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PalI domain protiens of Saccharomyces cerevisiae and Candida
albicans
Yan, Lan; Côte, Pierre; Li, Xing-Xing; Jiang, Yuan-Ying; Whiteway, Malcolm
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ContentslistsavailableatSciVerseScienceDirect
Microbiological
Research
j o ur na l ho m e p a g e :w w w . e l s e v i e r . d e / m i c r e s
PalI
domain
proteins
of
Saccharomyces
cerevisiae
and
Candida
albicans
Lan
Yan
a,1, Pierre
Côte
b,c,1, Xing-Xing
Li
a,
Yuan-Ying
Jiang
a,∗,
Malcolm
Whiteway
b,c,∗∗aCenterforNewDrugResearch,DepartmentofPharmacology,SchoolofPharmacy,SecondMilitaryMedicalUniversity,325GuoheRoad,Shanghai200433,PRChina bBiotechnologyResearchInstitute,NationalResearchCouncilofCanada,6100RoyalmountAve.,Montreal,Quebec,CanadaH4P2R2
cDepartmentofBiology,McGillUniversity,Montreal,Quebec,CanadaH3A1B1
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received2December2011 Receivedinrevisedform 19December2011 Accepted30December2011 Keywords: PalIdomain Candidaalbicans Saccharomycescerevisiae Environmentalstressresponse
a
b
s
t
r
a
c
t
TheRim9/PalIgroupsofproteinsaremembersoftheSur7family,allofwhichcontainasignalsequence andablockofthreepotentialtrans-membranehelices.Multi-proteinsequencecomparisonsamongfungi suggestthattherearetwoclassesofRim9/PalIproteins;longerproteinslikePalIthatcontainaSur7 domainandaC-terminalextension,andshorterproteinslikeRim9thatcontainessentiallyonlythe Sur7domain.Wehaveexaminedpossiblerolesofthelonger,PalI-likeproteinsofbothSaccharomyces cerevisiae(Yol019w)andCandidaalbicans(Orf19.1510/Srd1),twospeciesthatalsocontainshortRim9 proteinsrequiredforalkaline-associatedstressresponses.Deletionsofthelongformgenesdidnotcreate anysignificantstressresponsephenotypeineitherS.cerevisiaeorC.albicans,nordidthedeletionsenhance anyoftherim9deletioneffectswhencombinedinadoublemutant.Furthermore,challengesinC.albicans showRIM9butnotSRD1isimportantforproperresponseandhyphalformation.Itappearsthatinfungal speciessuchasAspergillusnidulanscontainingonlyalong-formPalI-likeprotein,thiselementfunctions intheprocessofstressresponse,whileinfungiwithbothversionstheresponsetostressfunctionis limitedtotheshort-formprotein.
© 2012 Published by Elsevier GmbH.
Introduction
The multiple genome sequences available for thefungi can
permithigh-resolutionbioinformaticalanalysesofprotein
fami-lies,andthiscanbehelpfulincharacterizingproteinswithasyet
poorlyunderstoodfunctions.TheSur7domainfamilyconsistsof
anintriguinggroupofproteinsthatallcontainasignalsequence
and atriple trans-membranedomain(3TMD).Inthe yeast
Sac-charomycescerevisiaethisfamilycontains9members;sixshorter
proteinsthatconsistoflittlemorethanthesignalsequenceandthe
potentialtrans-membranehelices,andthreelongerproteins,one
withaCterminalextensionandtwowithN-terminalextensions
relativetothe3TMDregion.Manyoftheseproteinshavepoorly
definedmolecularroles.Althoughtheirrolesintheformationof
suchstructuresareunclear,thegroup-definingproteinSur7along
withtwootherfamilymembersYnl194cpandYdl222cp(Fmp45p)
haverecentlybeencharacterizedaselementsoftheMCC
(Mem-braneCompartmentofCan1),whichisalateralmembranedomain
∗ Correspondingauthor.Tel.:+8602181871357;fax:+8602165490641. ∗∗ Correspondingauthorat:BiotechnologyResearchInstitute,NationalResearch CouncilofCanada,6100RoyalmountAve.,Montreal,Quebec,CanadaH4P2R2. Tel.:+15144966146;fax:+15144966213.
E-mailaddresses:jiangyysmmu@sina.com(Y.-Y.Jiang),
malcolm.whiteway@cnrc-nrc.gc.ca(M.Whiteway).
1 Theseauthorscontributedequallytothework.
associated with furrow-like invaginations in the yeast plasma
membrane(Stradalovaetal.2009;Malinskyetal.2010).These3
proteinshavearelativelycloseevolutionaryrelationship.Ydl222cp
andYnl194cparelikelyparalogsarisingthroughthewholegenome
duplicationasthereisonlyoneequivalentprotein(Orf19.6489p)
inthepre-duplicationC.albicans;thecloselyrelatedbutdistinct
Sur7phasa singleequivalentacrosstheascomycetes, including
Orf19.3414p in C. albicans.Ylr414cp/Pun1p hasbeen identified
as a plasma membrane protein localizingto an ergosterol-rich
membranecompartmentrequiredforcellwallintegrity(Hosiner
etal.2011).However,thetwo paralogswithN-terminal
exten-sions,Ylr413wpandYkl187cp,havenotbeencharacterized,andthe
molecularfunctionofYfr012wp/Dcv1phasnotyetbeendefined.
Structurally, Pun1p is most similar to the Ylr413wp/Ykl187cp
pair, but while homologs of Pun1p are found throughout the
ascomycetes, includingOrf19.6741 inC.albicans,theduplicated
genesYLR413and YKL187appear uniquetoS.cerevisiaeandits
closerelatives,andthustheyappeartohavearisenandundergone
aduplicationeventaftertheWGD.Thefinalshort-formprotein
Rim9ispartoftheRim101signalingpathwayinvolvedin
sporula-tion,temperatureresponse,andinvasivegrowthinyeast(Suand
Mitchell1993;LiandMitchell1997);theotherfamilymember,
Yol019wp,isstructurallysimilartoRim9pbutcontainsaCterminal
extension,andhasnodefinedcellularfunction.
Rim9phasorthologsinotherspecies.ThereisaclearRim9
pro-teininthefungalpathogenCandidaalbicans,whilethePalIprotein
0944-5013/$–seefrontmatter © 2012 Published by Elsevier GmbH. doi:10.1016/j.micres.2011.12.005
ofAspergillusnidulanshasstructuralsimilaritytoRim9pandisa
componentofthePalCpathway,theequivalentpathwaytothe
Rim101pathway ofyeast.Whenmultiplefungal sequencesare
compareditisapparentthatthisRim9groupofproteinsfallsinto
twoclasses,theshort(225–399aminoacids)Rim9-likeproteins,
andthelonger(476–756aminoacids)PalI-likeproteins.In
gen-eral,specieswiththeshortversionproteinalsohavethelonger
versionprotein,butsomespecies,likeA.nidulans,onlyhavethe
longerprotein.
Rim9andPalIproteinsplayapparentlyanalogous,butnotfully
understood,rolesintheRim101andPalCpathwaysrespectively.
Thesepathwaysareinvolvedinavarietyoffunctions,including
responsetoaselectionofstresses.Fungihaveadaptedtoadiversity
ofnichesincludingthecolonizationofanimalsandplants,andover
millionsyearsofevolutiontheyhavedevelopedwaystosurviveand
proliferateinthesecontinuouslychanging,andoftenhostile,
envi-ronments.Mostcommonenvironmentalstresses originatefrom
nutrientlimitation,temperature,osmolarity,pHandexposureto
toxins(Gasch2007).Inaddition,pathogenicspeciesmustconfront
hostdefensemechanisms,inparticularthereactiveoxygenand
nitrogenspeciesgeneratedbytheimmunesystem(Baeuerleetal.
1996;Diez-OrejasandFernandez-Arenas2008).
ExtracellularpHrepresentsanimportantfactorinfluencingcell
physiologyandgrowth.AtthewrongpH,proteinsareoftenpoorly
functionalorevennon-functional;chargedmoleculescanbecome
inappropriatelycharged,whichcandestabilizetheirstructureand
function,andnutrientuptakecanbecomprised.Forexample,the
changeiniron ionsfromtheirinsolubleferricformFe3+tothe
solubleferrousformFe2+ispH-dependent,andthuslowpH
con-tributestomaintainingthebioavailabilityofthisessentialmetal
(Bensenetal.2004).Whileacidicenvironmentsarerelativelywell
toleratedbyfungi,theabilitytoproliferateinanalkalinepH
envi-ronmentisacharacteristicsharedbymany,ifnotall,pathogens
(Davis2003).
Theabilityoffungitosenseandrespondtoalkaline
environ-mentsiscontrolledinalargepartbytheRim101/PalCpathway
(Tilburnetal.1995;Davis2003;Penalvaetal.2008).High
ambi-entpHis apparentlysensedbytheplasmamembrane complex
composedofRim9p/Rim21p(inyeast)orPalI/PalH(inA.nidulans)
which,ifinthepresenceofalkalineenvironment,willbe
endocy-tosedtogetherwithphosphorylatedandubiquitylatedRim8p/PalF.
Onceinternalized,theseproteinsaretransmittedtoan
endoso-malmembranecomplexcontainingrespectivelyRim20p/Rim13p
orPalA/PalB. Then,Rim13p(PalB)activatesthezincfinger
tran-scriptionfactor Rim101p(PacC) to stimulate the expression of
alkaline-dependentgeneswhile repressing those that are
acid-dependent(Davis2003;Penalvaetal.2008).Thishasaprofound
effecton gene expression; DNA microarrays analysis identified
morethan500genesmodulatedwhenC.albicanscellswere
trans-ferredfromanacidic(pH4)toanalkaline(pH8)medium(Bensen
etal.2004).
Because of the structural and functional relationship of
Rim9p/PalI,sequencesimilaritytoRim9phasbeenusedto
iden-tifyproteinspotentiallyimplicatedinpHresponseinotherspecies
(Denisonetal.1998;Cornetetal.2009).However,whilelossof
Rim9pinC.albicanshassignificanteffectsonthecellularresponse
toalkalistress(Denisonetal.1998;Cornetetal.2009),andlossof
PalIcompromisesgrowthinhighpHinA.nidulans(Denisonetal.
1998),thedeletionoftheRim9-likeproteininYarrowialipolytica
causeslesssignificanteffects(Gonzalez-Lopezetal.2006).Asyet
therelationshipofalkalistresssensitivityandS.cerevisiaeRim9p
hasnotbeenreported.
WhileA.nidulanscontainsonlythelongPalImemberofthe
fam-ily,bothC.albicansandS.cerevisiaecontain,inadditiontotheRim9
orthologs,uncharacterizedgenesencodinglonger,PalI-likefamily
members.HerewereportthephenotypicalcharacterizationinC.
albicansandS.cerevisiaestrainsdeletedforonlyPalI-likeproteins
(Orf19.1510and Yol019wrespectively), andfor both Rim9 and
thePalI-likeproteins.WefindthatbothYol019wandOrf19.1510,
whichwehavedesignatedSRD1forSimilartoRim9pDomain,are
notimplicatedinstressresponsiveness.
Materialsandmethods
Mediaandgrowthconditions
YeaststrainsweremaintainedinYPDmedium(1%yeastextract,
2%peptone,and2%dextrose)orYPDagar(2%).Unlessindicated
otherwise, all strainsweregrown routinely in YPDmedium at
30◦C withshaking overnight, dilutedto anOD600=0.1–0.2the
nextmorning,growntologarithmicphaseandusedforthe
exper-iment. Whereindicated, synthetic complete medium (SC; 6.7g
yeastnitrogenbaseand2%dextrose)wassupplementedwith
his-tidine(20g/ml), leucine (60g/ml), or arginine (40g/ml)as
appropriate.To inducehyphalgrowth,overnightgrownC.
albi-canscellsweresubculturedat37◦CineitherYPDsupplemented
with10%heat-inactivatedfetalbovineserum(FBS;Sigma),inLee’s
medium(Lee etal.1975), inspidermedium(Liuetal.1994)or
inM199medium(Invitrogen)bufferedwith50mMglycine–NaOH
atpH9.2.ToassesssensitivitiesintheC.albicansandS.cerevisiae
mutants,differentstressesweremonitoredbyspotassays.Mid-log
phasecellswereadjustedto107cells/ml,5-foldseriallydiluted,
andspottedontoplates,whichweresupplementedvariouslywith
4g/mlfluconazole,1g/mlketoconazole,1g/ml miconazole,
4g/mlamphotericinB, 15g/mlbrefeldinA,500g/mlcongo
red,500g/ml calcofluorwhite,1.5MLiCl, 1.5MNaCl,250mM
CaCl2,2.5mMglycerol,3mMsorbitol,4mMH2O2,0.02%methyl
methanesulfonate(MMS), or 30mM hydroxyurea (HU),
respec-tively.TheeffectofpHwastestedwithYPDmediumadjustedto
acidicoralkalineconditionswith150mMHEPESbufferor50mM
glycine–NaOH.
Bioinformaticalanalyses
Multi-proteinsequencealignmentswereperformedusingthe
MAFFT web application (default settings; Katoh et al. 2005)
and visualizedusing Jalview (version 2.4.0b2, default settings;
Waterhouse et al. 2009). Protein homologs were retrieved by
either BLASTP or TBLASTN from the Fungal Genome Search
(www.yeastgenome.org). Identity percentages were quantified
throughGeneDoc(version2.6.002;Nicholasetal.1997).
Candi-dategenesinbothS.cerevisiaeandC.albicanswerescreenedbythe
proteinstructurepredictionprogramInterproscan(Zdobnovand
Apweiler2001).
Strainconstructions
C.albicansandS.cerevisiaestrainsusedinthisstudyarelisted
inTable1;primersusedinstrainconstructionarelistedinTable2.
Theentirecodingsequenceoftargetgenesweredeletedfrom
wild-typestrainSN152bytwo-stephomologousrecombinationusinga
fusion-PCR-basedstrategy(NobleandJohnson2005).Briefly,the
auxotrophic markers Candida dubliniensis HIS1or Candida
mal-tosaLEU2 wereobtainedfrom plasmids pSN52and pSN40and
usedtogeneratetheknockoutPCRcassettessrd1::C.d.HIS1and
srd1::C.m.LEU2.Thesrd1nullmutantwasgenerated by
trans-formingSN152withsrd1::C.d.HIS1(creatingCaLY86),followedby
srd1::C.m.LEU2(creatingCaLY202)(Fig.1AandB).Toconstruct
theSRD1reconstitutedstrain(CaLY351),a2611bpBsiWI-BamHI
fragmentcontaining theentireSRD1 coding sequence wasfirst
clonedinto theplasmidpFA-ARG4(Schaubetal. 2006)to
Table1
Strainsusedinthisstudy.
Strain Relevantgenotype Reference
Candidaalbicans
SN152 arg4/arg4leu2/leu2his1/his1URA3/ura3::imm434IRO1/iro1::imm434 NobleandJohnson(2005)
CaLY335 (Wildtype)
arg4::ARG4/arg4leu2::LEU2/leu2his1::HIS1/his1URA3/ura3::imm434IRO1/iro1::imm434 Thisstudy
CaLY86 (SRD1/srd1)
srd1::C.d.HIS1/SRD1arg4/arg4leu2/leu2his1/his1URA3/ura3::imm434
IRO1/iro1::imm434
Thisstudy CaLY202
(srd1/srd1)
srd1::C.d.HIS1/srd1::C.m.LEU2arg4/arg4leu2/leu2his1/his1URA3/ura3::imm434
IRO1/iro1::imm434
Thisstudy CaLY351
(srd1/srd1SRD1)
srd1::SRD1::C.d.ARG4/srd1::C.mLEU2arg4/arg4leu2/leu2his1/his1 URA3/ura3::imm434IRO1/iro1::imm434
Thisstudy CaLY60
(SRD1-GFP)
SRD1::GFP::C.m.ARG4/srd1::C.d.HIS1arg4/arg4leu2/leu2his1/his1 Thisstudy CaLY204
(RIM9/rim9)
rim9::C.m.LEU2/RIM9arg4/arg4leu2/leu2his1/his1URA3/ura3::imm434
IRO1/iro1::imm434
Thisstudy CaLY207
(rim9/rim9)
rim9::C.m.LEU2/rim9::C.d.ARG4arg4/arg4leu2/leu2his1/his1URA3/ura3::imm434
IRO1/iro1::imm434
Thisstudy CaLY359
(rim9/rim9RIM9)
rim9::RIM9::SAT1-FLIP/rim9::C.d.ARG4arg4/arg4leu2/leu2::LEU2his1/his1 URA3/ura3::imm434IRO1/iro1::imm434
Thisstudy CaLY217
(srd1/srd1rim9/rim9)
srd1::C.d.HIS1/srd1::C.m.LEU2rim9::C.d.ARG4/rim9::SAT1-FLIParg4/arg4leu2/leu2 his1/his1URA3/ura3::imm434IRO1/iro1::imm434
Thisstudy CaLY247
(srd1/srd1rim9/rim9)
srd1::C.d.HIS1/srd1::C.m.LEU2rim9::C.d.ARG4/rim9::FRTarg4/arg4leu2/leu2 his1/his1URA3/ura3::imm434IRO1/iro1::imm434
Thisstudy Saccharomycescerevisiae
ScLY4 MAT␣his31leu20lys20ura30 Thisstudy
ScLY6 MAT␣ his31leu20met150ura30yol019w Thisstudy
ScLY7 MATahis31leu20ura30rim9 Thisstudy
ScLY8 MAT␣his31leu20lys20met150ura30rim9yol019w Thisstudy
ScLY5 MAT␣YOL019W::GFP::ARG4 Huhetal.(2003)
fragmentwasthenclonedintothepLY09.Theresultingplasmid
pLY15waslinearizedbydigestionwithSacIIandintegratedinto
at theSRD1 promoter of thesrd1 null mutant (Fig.1C and D).
TheC.albicansstrain CaLY60(SRD1-GFP) wascreatedby
trans-formingCaLY86withaPCRcassettegeneratedfromtheplasmid
pFA-GFP-ARG4(Schaubetal.2006)and using100bphomology
forgenomicrecombination(Golaetal.2003)(Fig.1EandF).
Aux-otrophic makers C. dubliniensis ARG4 or C. maltosa LEU2 were
obtainedfromplasmidspSN69andpSN40andusedtogenerate
theknockoutPCRcassettesrim9::C.d.ARG4andrim9::C.m.LEU2.
Therim9nullmutantwasgeneratedbytransformingSN152with
rim9::C.m.LEU2(creatingCaLY204),followedbyrim9::C.d.ARG4
(creatingCaLY207)(Fig.2AandB).ToconstructtheRIM9
reconsti-tutedstrain(CaLY359),a1581bpKpnI-XhoIfragmentcontaining
theentireRIM9codingsequencewasfirstclonedintotheplasmid
pSFS2A(Reussetal.2004)toproducetheplasmidpLY16.3′
flank-ingsequenceof394bpSacII-SacIfragmentwasthenclonedinto
thepLY16.TheresultingplasmidpLY21waslinearizedby
diges-tionwithKpnIandintegratedintoattheRIM9promoteroftherim9
nullmutant(Fig.2CandD).TheC.albicansSAT1-flippercassettewas
obtainedfromplasmidspSFS2Aandusedtogeneratetheknockout
PCRcassettesrim9::C.a.SAT1-FLIP.Thesrd1rim9doublemutant
wasgenerated bytransformingthesrd1null mutant (CaLY202)
withrim9::C.d.ARG4,followedbyrim9::C.a.SAT1-FLIP(creating
CaLY217).TheSATmarkerwassubsequentlyloopedoutofthefinal
double mutant strain(CaLY247)as described previously (Reuss
et al.2004).Proper integrationofall thetarget constructswas
confirmedbygenomicPCR(Fig.3AandB).
InS.cerevisiae,theparentalstrainBY4742,theyol019wmutant
andtherim9mutantwereobtainedfromtheS.cerevisiaeKnock-Out
collection(Winzeleretal.1999).Tocreatethedoublemutant,the
MATarim9deletedstrainwascrossedtotheMAT␣yol019wdeleted
strain, and diploids selected by micromanipulation of zygotes.
Thesediploidsweresporulatedandascidissectedby
micromanip-ulation;tetra-typetetradswereidentifiedascontainingasingle
G418sensitivesporecolony,andthegenotypeofthesporeswas
identified by PCR analysisof the rim9 and yol019w loci. The 4
colonies fromone tetratype tetradwere analyzed toprovide a
matchedsetofWT,singlemutantanddoublemutantstrains.AGFP
fusedYol019wpwasobtainedfromGFP-collectioninS.cerevisiae
(Huhetal.2003).
Stainingprocedures
Vacuolemorphologyinyeastcellswerevisualizedusingthe
flu-orescentdyeFM4-64(MolecularProbes)asreportedbeforewith
minormodifications(VidaandEmr1995).ForGFPfluorescence,
cellsinmid-logphasegrowninYPDmediumwerewashedtwicein
phosphate-bufferedsaline(PBS)andvisualizedinSCmedium.Cells
wereexaminedwitha63×oilimmersionlensunderaTCSSP5
con-focallaser-scanningmicroscope(LeicaMicrosystemsTradingLtd.,
Shanghai,China)andmicrographpictureswereacquiredusingLAS
AFLiteprogram(Version2.1.1build4443,LeicaMicrosystems).
Results
StructuralconservationofPalIproteinsinfungi
SRD1andYOL019Wareessentiallyuncharacterized,cellcycle
regulated genes predicted to code for structurally related 626
and551aminoacidlongproteinsinC.albicansandS.cerevisiae
respectively(CandidaCellCycledatabase,C3db;Coteetal.2009).
We performed a comprehensive survey of SRD1/YOL019W-like
proteins, and foundthatthis proteinfamily couldbeidentified
throughoutthefungi(Fig.4A).Overall,theevolutionary
conserva-tionofthisproteinfamilyisrelativelylowbetweenrepresentative
species,withanaverageof 30%sequencesimilarity amongkey
representativespecies suchasS.cerevisiae,C.albicans,Y.
lipoly-tica, A. nidulans,S.pombe and U. maydis. However,when these
proteinswereanalyzedinmulti-proteinalignments,weobserved
conservedproteindomainsandmotifspresentthroughout(Fig.4B).
Secondarystructureanddomainanalysisidentifiedthepresence
attheN-terminusofbothasignalpeptideandapotential3TMD,
locatedbetweenaminoacidpositions95and175.Thestructural
organizationofthe3TMDregionstronglyresemblesaPalIdomain
Fig.1.Strainconstruction.Fordetailedexplanationsofthetechniques,pleaseseematerialsandmethodssection.Smallarrowsrepresentorientationandapproximate positionofoligonucleotides(Table2)usedforfusionPCRandconfirmationofthedisruption.(A)Fusion-PCR-basedcassettemethodfordisruptionofSRD1intwosteps. (B)PCRconfirmationofthedisruptionofSRD1bygenomicDNA.Thesrd1mutant(srd1/)andthewildSN152wereanalyzedbygenomicDNAamplifiedwiththe oligonucleotidesindicatedatthebottomofthefigure.(C)ConstructionoftheSRD1reintegrantstrain.(D)There-introductionofSRD1wasverifiedbygenomicDNAand amplifiedwiththeoligonucleotidesindicatedinthebottom.(E)PCR-basedcassettemethodforconstructionoftheSRD1-GFPfusionstrains.(F)Properintegrationofthe constructattheSRD1locusinthestrainCaLY60(SRD1-GFP)wasverifiedbygenomicDNAamplifiedwiththeoligonucleotidesindicatedatthebottomofthefigure.
C-terminus,weidentifiedthepresenceoftwoextensiveconserved
regionswhoseconsensussequencedidnotmatchanycurrently
knownmotifsorfoldingdomains(Fig.4B,reddashedboxes).2As
the“RIM9 ortholog”designationfor otherspecies wasbasedon
thepresence of thePalI-domain,interpretations about
species-specificpHsensingpathwaysassociatedwithRim9/PalIdeletionin
2 Forinterpretationofthereferencestocolorinthistext,thereaderisreferredto
thewebversionofthisarticle.
fungi(Blanchin-Rolandetal.2008)havenottakenintoaccountthe
presenceofdistinctclasseswithintheproteinfamily.Thepresent
identificationoftwo distinctPalI-containingproteinclasses,the
Rim9pgroupandtheC-terminalextendedPalIgroup,suggestthat
Y.lipolyticaandseveralpezizomycotinaspecies(Fig.4A,bluegroup)
includingA.nidulansandA.fumigatus,possessasingle
representa-tivebelongingtothePalIproteinclassandthereforelackatrue
Rim9portholog.Intriguingly,thispatternisessentiallyconserved
betweenthepre-andpost-wholegenomeduplicationspecies,with
Fig.2. Strainconstruction.Fordetailedexplanationsofthetechniques,pleaseseematerialsandmethodssection.Smallarrowsrepresentorientationandapproximateposition ofoligonucleotides(Table2)usedforfusionPCRandconfirmationofthedisruptions.(A)Fusion-PCR-basedcassettemethodfordisruptionofRIM9involvedtwosteps.(B)PCR confirmationofdisruptionofRIM9.Therim9mutant(rim9/)andthewildtypeSN152wereanalyzedthroughgenomicDNAamplifiedwiththeoligonucleotidesindicated atthebottomofthefigure.(C)ConstructionoftheRIM9reintegrantstrain.(D)There-introductionofRIM9wasverifiedbygenomicDNAamplifiedwiththeoligonucleotides indicatedatthebottomofthefigure.
Fig.3. Strainconstruction.Fordetailedexplanationsofthetechnique,pleaseseematerialsandmethodssection.Smallarrowsrepresentorientationandapproximateposition ofoligonucleotides(Table2)usedforfusionPCRandconfirmationofthedisruption.(A)Fusion-PCR-basedcassettemethodfordisruptionofSRD1andRIM9infoursteps.(B) PCRconfirmationofdisruptionofSRD1andRIM9bygenomicDNA.Thesrd1rim9mutantswithorwithouttheSATmarker(srd1/rim9/)wereanalyzedbygenomic DNAamplifiedwiththeoligonucleotidesindicatedatthebottomofthefigure.
Fig.4.EvolutionaryconservationofPalIcodinggenesinyeasts.(A)HomologousPalI-containingproteinsinfungiaredisplayedaccordingtothephylogenictreerepresentation. :Indicatesmissingcandidatehomologousgene.*:ProteinsequencesoriginatingfrommultipleDNAsequencedfragmentsthatweremanuallyalignedandfusedtocreate thefull-lengthprotein.(B)Multi-proteinsequencealignmenthighlightingtheoverallproteinconservationandproteinsecondarystructure.Aschematicrepresentationof bothC.albicansSrd1andRim9proteins(attheirapproximatelength)isdisplayedaboveeachofthetwoproteinfamilies.Theapparentdiscrepancybetweentheschematic representationresultsfromthepresenceofgapsthathavetobeincludedinthemulti-proteinsequencealignmentdisplay.
inthecaseofS.cerevisiaeitispossiblethatYFR012Wrepresentsa
secondRim9-likeprotein.
OnlyRim9proteinsarerequiredforpHresponse
Rim9/PalI-likeproteinsinfungihavetypicallybeenfoundtobe
apartofapHsignaltransductioncascade(PenalvaandArst2002;
Penalvaetal.2008).Therefore,wetestedwhetherthedeletionof
theirrespectiveproteinswouldinfluencethepHresponseineither
C.albicansorS.cerevisiae.AsshowninFig.5,theC.albicanssrd1
mutant,rim9mutant,andthedoublemutantstrainsofC.albicans
grewwellinacidicmedium(pH4.9),similartothewildtype.In
con-trast,therim9mutantwassensitivetopH8.8andshowedmarked
growthreductionathigherpH(pH9.7and10.0).Complementation
oftherim9mutantrestoredsensitivitytowild-typelevel.
How-ever,thesrd1mutantwasasresistantaswildtypeuptopH10.0.
Thesrd1rim9doublemutantshowedthesamegrowthdefectto
alkalinepHastherim9mutant.Similarly,inS.cerevisiae,therim9
mutanthadagrowthdefectatpHabove7.8,whilenodifferences
wereobservedbetweenwild-typeandtheyol019wnullstrains;
thedoublemutantwasassensitivetoalkalinepHastherim9
sin-gleknockoutstrain.Together,thesefindingsestablishthatRim9
proteinsbutnotSrd1p/Yol019wpareimportantforhighalkaline
pHtoleranceinbothC.albicansandS.cerevisiae.
OnlyRim9proteinsarerequiredfortheadaptationtootherstress
responses
To investigate whether the PalI homologs in the
Rim9p-containing species are involved in other stress responses, we
subjectedtheC.albicansandS.cerevisiaemutantstrainstoa
vari-etyof challenges. Growthof thesrd1 mutant wasnot severely
affectedinresponsetoallthetestedstressesincludingionstress
Fig.5.DeletionofRIM9compromisesbothC.albicansandS.cerevisiaetoalkalinepH.C.albicans(uppanel)andS.cerevisiae(downpanel)cellswereallgrowntomid-log phaseandthenspottedin5-folddilutionsontoYPDplateswithdifferentpHasindicated.Growthdifferencesweredetectedaftera48hincubationperiod.
whiteand congored),antifungalagents (fluconazole,
ketocona-zole,miconazole,amphotericinB,andbrefeldinA),DNAdamaging
agents(MMSandHU),andoxidativestress(H2O2)(Fig.6A).
How-ever,incomparisontothewildtype,theC.albicansrim9mutant
washighlysensitivetoLiCland,toalesserdegree,amphotericin
B.Incontrast,therim9mutantexhibiteddecreasedsensitivityto
fluconazole,ketoconazole,HU,H2O2,calcoflourwhite,andcongo
red.TheRIM9reintegrantcellsrestoredtheabovesensitivityto
thewild-typelevel.Inaddition,noparticulargrowthdeficiency
couldsignificantlydifferentiatetherim9mutantfromtheparental
strainwhenthestrainsweretreatedwithNaCl,CaCl2,miconazole,
brefeldinA,orMMS.Furthermore,thesrd1rim9doublemutanthad
thesamegrowthbehaviorastherim9mutantinresponsetothe
testedstresses(Fig.6A).Intheotherhand,theS.cerevisiaerim9
singlemutant,togetherwiththerim9yol019wdoublemutant,was
highlysensitivetoLiCl,NaCland,toalesserextent,miconazoleand
KClincontrasttotheparentalcontrol(Fig.6B);nodistinguishable
growthdifferencewasobservedinthewildtypewhentreatedupon
fluconazole,ketoconazole,brefeldinA,amphotericinB,H2O2,
glyc-erol,andsorbitol(datanotshown).LossofYOL019Wdidnotaffect
Fig.6. DeletionofRIM9compromisesC.albicansandS.cerevisiaeadaptationtovariousstresses.(A)C.albicanswild-typeSN152,thesrd1mutant(srd1/),SRD1reconstituted strain,therim9mutant(rim9/),RIM9reconstitutedstrain,andthesrd1rim9mutant(srd1/rim9/)wereallgrowntomid-logphaseandthenspottedin5-fold dilutionsontoYPDplateswithorwithoutchemicalagentsasindicated.(B)S.cerevisiaewild-typeBY4742,theyol019wmutant(yol019w),therim9mutant(rim9),andthe yol019wrim9mutant(yol019wrim9)wereallgrowntomid-logphaseandthenspottedin5-folddilutionsontoYPDplateswithorwithoutchemicalagentsasindicated. Growthdifferencesweredetectedaftera48hincubationperiod.
Table2
Oligosusedinthisstudy.
Oligo Sequence LY11 TCCCAAATCAAGGGCCTGTACCTG LY12 TAATGCCAAGGTAGTATTCATCAC LY105 TCTGATTGGAGGACCAGGTAATAAGAATCAAAATGCTAGTGCTTTAAT GGGAAGAAGAGAAAATACTGGACCTAGAAGTGGTCCTTATGGTATTA CTAGAggtgctggcgcaggtgcttc LY106 TGTGTATATATATTTATGTATATAAAACATAGACGAATTGAAATAAACT AAACTAAAATAAAATAAAATCAAATAATATTATAAACTCTAACAT AAATCAtctgatatcatcgatgaattcgag LY224 TTTCTTCTTCCTCTTTTACTTTTTCC LY230 gtcagcggccgcatccctgcGGGGGAGTACCAATGTTGT LY231 TGATTAGGACGCAAAAATCG LY232 ccgctgctaggcgcgccgtgAGCTCGGATCCACTAGTAACG LY233 gcagggatgcggccgctgacGCCAGTGTGATGGATATCTGC LY238 TTACAAGTATGAAAGGAGGGG LY239 CTTCAACCTTTCAAACGATGC LY240 ACGCTAACTAACAAACTCAGTC LY243 ATCATCTCCCTTCCTCTACAC LY244 TTAGCACCTATTGCCCTTGG LY245 AGCTCCAGCTGCCACTAAAG LY246 CAACGACCACCTAATCCTCAA LY247 TGTTGTTGACCACCACGACT LY330 AATGGATCAGTGGCACCGGTG LY331 GGGCCCATTGGTTAAGTTCATATGC LY341 GCCcgtacgCTGTTTCCCGCGTAACTTC LY342 CGCggatccAGGACGCAAAAATCGAGC LY343 GCGTGTTAgagctcGGTGTAAACCAGTGTGTGATGAA LY344 TCCccgcggCGGTCAATCAGTTCCGTTCT LY364 TCAAGCCCTGTAGCTCCATT LY365 TCCGCTCATTTGATTTCCTC LY366 GCACGCCGTTACAGGAGTTA LY367 GAAGTTGGTGACGCGATTGT LY383 TTGAATATCTTGCAGATACAAACTG LY384 cacggcgcgcctagcagcggCTAATGGATCTGGATTTCATTTAG LY385 gtcagcggccgcatccctgcACAGATCTCCGCCAAATCTGTC LY386 GATGTGTGCCCTTCAGAAACC LY387 CTCTCTGTGGGGCTGACAAT LY388 CACGGAGATATCCCACCATC LY389 TTGCCTTTTGCTTTTCGAGT LY390 CAGCTAAGAAAGCCAACAACG LY391 cacggcgcgcctagcagcggGAATGAATGTATGTATGCGTG LY451 CTGGTGTTGGATCAGGAGGT LY452 TGTTGTTGAGGTGGTTGTGG LY497 ccgctgctaggcgcgccgtgTACCGGGCCCCCCCTCGAGGAA LY498 gcagggatgcggccgctgacAGCTCCACCGCGGTGGCG LY499 CGGggtaccCTCTCTGTGGGGCTGACAAT LY500 CCGctcgagCAGATTTGGCGGAGATCTGT LY501 TCCccgcggACAGATCTCCGCCAAATCTGTC LY502 GCGgagctcGATGTGTGCCCTTCAGAAACC LY503 TTGCCGGTCCTATTTACTCG LY504 GACGCTCAGTGCACACAACT
thegrowthinthesingleorthedoubledeletioncells.Overall,these observationsindicatethatRim9proteins,butnotSrd1p/Yol019wp, contributetoa varietyofstressresistancesin C.albicansandS. cerevisiae.
OnlyRim9proteinscontributetotheyeast-hyphaltransition SeveralstudieshavereportedthatRim101pathwaymembers playanimportantroleinalkalinepH-inducedfilamentation(Davis
2003).Therefore,thehyphalinductionresponsesoftheC.albicans
rim9andsrd1null strainsweretestedonavarietyof
filament-inducingmedia.AsshowninFig.7,thesrd1mutantwasabletoform
hyphaein10%inactivatedFBS,Lee’s,Spider,andM199medium(pH
9.2).Theroughnessofthesrd1coloniesandthehyphaearound
thecolonieswerethesamepatternasthoseofthewildtype.
How-ever,therim9mutantwasunabletoformhyphaeinspider(pH
7.2)or M199medium (pH 9.2);its coloniesappeared flatwith
nohyphaearoundthecolonies,whereasthoseoftheWTcontrol
werehighlyfilamentous.Althoughthecoloniesoftherim9mutant
appearedwrinkledandunevenwhengrownonserumcontaining
solidmedium(pH6.7),theperipheryofthecolonieswereflatter
thanthoseofthewildtypereference.Additionally,rim9mutants
failedtoformfilamentsaroundthecoloniesinLee’smedium(pH
6.7),thoughthesecolonieswerewrinkledandunevenlikethewild
type.Moreover,thedeletionofSRD1didnotaffectthe
morphogen-esischangeoftherim9singleknockoutcells.Thesefindingssuggest
thattherim9mutantwasdefectiveforfilamentationinC.albicans,
independentoftheextracellularpH.
NeithertheRim9northelongPalIproteinsinfluencevacuole
biogenesis
Based on their hypothesized domain structures, Srd1p and
Yol019wpwerepredictedtobemembraneassociated.To
deter-mine the localization of the Srd1-like proteins, we used the
GFP-collectionavailableinS.cerevisiae(Huhetal.2003)and
cre-atedaGFPfusedproteininC.albicans.InC.albicans,theSrd1-GFP
proteinprimarilylocalizedwiththevacuolarmembranestaining
agentFM4-64inboth yeast(Fig.8A, Cand D)andhyphal cells
(Fig.8B).Inparticular, theSrd1-GFP proteinlocalized
predomi-nantlyatnewbudsandhyphaltips,whichwasconsistentwiththe
SRD1transcriptionpeakoccurringattheG1toSphasetransition
ofthecell-cycle(Coteetal.2009).TheS.cerevisiaeYol019wp-GFP
localizedtothevacuoleaswell,mostintensivelyinthenewbud,
whichalsomatchedwithitstranscriptionallyregulatedexpression
duringtheG1/Stransition(Fig.8A).
Asshownabove,bothSrd1p-GFPandYol019wp-GFPlocalize
tovacuolecompartments,soweexaminedwhethertheirremoval
influenced vacuolarstructure. In C. albicansthewildtype, srd1
mutant,rim9mutant,andthedoubledeletionstrains,afteran
ini-tialpulseandchaseof1hFM4-64stainedbetweenonetothree
largering-likesub-compartmentswithclearbordersthatclustered
tooneregionofthecytoplasm(datanotshown).SimilarlyinS.
cerevisiae,wedonotobservesignificantdifferenceinthevacuolar
morphologybetweenthewild-typeBY4742,therim9mutant,the
yol019wmutant,andthedoublemutant(datanotshown).
There-fore,weconcludethatthedeletionofeitherRIM9orSRD1/YOL019W
inbothC.albicansandS.cerevisiaedidnotdetectablychange
vac-uolebiogenesis.
Discussion
Sequence alignmentsshow that thePalI-domain proteinsof
fungifallintotwodistinctclasses.Thelongerproteins,suchasA.
nidulansPalI,C.albicansSrd1p,S.cerevisiaeYol019wpandS.pombe
mac1p,containasignificantlylongC-terminalregioninaddition
tothePalIdomainandthesignalsequence,whilethesmaller
pro-teins,suchasS.pombemug33p,andRim9pofS.cerevisiaeandC.
albicans,consistofshortsequencesfeaturingalmostexclusivelya
PalIdomainandasignalsequence.Fungalcellsappeartohavemade
differentialuseofthesetwoclassesofPalI-domainproteins.InC.
albicans,S.cerevisiae,andpotentiallyS.pombeandUstilagomaydis,
theRim9pfamilymemberaloneiscriticalforpHandotherstress
responses(thisstudy,LiandMitchell 1997;Cornetetal.2009),
whiletheSrd1porthologhasnoapparentroleinanystress
adapta-tion(thisstudy,GrandinandCharbonneau2002;Cervantes-Chavez
etal.2010).Severalspecies,suchasA.nidulansandA.fumigatus,as
wellasY.lipolytica,lackaclearorthologofRim9p,butmakeuse
oftheirSrd1-typefamilyproteininpHstressresponse(Arstetal.
1994;Gonzalez-Lopezetal.2006).A.nidulansPalIandY.
lipolyt-icaRim9p,twoclearmembersoftheSrd1-typefamilyaccording
toourclassification(Fig.1),arefunctionallyimplicatedinthepH
stressresponseoftheirrespectivefungi.Interestingly,thelong
C-terminaltailofRim9pofY.lipolyticahadnoeffectonambientpH
Fig.7.C.albicansrim9mutantcellsaredefectiveforfilamentousgrowth.C.albicanswild-typeSN152,thesrd1mutant(srd1/),SRD1reconstitutedstrain,therim9mutant (rim9/),RIM9reconstitutedstrain,andthesrd1rim9mutant(srd1/rim9/)weregrownonsolidfilament-inducingmediaat37◦Cfor5days,andthenphotographed.
Fig.8.Srd1p/Yol019wplocalizestothevacuolarcompartment.C.albicanscellsexpressingSrd1p-GFPandS.cerevisiaecellsexpressingYol019wp-GFPwerestainedwith FM4-64tolabelvacuolesandchasedinYPDmediumfor2hat30◦C(A),andinYPDplus10%FBSmediumfor2hat37◦Ctoinducehyphalmorphogenesis(B).Cellswere
thenobservedbybrightfieldmicroscopyandfluorescencemicroscopywithappropriateexcitationlasersandemissionfiltersforGFPandFM4-64.Dashedrectangleswere presentedatahighermagnificationinCandD.Bar,5m.
Fig.9.C.albicanseffectspHofhyphal-inductionmedia.C.albicanswild-typeSN152 wasgrowninLee’s,spider,andYPDcontaining10%FBSmediaat37◦Cwithshaking.
ThepHofeachculturewasmeasuredattheindicatedtimepoints.
protein(Blanchin-Rolandetal.2008).Itistemptingtoproposean
evolutionarymodelinwhichthepHsensingandstressresponses
functionareprimarilyundertakenbyashort,Rim9-typeprotein
unlessnosuchmemberexists.InthesecasestheuniquePalIdomain
proteinbelongingtotheSrd1-typefamilyisconnectedtothepH
sensingandstressresponsepathway.Itremainsunclearwhatrole
theSrd1-typeproteinsplayincelltypescontainingtheshort-form
proteins,butitisintriguingthatthey,togetherwiththeirS.pombe
homologmac1,haveretainedG1/Scellcycleperiodicity(Coteetal.
2009).Deletionsofthesegenesdonotaffectthegrowthrateofthe
mutantcells,andremovaloftheCterminal452aminoacidsofSRD1
alsodidnotgenerateanydetectablephenotype(datanotshown).
It is apparentthat Rim9pfunction goesbeyond alkalinepH
stressadaptationandLiClhypersensitivityinC.albicans(thisstudy,
Cornetetal.2009).Inageneralsurveyofvarioussourcesof
envi-ronmentalstressinourstudy,therim9mutantwasfoundtobe
resistant tocertain antifungal agents, cell walldisruptors, and
oxidativestress.PreviousworkhassuggestedthattheRim
path-wayisrequiredforalkalinepH-inducedfilamentation(Davis2003,
2009).HereweshowthatRIM9deletionisdefectiveinforming
hyphaenotonlyinalkalinemedia(M199,Lee’s,spider),butalso
inneutral induction medium(YPD containing10% FBS).Recent
workhasrevealedthatnutrient-deprivedCandidacellsareableto
raisetheenvironmentalpHandstimulatemorphogenesis(Vylkova
etal.2011),whichisconfirmedinourstudy.Lee’sandspiderwere
initiallyneutral,buttheirpHvalueswereraisedtoweakalkaline
(spider)orevenhigherthan8(Lee’s)afterthreedaysculturewith
strains(Fig.9).Intheotherhand,CandidacellsraisedthepHof
richYPDmediumplus10%FBSfromacidtoneutral(Fig.9).This
suggeststhattheRim101pathwaymaygoverntheexpressionof
proteinsrequiredforbothalkaline-dependentand-independent
filamentation.
AlthoughtheRim9proteinsofyeastandC.albicansplay
orthol-ogousroles,theknock-outphenotypesin thetwo cellsare not
identical.BothorganismsrequireRim9pforpHresponse,buttheir
degreeofsensitivitytoalkalinepHisdifferent;theC.albicansrim9
mutanthadagrowthdeficiencyata pHabove8.8,whiletheS.
cerevisiaerim9mutantwassensitivetoapHabove7.8.TheC.
albi-cansrim9mutantwassensitivetoamphotericinB,butresistant
tofluconazole,ketoconazole,andH2O2,whiletheS.cerevisiaerim9
mutantshowedwildtyperesponsetothesestresses.Bycontrast,C.
albicansRim9pwasnotneededforresponsetoNaClormiconazole,
whileS.cerevisiaerim9mutantwassensitivetothesecompounds.
Theseobservationssuggestthatthedistinctphysiologiesofthetwo
yeastshaveconnectedthegeneralRim9pfunctiontobothcommon
andcellspecificfunctions.
Overall,thesePalI-domainfungalproteinsrepresentan
inter-estingclassofmolecules.Theyfallintotwo generalgroups,the
shortversions,exemplifiedbyRim9pinS.cerevisiae,andthelong
versions,exemplifiedbyPalIinA.nidulans,whichhavebothbeen
linkedtopHresponse,aidingthefunctionofa7-TMDpHsensor,
andtootherstressresponse.Incellsthatcontainonlyalongform
PalIproteinthiselementfunctionsincontrolofstressresponse;
incellsthatcontainbothversionstheresponsetostressresponse
islimitedtotheshortformprotein.Furtherworkwillbeneeded
tounderstandthecellularroleoftheSrd1-classofproteinsinthe
Rim9-classcontainingcells.
Acknowledgments
Wewould liketothankDr.SuzanneM.Noble(Universityof
California-SanFrancisco)fortheC.albicansSN152,plasmidpSN40
and pSN52. We thank Dr. Jackie Vogel (McGill University) for
theS. cerevisiaeYOL019W-GFP clone. We alsothank Dr. Jürgen
Wendland(CarlsbergLaboratoryinDenmark)forproviding
plas-midpFA-ARG4andpFA-GFP-ARG4andDr.JoachimMorschhauser
(UniversityofWurzburg)forproviding plasmidpSFS2A.Finally,
we thankall membersof theWhiteway lab forhelpful
discus-sions. This is NRC publication number 50696. This work was
supportedby CIHR MOP42516 toMW, China NSFC(30825041,
30630071) toYJ, ChinaNSFC(31000079) toLY,ChinaNational
973Program(2005CB523105)toYJ,ChinaNational863Program
(2008AA02Z187),andShanghai KeyScientificandTechnological
Program(10JC1417500).PCwassupportedbyaCHIRSystems
Biol-ogyScholarship.LYwassupportedbya scholarshipfromChina
ScholarshipCouncilonMOE-NRCResearchandPost-doctoral
Fel-lowshipProgram.
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