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Introduction

1.1TheImportanceofHomeostasisinMaintainingCellularFunction.

Organismsneedtobeabletomaintainnearlyconstantinternalenvironmentsinordertosurvive,

growandfunctioneffectively(Guyton&Hall,2006).Homeostaticmechanismsresistchangesto

theorganism'sinternalenvironment.Thesecomplexmechanismsarespecifictoeachindividual

factor,andactviaoneoftwodistinctpathways:positiveandnegativefeedback.Intheyeast

species,InternalpH,waterpotentialandtemperature(Walker,1998)areamongthemanyfactors

wherebyhomeostaticmaintenanceisvitalfornormalcellfunction.

Likeotherorganisms,yeastshavevariousphysiologicalrequirementstopermitnormalgrowth

andsurvival.Yeastrequiressubstratesandenzymesforcellfunction,andthesearefoundin

aqueoussolution(Walker,1998).Withouthighwaterconcentrations,enzymaticactivitywould

ceaseandthereforeimpairnormalcellfunction.Differentyeaststrainsexhibitdifferenttolerance

levelstochangesinwaterpotentialoftheyeastgrowthmedia.Forexample,Saccharomyces

cerevisiae(S.cerevisiae)requiresawaterpotentialbetween-5to-20(Mpa)fornormalcell

growth(Jennings,1995).

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InvestigatingtheRegulationoftheHeatShockResponseinSaccharomycesCerevisiae.

Yeastgrowthmediawithhighlevelsofexternalundissociatedorganicacidshaveaninhibitory

effectonyeastgrowthasaresultoftranslocationacrossthecellmembrane,loweringthe

intracellularpH(Walker,1998).YeastcellsinstationaryphaseareabletomaintainaconstantpH

whenextracellularpHisdecreasing(Vallietal,2004).

Oneofthemostimportantphysiologicalfactorsinfluencingyeastgrowthistemperature(Walker,

1998).Yeastspeciespossessoptimumgrowthranges,withbothminimumandmaximumgrowth

temperatures.Deviationfromthesephysiologicalparametersleadstotheonsetofmechanisms

thatmaintainhomeostasis,thusprotectingtheorganismfromirreversibledamage.

1.2TheUniversalEukaryoticHeatShockResponse

Itiswellknownthatlivingeukaryoticcellspossessmechanismstoprotectthemselvesagainst

changesinexternalenvironment(Mageretal,1993).Theseeukaryoteselicitacomplexresponse

tothermalstressinordertoprotectthecellproteomefromdegradation.Firstobservedin

DrosophilaMelanogasterbyRitosain1962,thisresponseisknownastheheatshockresponse

(HSR).Wheneukaryoticorganismsaresubjectedtothermalstressi.e.temperaturesabove

optimumgrowthandsurvival,cellularproteinsandnucleicacidsbecomedamagedashydrogen

bondingandhydrophilicinteractionsaredisrupted(Walker,1998).

Cellssubjectedtosub-lethaltemperaturesrespondbyincreasingthesynthesisofheatshock

proteins(Craig,1985)duetoincreasedtranscriptionofheatshock(HS)genesTheseHSgenesare

presentinalllivingorganisms(Khalawanetal,1997).HSgenesareinducedbytheactivationof

heatshocktranscriptionfactors(HSF)(Sorger&Pelham1988)thatbindtoheatshockelements

(Pelham&Beinz1982)atthepromoterregionsoftheHSgenes(Pelham,1982).Anumberof

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otherstressingagentsarefoundtoinducemanyofthesameHSgenes,includingexposureto

increasedethanolconcentrations,exposuretoheavymetal-ionconcentrations,(Parsell&

Lindquist,1993)oxidativeandosmoticstress(Mager,Ferreira,1993).

TheseHSgenesencodehighlyconserved,ubiquitousheatshockproteins(Hsps)thatactas

molecular'chaperones'.TheseHspsbindtocellularproteinstocopewithdenaturationinduced

bythermalstress(Federetal,1999).Theyactby"folding,trafficking,maturing,anddegrading"

cellularproteins(Hashikawa,etal2004).Heatshockproteinsdisplayavarietyoffunctions.Hsps

functiongenericallyby"assistingpolypeptidestoself-assemblebyinhibitingalternativeassembly

pathwaysthatproducenonfunctionalstructures"(Ellisetal,1991).Hspsacttomaintainnormal

cellularfunctions,andareinducedatdifferenttemperaturesindifferentorganisms(Lindquist,

1986).

1.3TheMolecularResponseInducedByHeatStress

Theheatshockfactors(HSF)andheatshockelements(HSE)involvedintheHSRarehighly

conservedtranscriptionalsequencespresentinnature(Hahnetal,2004).S.cerevisiaecontains

manyofthesameHSgenesasotherorganisms.Thehsp70geneappearstohavebeenconserved

throughoutevolutionandispresentamongstvariousorganisms,includingbothDrosophilaandS.

cerevisiae(Lindquist,1984).S.cerevisiaehasthereforebeenutilisedasamodeleukaryotic

organismtoattempttounderstandthemolecularmechanismsinvolvedinactivation,durationof

responseandthefunctionsofheatshockproteins.

Theheatshockresponseiscontrolledatthetranscriptionallevel.InS.cerevisiae,themolecular

responsehasbeenhighlycharacterised.Investigationsonamolecularlevelhavehighlighteda

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numberofcomplexprocessesandeventsdetailingthewholeresponse.Thephosphorylationstate

oftheheatshocktranscriptionfactorinS.cerevisiae(ScHsf1)isfoundinthenucleusboundtoHSE

undernormalconditions(Pelham&Jakobsen,1988).TwotranscriptionaldomainsnamelyAR1

andAR2locatedontheCandN-terminalofScHsf1haveactivationdomainsthatarerepressed

undernormalconditions.Ithasbeenfoundthathyper-phosphorylationoftheC-terminalofthese

activationdomainsresultsinactivationofScHsf1andsoinductionofHSgenes(Hashikawaetal,

2004).ScHsf1belongstoafamilyofwingeddomainproteins(Littlefieldetal,1999).These

'wingeddomains'areknowntointeractwithDNA.Morespecifically,'wingeddomains'interact

upstreamofthetranscriptionstartsitesofgenesencodingHsps;thesepromotersarecalledHSE.

ThesewingeddomainsareimportantforoptimalbindingtoHSEs(Ciceroetal,2001).Anincrease

intheexpressionofHspgenes,resultinaconcomitantincreaseinHspmRNAs,whichmaythen

subsequentlybetranslatedintoHsps.ThesynthesisofHspmRNAandHspspeak10-20minutes

aftercellsaresubjectedtoheatshock(Broachetal,1992).

Hspspossessavarietyoffunctionsinprotectingthecellfromproteindegradation.Thenamesand

functionsofthemajorHspsinS.cerevisiaehavebeensummarizedinthetablebelow.

FunctionsoftheMajorHeatShockProteinsinS.cerevisiae.

HeatShockProtein Hsp104

Hsp83 Hsp70Family

Hsp60

ProposedPhysiologicalFunction Acquisitionofstresstolerance.Constitutivelyexpressed inrespiring,notfermentingcellsandonentryinto stationaryphase. Chaperone(s)function. Interactwithdenatured,aggregatedproteinsandassists insolubilisingthemwithsimultaneousrefolding(i.e. chaperones(s)function).Alsoinvolvedinpost- translationalimportpathways. SimilartoHsp70,Thischaperoninfamilyfacilitatepost- translationalassemblyofproteins.Hsp60facilitatesthe

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foldingandassemblyofunfoldedproteinsinanATP- dependentmannerbydirectlybindingtotheunfolded protein(Craig,1994). Cellularrolestillelusive,butmaybeinvolvedinentry intostationaryphaseandtheinductionofsporulation. Hsp30mayregulateplasmamembraneATPase.

SmallHsps Hsp30 Hsp26 Hsp12

Table1.FunctionsofmajorheatshockproteinsinS.cerevisiae.Adaptedfrom:Walker,G

(1998).YEASTPhysiologyandBiotechnology.WestSussex,England:Wiley&SonsLtd.151

DespitedetailedmolecularcharacterisationoftheresponseinS.cerevisiaethetriggerfor

activationofthetranscriptionfactorhasnotyetbeendiscovered.Asaresult,theregulatorofthe

molecularHSRisunknown.

1.4TemperatureastheDirectInduceroftheHeatShockResponse.

TheHSRisaresponsetosub-lethaltemperatureasameansofprotectionfromthermaldamage

(Sorger&Pelham1988).S.cerevisiaecharacterisesanormaltemperaturerangebetween35-43°C

(Walker,1998)containingminimum,maximumandoptimumtemperatures(Tmin,Tmax,andTopt).

Itisthereforereasonabletoinferthatthermalstressingagentsareresponsibleforinductionof

theHSR.Exposuretosub-lethaltemperatures,resultinproteindenaturationanddamage.Cells

respondtothepresenceofthermallydenaturedproteinsandinducearesponsetosynthesise

Hsps.Untilthelateeightiesthe'classical'viewwasinfactthattemperatureitselfwasthoughtobe

thedirectinduceroftheHSR(Lindquist,1986).Severalstudiesprovidedevidenceinfavourof

this.IfthereweresecondarymessengersinvolvedinHsptranscriptionotherthantemperature,

thentemperaturewoulddamagethesesecondarymessengersandhenceaffecttranscription.

VariousstudiesdemonstratedthelackofsecondarymessengersininductionoftheHSR.When

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heatshockedcellsaresubjectedtonormaltemperatures,theydonotimmediatelyceaseto

synthesiseHsps(Lindquist,1981).Thelackofasecondarymessengerintranscriptional

regulationcontributedtotheideaoftemperatureastheprimarytranscriptionaltrigger.

1.5TheTransientNatureoftheHeatShockResponse,andtheAbilitytobeinducedby

OtherStressAgents.

Despitethereasonableargumentthattemperatureactsasthetrigger/induceroftheheatshock

response,thediscoveryofakeyphenomenonintheHSRcontradictedthis'classicallyheld'view

(Lindquist,1986).TheHSRwasdiscoveredtobehaveintransientmanner(Miller,etal,1990).

Whensubjectedtosub-lethaltemperatures,theHSRwasfoundtodiminishwithinonehourof

inductionandreturntonormalproteinsynthesis(Milleretal,1990).MorespecificallyintheS.

cerevisiaetranscriptionfactor(ScHsf1)theN-terminalregionoftheAR2domainofScHsf1was

foundtohaveseparablepropertiestotheC-terminalregion,inthatitwasabletoinducea

transientresponse(Sorger,1990).Thediscoveryofthisphenomenonchallengedtheideaof

temperatureasadirecttriggeroftheHSR.TheveryfactthatthenatureoftheHSRistransient,

suggeststhatthetriggerfortheHSRis'short-lived'.

Temperature,bydefinitioncannotactasthetriggerfortranscriptionalactivationoftheHSR.

TemperatureisaconstantfactorintheHSR.Ifindeedtemperaturedoesactasadirect

transcriptionaltrigger,itshouldlogicallyresultinaconstantheatshockresponse,andtherefore

nodiminutionoftheresponseshouldbeobserved.However,thisisnottheobservedresponse

(Miller,etal1979).Asaresult,itislogicaltosuggesttemperaturepossiblyplaysanindirectrole

inHSRinductionbutnotadirectrole.Thisdiscoverychallengedexistingideasandfurthermore

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developedresearchtargetswiththeaimofdiscoveringthe'directtrigger'ofthisuniversalstress-

response.

(Milleretal,1979)presentedevidencetocharacterizeatransientHSR.Itcanthereforebeargued

thatthemechanismthatsensestemperaturebecomesdesensitizedovertime.Another

phenomenondescribed,indicatesthatorganismsexposedtomildheatshockexhibitinduced

thermotolerance(Parsell&Lindquist,1993).S.cerevisiaecellssubjectedtobriefheatshockat

mildtemperatures,exhibitresistancetothermaldenaturationatotherwiselethaltemperatures.

CellssubjectedtoHSat37°CdisplayatransientHSR.Whenbrieflyexposedtolethal

temperatures,thesecellsexhibitresistancetothermaldamageasaresultofinduced

thermotolerance(McAlister&Finkelstein,1980)

Furthermoreinthepresenceofethanol,theHSRincreasesinsensitivityasthetemperature

requiredformaximalHSinductionisdecreased(Curran&Khalawan,1994).Plasmamembrane

ATPaseactivityinfluencestheHSR(Panaretou&Piper,1990)aswellasosmoticstress(Varelaet

al,1992).MorespecificallyHsp-104hasshowntoplayanimportantroleinthermotolerance,

includingcellswithmutatedHsf1.(Lindquistetal,1996).Neitherthetransientresponseor

ethanolsensitivephenomenonsupportstheclassicalideathatorganismsresponddirectlyto

thermallydenaturedproteins,toinducetheHSR.Instead,theseconceptssuggestthatthe'primary

sensor'thatdetectscellularproteindenaturationisanactive,adaptabletrigger(Chatterjeeetal,

1997)thatisabletochangetovarysensitivityoftheHSRtothesametemperature

(thermotolerance).

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1.6Evidencetosuggesttheheatshockresponseislipidmediated(S.cerevisiae).

InductionoftheHSRinvolvesphosphorylationandthereforeactivationofSchsf1.HSisalso

inducedattemperaturesspecifictoeachindividualcell(Carratuetal,1996).Howeverthe

phosphorylatingagentisunknown.ThemolecularstructureofthecellmembraneinS.

cerevisiaewasinvestigatedasapotentialregulatorofHSRinduction.Commonphysiological

responsestoachangeinexternalenvironmentalconditionsconsistoflipidmembrane

reorganizationandmodification(Rogers&Glasser,1993).Responsestotemperaturechanges

areknowntoinvolvefattyaciddesaturation.(Lee&Cossins,1990).S.cerevisiaewasuseda

modeltodeterminewhethertheHSRislipidmediated.Exposureofatemperaturesensitive

strainofS.cerevisiaetosaturatedfattyacids(SFA)resultedinanincreasedtranscriptionofHS

genetranscriptionat37°C,andadditionofunsaturatedfattyacids(UFA)downregulatedHS

genetranscription(Carratuetal,1996).

Furtherinvestigationsdevelopedthesefindingstolinkcellularlipidconformationsas

responsiblefordesensitisationandthereforethetransientnatureoftheHSR.Unsaturated

fattyacid(UFA)levelsweremonitoredduringsub-lethalheatshockatvaryingtemperatures

(Chatterjeeetal,1997).Followingashiftintemperatureofyeastcellsfromoptimumtosub-

lethaltemperatures,anincreaseincellularfattyacidunsaturationisassociatedwithan

increasedtemperatureatwhichmaximalHSoccurs.Unsaturatedfattyacidlevelsandthe

maximalHSRbothdeclinewhenreacclimatizedfromsub-lethaltooptimumtemperatures

(Khalawanetal,1996).ThesefindingsdemonstratedthatdensitisationoftheHSRwaslinked

toUFAcellularlevels.FurthermorefindingssuggestedthekineticsofUFAlevelswere

consistentwiththekineticsfordownregulationofHSgeneinduction,thetransientresponse

(Khalawanetal,1996).

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WithevidenceproposinglinksbetweenUFAlevelsandHSinduction,itcanbereasonedthat

UFAlevelsplayaregulatoryroleintheHSR.InS.cerevisiaetheOLE1geneencodesthedelta-9

desaturaseenzymeresponsibleforfattyaciddesaturation(Stukey,etal,1990).IfUFA

desaturationactsasthe'primarysensor'inHSgenetranscription,knockoutoftheOLE1gene

wouldresultinaninabilityoffattyacidstobecomeunsaturated.Ifthecellularlipidsremain

saturatedandifdesaturationisthetriggerforHSdownregulation,aconstantlysaturatedFA

cellularprofilewouldresultintheinabilityoftheHSRtobedownregulated.TheHSRwould

remainconstant,thereforehighlightingUFAlevelsasresponsibleformediatingthetransient

natureoftheHSR.

1.7YeastCharacteristicsandCellMembranePhysiology

"Yeastsareascomycetousorbaidomycetousfungithatreproducevegetativelybybuddingor

fission,andthatformsexualstateswhicharenotenclosedinafruitingbody."(Boekhoutand

Kurtzman,1996).Yeastcellmembranesactsasimpermeablebarriersagainsthydrophilic

moleculestopreventthemixingofthecytoplasmandexternalenvironment.Around7.5nthick,

thecellmembraneiscomposedofalipidbilayer(Walker,1998).Aswithalleukaryotic

membranes,thelipidbilayercontainsglobularproteinsdispersedthroughoutalipidmembrane,

toformafluidmosaicstructure(Nicholson&Singer,1972).Thecellmembraneconsistsof

discontinuousamphipathiclipidbilayers,withthepolarhydrophobictailsfacinginternally,and

theirnon-polarhydrophilicheadsfacingoutwards.

Bothintegralandmembranespanningproteinsarefounddispersedthroughoutthemembrane.

Proteinsplayaroleintransportthroughactingascarrierorchannelproteins(Guyton&Hall,

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2006).Forexample,theprotonpumpingplasmamembraneATPase,utilizesATPtoexpelcellular

proteinstocreateanelectrochemicalgradientfornutrientuptake(Cooteetal,1994).Thelipid

bilayerisprimarilycomposedofphospholipids,mainlyphosphatidylcholineandsterols(Walker,

1998).Thephosphatidylcontentincreasedbyapproximately10-foldinS.cerevisiae(Walker,

1998).Sterolsstabilizethelipidbilayerwhereasphospholipidsaddfluidity(Walker,1998).The

selectivepermeability,mediatedbycertainproteinsandlipidsexhibitinghydrophobicand

hydrophilicinteractions(Nicholson&Singer,1972)ofthesemembranesservetocontrolwhat

canenterandleavethecell.

LipidComponentsOfThePlasmaMembrane

Figure1.Lipidcomponentsoftheplasmamembrane.Theouterleafletconsistspredominantlyof phosphatidylcholine,sphingomyelin,andglycolipids,whereastheinnerleafletcontains phosphatidylethanolamine,phosphatidylserine,andphosphatidylinositol.

Cholesterolisdistributedinbothleaflets.Thenetnegativechargeoftheheadgroupsof phosphatidylserineandphosphatidylinositolisindicated.

Adaptedfrom:Cooper,GM(2000).StructureofthePlasmaMembrane,TheCell:AMolecular Approach.2nded.SunderlandMA:SinauerAssociates.1.

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Thecellmembranehasavastrangeoffunctions,ofwhichthemainonesarementionedbelow

withrelevantexamples.

MainFunctionsoftheCellMembraneinS.cerevisiae.

RelevantExamplesWhereThisIs Shown AphysicalbarriertopreventthemixingofPlasmamembraneprotonpump(ATPase) aqueousandcytoplasmicmolecules,andtoisvitalinexpellingcellularproteinsin controlwhatentersandleavesthecell.ordertocreatetheelectrochemical Membraneproteinsmediatethisresponse.gradientneededforuptakeofessential solutes(e.gH+K+Ca2+ CellSignalling.InS.cerevisiaePhosphatidylinositol4,5-bisphosphateis phosphoinositidephopsphorylationformsresponsibleforrolesincellular moleculesthatarefurtherbrokendowntoproliferation. formmoleculesthatactassecondary messengersinmajorsignalingpathways.

Exocytosis Secretoryvesiclesaresecretedbythe GolgiapparatusandtheEndoplasmic Reticulum.Thesevesiclesfusewiththe plasmamembranetoexpeltheunwanted molecule. Endosomesinternalizestructures requiredbythecell.Invaginationsfrom thecellmembraneare'pinched'awayto formvesiclesthatarethentransported throughthecytoplasm.InS.cerevisiae thisprocessisimportantininternalizing matingpheromes.

FunctionsOfTheYeastCellMembrane

Endocytosis

Table2:MainFunctionsoftheCellMembraneinS.cerevisiae..Source:Walker,G(1998).YEAST

PhysiologyandBiotechnology.WestSussex,England:Wiley&SonsLtd.19-21.

1.8PrinciplesofOsmosis

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Osmosisiscommonlyknownasthemovementofparticlesthroughasemi-permeablemembrane,

fromareasofhighwaterpotential(lowsoluteconcentration)toareasoflowwaterpotential(high

soluteconcentration).Thefactthatthecellmembraneissemi-permeableallowstheexistenceof

thisphenomenon.(Roseetal).

Duetothesmallsizeofwatermolecules,theyexistathighconcentrationsinsolution.Purewater

existsataconcentrationof55.4Mat20°C)(Roseetal).Thishighconcentrationdoesn'tappearto

bedramaticallyalteredinsolutionsmixedwithothersolutes.Itistheassociationofwater

moleculeswiththesesolutesthatchangethestateofwater.(Roseetal).Achangeinthestateof

wateraffectstheamountofthermodynamicallyavailablewater.Thewaterpotentialisdefinedas

"thefunctionoftheconcentrationofsoluteparticles."(Roseetal).

OsmoticpotentialsinS.cerevisiaewerededucedbydegradingthecellwalltoformprotoplasts.

Investigationshighlightedthataprotoplastconcentrationof0.5M,equivalenttoawaterpotential

of-1.5mpawassufficienttomaintainnormalturgorpressure,andnormalcellularstructure.(Rose

etal).Externalwaterpotentialandcellularosmoticpotentialformthebasisofturgorpressure:

pressureofthecellconstituentsagainstthecellwalloftheorganism.

1.9UsingOsmosistoInduceStructuralChangestoYeastCellMembranes

Evidence(seesection1.7)clearlyillustratesthatthecellmembraneofyeasts,andalleukaryotes

arevitalinperformingessentialcellularfunctions.Lipidsformanintegralpartofthecell

membrane,structureandfunction.Evidence(seesection1.6)suggeststhetransientHSRislipid

mediated,(Chatterjeeetal,1997).Changingthestructureoftheyeastcellmembranewould

disruptlipidstructureandmetabolism.IftheHSRislipidmediated,thenchangestocellularlipid

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content,wouldhaveaneffectontheHSR.Toexperimentallyinducethesestructuralchanges,cells

couldbesubjectedtoosmoticshock,followedbyHStomonitorchangesintheHSR.

1.10UtilisingGeneDeletionTechniquesToMeasureHeatShockRegulation

Homologousrecombinationistheabilityofanorganismtoexchangenucleotidesequences

betweensimilarsectionsofDNA.Thischaracteristichasbeenexploitedtoformthebasisofgene

deletiontechnology.Homologousrecombinationcanbeutilisedtopinpointregulationofcellular

responsesonamolecularlevel.MethodsweredevelopedtoenablefragmentsofDNAtobe

integratedintothegenomeofcells(Tropp,2004)toknockoutaparticulargene:eitherviagene

replacementorgeneinsertion(Tropp,2004).Genereplacementinvolvesthereplacementofthe

wholecodingsequenceofatargetgenewithaselectablemarker.Thisresultsingeneknockout

anddisruptionofgenefunction.Knockoutgenesarereplacedwithacodingsequencethatcanbe

detected.SequencehomologybetweentherecipientgeneandtheDNAfragmentenabletheDNA

constructtointegrateatthegeneyouwishtoreplace.

ConstructionoftheDNAfragmentcanbepreparedviaPCRmethods.Primerscontain

approximately'50bpofhomologytothegeneofinterestand20bofhomologytotheselectable

marker'(Tropp,2004)resultinginaPCRproductwith50bpofsequencehomologytothegene

targetedforknockout.

TheOLE1genecanbesubjectedtogenedeletionviathesamemethodology.Usingtheamplified

HIS4genethathasbeendesignedtosharesequencehomologywiththeOLE1gene,theOLE1gene

canbedeleted.InsertionoftheHIS4DNAfragmentintotheS.cerevisiaeDBY747lacZstrainresults

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inhomologousrecombinationtoreplacetheOLE1gene.TheHIS4geneencodestheaminoacid

histidine.TheoriginalDBY747lacZstraincannotgrowonmedialackinghistidine,leucine,or

tryptophanastheplasmiddoesnotcontainthegenesabletosynthesisetheaminoacids.

However,replacementoftheOLE1genewithHIS4resultsintheabilityoftherecombinant

plasmidtosynthesishistidine.Thisresultsinthefollowingphenotypes:

1.Wild-typeDBY747lacZphenotype:His-Leu-Tryp-

2.Mutant-DBY747lacZphenotype:His+Leu-Tryp-

HIS4isthenusedasadetectablemarkerasitisknownthatthemutant-DBY747lacZstraincan

synthesiseitsownhistidine,andcanthereforegrowonmedialackinghistidine,onlyrequiring

leucineandtryptophan.

HavingsuccessfullyknockedouttheOLE1gene,themutantstaincanbetestedforexpected

behaviourstoindicatewhetherofnotthetransientnatureoftheHSRislipidmediated(see

section1.6).

1.11AimsandHypotheses

Theaimsofthisprojectaretwofold.Evidencesuggeststhatthetransientnatureoftheheatshock

responseappearstobelipidmediated.'Knockingout'theOLE1generesponsibleforthistransient

natureandmonitoringforanexpectedchangeinheatshockactivity,couldestablishwhetherthis

isthecase,onamolecularlevel.Secondly,cellsunderosmoticstressundergophysiological

changesacrossthecellmembraneandinothercellcomponents.Iftheheatshockresponseislipid

mediated,structuralchangestotheselipidsasaresultofosmoticstressshouldinduceachangein

thedynamicsoftheHSR.

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Theprojectaimsleadstothefollowingtwohypotheses:

1.DoestheknockoutoftheOLE1geneaffecttheheatshockresponse?Ifso,istheOLE1gene

responsibleforthetransientnatureoftheHSR?

2.IstheHSRaffectedwhenlipidstructureandmetabolismaredisruptedinS.cerevisiaevia

osmoticpressurechanges?

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