrip-tional profiling using a DNA microarray, the DAN/TIRcell wall mannoprotein genes were dramatically in-duced by high pressure and low temperature (Abe2007a). Accordingly, the expression of these cell wallprotein genes might be regulated by Paf1 and Ccr4 inresponse to high pressure and low temperature.

Ribosomal proteins: The assembly of ribosomes isknown to be impaired at low temperature in the absenceof certain ribosomal proteins. The rpl39D mutationcauses an increased translational error frequency at lowtemperature and thereby confers low temperature-sensitive growth (Dresios et al. 2000). However, noribosomal protein mutant has been examined underhigh-pressure conditions. We found that the rpl1bD andrpl21aD mutants were defective in growth at both highpressure and low temperature, but the rps30bD mutantexhibited growth defects only at low temperature (Table1 and Figure 2H). Oligomerization of proteins is atypical high-pressure-sensitive process because it is usu-ally accompanied by large volume changes. Because theribosome is an enormously large structure, its assemblyis likely to be sensitive to high pressure. Rpl1b andRpl21a could function to stabilize ribosomal structuresand subunit association at high pressure. Althoughbiochemical analysis is required to identify the role ofthese ribosomal proteins, our present data suggest thatRps30b has a role different from that of Rpl1b andRpl21a in ribosomal functions in terms of differentialresponses to high pressure and low temperature.

Other genes and interaction networks: Deletionmutants for PLC, ARG82, and KCS1 exhibited growthdefects at high pressure and low temperature (Table 1and Figure 2I). Plc1, Arg82, and Kcs1 catalyze steps ininositol polyphosphate metabolism and play roles in var-ious cellular functions including negative regulation ofthe phosphate signal transduction PHO pathway, remod-eling of the PHO5 promoter chromatin, arginine me-tabolism, salt stress, and cell wall integrity (Auesukareeet al. 2005). Genes involved in chromatin remodeling(IES1), telomere maintenance (ARD1), telomere uncap-ping and elongation (CGI121), and histone acetylation(YAF9) were obtained although the effects were moder-ate (Table 1 and Figure 2J). Although their contributionto growth deficiency is evident, the role of these genes isunaccountable. We recently reported that Hsp31 has arole in high-pressure growth and obtained the sameresult in this study (Miura et al. 2006). In addition, YDJ1


that encodes a chaperone for the regulation of HSP90and HSP70 functions (Schumacher et al. 1996) was ob-tained in this study (Table 1 and Figure 2K). Ydj1/Hsp40and Ssa1/Hsp70 are known to cooperate with Hsp104to unfold and reactivate denatured, aggregated proteins(Glover and Lindquist 1998). Hsp104 in associationwith trehalose is thought to play a role in unfolding ofprotein aggregates caused by hydrostatic pressure al-though the range of pressure (�150 MPa) is muchhigher than that under pure experimental conditions(Iwahashi et al. 1997, 2000). Hsp31 and Ydj1 may act tounfold some misfolded proteins caused by high pressureand low temperature.

We identified 12 unknown or uncharacterized genesin this study (Table 1 and Figure 2L). YDL172C andYDL173W are mutually overlapping in the opposite DNAstrand. Thus, the deletion of one gene means that theother is also lost. We found that the ydr442wD mutant hadtryptophan auxotrophy in our BY4742 genetic back-ground and thereby exhibited growth defects at highpressure and low temperature. However, the ydr442wDmutant was tryptophan prototrophic in the BY4741genetic background and thereby grew at high pressureand low temperature. Therefore, YDR442W is a ques-tionable ORF. The remainder of the mutants obtained inthis study are tryptophan prototrophs.

Figure 7 indicates physical and/or genetic interac-tions among genes responsible for high-pressure and/or low-temperature growth identified in this study ac-cording to the Saccharomyces Genome Database. Nota-bly, 40 of the 80 genes have some interactions amongthemselves. This interaction map provides clues to elu-cidate the consequences of individual gene deletions and

reveal the significance of cellular functions on growth athigh pressure and low temperature.

Perspective in the study of intracellular processes asa function of pressure and temperature: Our findingsdescribed here reveal unpredicted genes and anticipatethe functional roles responsible for the growth of yeastat high pressure and low temperature, and would providea clue to understand the survival strategies employed bydeep-sea organisms. Three major classes of function weidentified are (i) amino acid biosynthesis, (ii) micro-autophagy, and (iii) mitochondrial function. In a man-ner analogous to tryptophan uptake, our results suggestthat the uptake of phenyalanine, tyrosine, threonine, andserine is also likely to be impaired by high pressureand low temperature, probably due to reduced activityand/or degradation of their permeases.

global screening of genes essential for growth in high ...tubes were subjected to high pressure of...

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