grantsosos

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Anecosystemundermore-intensivedisturbanceisexpectedtohaveamorestabilizingcompositionofinteractiontypes.
Speciesdiversityhasbeenofprimaryinter-estinunderstandingtheroleofbiodiversityinecosystemmaintenance(24,27),andamajorfocusofbiodiversityconservation.
Thepresentstudyshedsnewlightonanotheraspectofbio-diversity:diversityininteractiontypes.
Wedeter-minedthatbiodiversityinspeciesandinteractionsisintegraltostabilizingbiologicalcommunities,whichhasimportantimplicationsforbiodiver-sityconservation.
Specieslossisofvitalconser-vationconcern;however,wemayalsoneedtoidentifytheinteractiontypeslostormaintainedfortwoprimaryreasons.
First,acomplexcommu-nitymaybeself-sustainingonlyinthepresenceofdifferentinteractiontypes.
Therefore,abiasedlossofoneinteractiontypemaycriticallydestabilizethecomplexecosystem.
Second,themultiplein-teractiontypescanchangethecomplexity-stabilityeffect.
Thepositivecomplexity-stabilityrelation-shipofhybridcommunitiesimpliesthatahybridcommunityisself-sustaining.
However,itshouldbenotedthatsuchaself-sustainingcommunityismorevulnerabletocascadingbiodiversitylosses;asimplifiedcommunitycandestabilizepopu-lationdynamicsandenhanceadditionalspecieslosses.
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Acknowledgments:ThisworkwassupportedbytheEnvironmentResearchandTechnologyDevelopmentFund(grantD-1102)oftheMinistryoftheEnvironment,Japan;aGrant-in-AidforScientificResearch(B)(no.
20370012);andaGrant-in-AidforChallengingExploratoryResearch(no.
23657019,30388160)oftheJapanSocietyforthePromotionofScience.
Theauthorsdeclarenocompetingfinancialinterests.
SupplementaryMaterialswww.
sciencemag.
org/cgi/content/full/337/6092/349/DC1SupplementaryTextFigs.
S1toS814February2012;accepted25May201210.
1126/science.
1220529LAAT-1IstheLysosomalLysine/ArginineTransporterThatMaintainsAminoAcidHomeostasisBinLiu,1,4*HongweiDu,2,3,4*RachaelRutkowski,5AntonGartner,5XiaochenWang4Defectivecataboliteexportfromlysosomesresultsinlysosomalstoragediseasesinhumans.
MutationsinthecystinetransportergeneCTNScausecystinosis,butotherlysosomalaminoacidtransportersarepoorlycharacterizedatthemolecularlevel.
Here,weidentifiedtheCaenorhabditiseleganslysosomallysine/argininetransporterLAAT-1.
Lossoflaat-1causedaccumulationoflysineandarginineinenlarged,degradation-defectivelysosomes.
Inmutantsofctns-1(C.
eleganshomologofCTNS),LAAT-1wasrequiredtoreducelysosomalcystinelevelsandsuppresslysosomeenlargementbycysteamine,adrugthatalleviatescystinosisbyconvertingcystinetoalysineanalog.
LAAT-1alsomaintainedavailabilityofcytosoliclysine/arginineduringembryogenesis.
Thus,LAAT-1isthelysosomallysine/argininetransporter,whichsuggestsamolecularexplanationforhowcysteaminealleviatesalysosomalstoragedisease.
Defectsinexportinghydrolyticdegrada-tionproductsfromlysosomescausely-sosomalstoragediseasessuchascystinosis,whichischaracterizedbyintralysosomalaccu-mulationoffreecystinebecauseofmutationsinthelysosomalcystinetransportergeneCTNS(cystinosin)(1–4).
Themosteffectivetherapeuticagentforcystinosis,cysteamine(anaminothiol),convertslysosomalfreecystinetocysteineandthemixeddisulfideofcysteine-cysteamine,whichisthoughttobeexportedfromlysosomesasalysineanalogthroughalysine/cationicaminoacidtransporter(5–7).
Themolecularidentityofthetransporterremainsunknown.
Althoughbio-chemicallydetected,mostmammalianlysosomalaminoacidtransportershavenotbeenmolec-ularlycharacterized(1).
FromaforwardgeneticscreenforCaeno-rhabditiselegansmutantswithincreasedembry-oniccellcorpses,weisolatedarecessivemutantqx42thataccumulatedmanyrefractilecorpse-likeobjectsandlysotracker-positivepuncta,suggestiveofabnormallysosomes(fig.
S1,AtoG).
UsingNUC-1::mCherry,whichlabelslysosomes(8,9),orlysotrackerstaining,wefoundthatqx42ly-sosomeswereonaveragetwicethevolumeofwildtype(1.
3versus0.
5mm3)(Fig.
1,AtoF′′′,andfig.
S1,HtoK).
Wenextexaminedwhetherqx42affectedly-sosomalcargodegradation.
Apoptoticcellsarephagocytosed,thendegradedinlysosomes.
Celldeathandcellcorpseengulfmentwerenormalinqx42mutants(fig.
S2).
However,degradationofapoptoticcellsinphagolysosomes(indicatedbyGFP::RAB-7orNUC-1::mCherry)asmeasuredbylossofHIS-24::GFPorH2B::GFP(whichlabelchromatininallsomaticandgermnuclei,includingcellcorpses,respectively)wasseverelyaffectedinqx42mutants,withHIS-24::GFPpersisting>4timesaslongasinwildtype(Fig.
2Aandfig.
S2,LtoO).
Yolklipoproteinisdegradedthroughoutembryogenesistonourishdevelopingcells(10,11).
Inqx42mutants,in-testinalsecretionofyolkreporterVIT-2::GFPanduptakebyoocyteswerenormal(fig.
S3,AtoB′).
However,qx42embryosaccumulatedsig-nificantlymoreVIT-2::GFPinenlargedpuncta,whichoverlappedwithNUC-1::mCherry,sug-gestingdefectivelysosomalyolkdegradation(Fig.
2,BtoD,andfig.
S3,CtoH′).
CellsurfaceproteinsCAV-1andRME-2,whichareinternal-1GraduatePrograminChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,China.
2StateKeyLab-oratoryofMolecularandDevelopmentalBiology,InstituteofGeneticsandDevelopmentalBiology,ChineseAcademyofSciences,Beijing100101,China.
3GraduateSchool,ChineseAcademyofSciences,Beijing100039,China.
4NationalIn-stituteofBiologicalSciences,No.
7ScienceParkRoad,ZhongguancunLifeSciencePark,Beijing102206,China.
5WellcomeTrustCentreforGeneRegulationandExpression,CollegeofLifeSciences,UniversityofDundee,DundeeDD15EH,UK.
*Theseauthorscontributedequallytothiswork.
Presentaddress:WalterandElizaHallInstituteofMedicalResearch,1GRoyalParade,Parkville3052,Australia.
Towhomcorrespondenceshouldbeaddressed.
E-mail:wangxiaochen@nibs.
ac.
cnwww.
sciencemag.
orgSCIENCEVOL33720JULY2012351REPORTSonAugust1,2012www.
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1.
laat-1mutantsaccumulateenlargedlysosomes.
(AtoF′′′)EnlargedlysosomesindicatedbyNUC-1::mCherry[(A)and(B),arrows]orlysotrackerred(LTR)[(E)to(F′′′),arrowheads]wereobservedinalaat-1(qx42)embryo(B)orcell[(F)to(F′′′)]butnotwildtype[(A)and(E)to(E′′′)].
Lysosomevolumesarequantifiedin(C)and(D).
Averagelysosomalvolumes(TSEM,n=100)indifferentstrainsareshownin(D).
**PTmutationinlaat-1thatcreatesaprematurestopcodonafterAsn127.
Otherindependentlyisolatedlaat-1mutantal-lelesalsocausedenlargedlysosomeandper-sistentcellcorpsephenotypes(figs.
S1,LtoR,andS2K).
laat-1wasexpressedinvariouscelltypesinembryos,larvae,andadults(fig.
S7).
GFPormCherryfusionofLAAT-1,whichfullyrescuedqx42defects(fig.
S6,AtoE),labeledmembranesofNUC-1–orlysotracker-positivestructuresandoverlappedwithlysosomalmem-braneproteinCTNS-1,theC.
eleganshomologofhumancystinosin(17),indicatingthatLAAT-1localizestolysosomalmembranes(Fig.
1,GtoH′′,andfig.
S7,AtoC′′).
LAAT-1(299-304)::GFP,whichlackstheC-terminaldileucine-basedlyso-somalsortingmotif(18),stainedplasmamem-branesinsteadoflysosomesandfailedtorescuelaat-1(qx42)mutantphenotypes,indicatingthatLAAT-1functiondependsonitslysosomallo-calization(figs.
S6,AtoF,andS7,DtoE′′).
WeexaminedlysosomespurifiedfromC.
elegansembryos(fig.
S8)andfoundthatlossofCTNS-1causedcystineaccumulation,suggest-ingthatCTNS-1mediatescystineeffluxfromlyso-someslikehumancystinosin(Fig.
3A).
Inlaat-1mutantlysosomes,cystinelevelswerenormal,butlysineandargininelevelswere16and8timesashigheraswildtype,respectively,suggestingthatLAAT-1exportslysineandargininefromlyso-somes(Fig.
3Aandfig.
S9A).
Macrophage-likecoelomocytesfromctns-1mutantscontainedhugegranules(>6.
5mmindiameter),whichaccumu-latedendocytosedcargoCherryandwerelabeledbylysosomalmembraneproteinCUP-5butnotendosomalproteinRME-8,indicatingthattheyareenlargedlysosomes(19,20)(Fig.
3,BandC,andfig.
S9B).
Mostwild-typeandlaat-1(qx42)coelomocytescontainedsmalllysosomes(0.
05.
Datain(A),(D),(E),(F),and(G)arerepresentativeofthreeindependentexperiments.
www.
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orgSCIENCEVOL33720JULY2012353REPORTSonAugust1,2012www.
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orgDownloadedfromwhichwasalmostcompletelyabolishedwhentheinvariantProinthefirstPQloopwasmu-tatedtoLeu(Fig.
3,FandG,andfig.
S6F).
Up-takeofhistidine,butnotalanine,glutamicacid,cystine,orcysteine,wasincreasedinLAAT-1-orPQLC2-expressingcells,suggestingspecifictrans-portofcationicaminoacids(fig.
S10).
laat-1lyso-somesdidnotsignificantlyaccumulatehistidine,indicatingthatLAAT-1isprobablynotthemajorhistidinetransporterinvivo(fig.
S9A).
laat-1mutantswereviablebutdevelopedslowly(Fig.
4A).
Externalsupplementsofbothlysineandargininecompletelyrescuedretardedembryonicdevelopment(Fig.
4Bandfig.
S11,AandB)butdidnotreversetheenlargedlysosomeordefectiveyolkdegradationphenotypesinlaat-1mutants(fig.
S11C).
Thus,lossoflaat-1af-fectslysosomalexportoflysine/arginine,whichlimitstheircytoplasmicavailabilityandtherebyretardsembryonicdevelopment.
Whendeprivedofaminoacids,eukaryoticcellstriggertheaminoacidresponse(AAR)pathwaythroughactivationofGCN2proteinkinase,leadingtorepressionofglobalproteinsynthesis(21).
Consistentwiththis,laat-1embryosshowedreducedproteinsynthesis,whichwasefficientlyrescuedbysupplementinglysineandarginine(Fig.
4Candfig.
S11D)(22).
TheAARpathwayisessentialforsurvivaldur-ingaminoaciddeprivation(23,24).
gcn-2(ok871)embryosdevelopednormallybutdiedwhenlaat-1wasdefective.
Thesyntheticlethalitywascompletelyrescuedbysupplyingbothlysineandargininebutnotglycine(Fig.
4D).
Thus,lossoflaat-1limitscytosoliclysineandarginine,causingembryoniclethalitywhentheGCN-2–mediatedAARpathwayisimpaired(fig.
S11E).
WehaveidentifiedLAAT-1anditshumanhomologPQLC2asthelysosomallysine/argininetransporter.
Cysteaminetreatmentsignificantlyreducedlysosomalfreecystineandefficientlysuppressedtheenlargedlysosomephenotypeinctns-1(lf)singlemutantsbutnotlaat-1(lf)ctns-1(lf)doublemutants,whichaccumulatedthelysineanalogmixeddisulfideofcysteine-cysteamineinlysosomes,suggestingthatLAAT-1(andprob-ablyPQLC2)maymediatecystinedepletionbycysteamine.
Itisthusimportanttoexaminewheth-erlossofPQLC2affectsmammalianlysosomefunctionandcauseslysosome-relateddiseases.
Ourfindingthatdefectivelysosomalexportoflysine/arginineleadstoretardedembryonicde-velopmentrevealstheroleoflysosomalaminoacidtransportersinmaintainingcytosolicaminoacidavailabilityduringembryonicdevelopment,providinginsightsintothepathogenesisofly-sosomaltransportdisorders.
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Acknowledgments:WethankB.
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Datadescribedinthepaperarepresentedinthemaintextandthesupplementarymaterials.
ThisworkwassupportedbyaMinistryofScienceandTechnologygrant(2010CB835201)toX.
W.
andaCR-UKCareerDevelopmentAward(C11852/A4500),aCR-UKProjectGrant(C11852/A5991),andaWellcomeTrustSeniorResearchFellowship(0909444/Z/09/Z)toA.
G.
SupplementaryMaterialswww.
sciencemag.
org/cgi/content/full/337/6092/351/DC1MaterialsandMethodsFigs.
S1toS11References(25–43)8February2012;accepted23May201210.
1126/science.
1220281Fig.
4.
LAAT-1maintainslysineandarginineavailabilityfornormalembryonicdevelopment.
(AandB)Retardedembryonicdevelopmentinlaat-1mutantsisrescuedbyexternallysineandargininesupplements.
Atleast88embryoswereexamined.
(C)Proteinsynthesisratesdeterminedbyfluorescencerecoveryafterphotobleachinginwild-type,laat-1(qx42),andlaat-1(qx111)embryosexpressingPlaat-1mCherrywithorwithoutexternallysuppliedlysineandarginine.
Atleast20embryoswerequantifiedineachstrainandtreatment.
(D)Lossoflaat-1andgcn-2causessyntheticembryoniclethality.
Theyaxisindicatesthepercentageofviableembryosineachstrainandtreatment.
Threeindependentexperimentswereperformedwithatleast95embryosexaminedineach.
Inpanels(C)and(D),dataareshownasmeanTSEM.
**P<0.
0001.
Inpanels(B)to(D),lysine(K)andarginine(R)weresuppliedat100mMeach,andglycine(G)wassuppliedat200mM.
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