TRAPPIII is responsible for the vesicular transport from early endosomes to the Golgi apparatus ...

41 Pages·2013·5.59 MB·English

Research Article 4963
TRAPPIII is responsible for vesicular transport from
early endosomes to Golgi, facilitating Atg9 cycling
in autophagy
Kanae Shirahama-Noda1, Shintaro Kira1, Tamotsu Yoshimori2,3 and Takeshi Noda1,2,*
1CenterforFrontierOralScience,GraduateSchoolofDentistry,OsakaUniversity,OsakaUniversity,565-0871Osaka,Japan
2GraduateSchoolofFrontierBioscience,OsakaUniversity,565-0871Osaka,Japan
3DepartmentofGenetics,GraduateSchoolofMedicine,OsakaUniversity,565-0871Osaka,Japan
*Authorforcorrespondence([email protected])
Accepted8August2013
JournalofCellScience126,4963–4973
(cid:2)2013.PublishedbyTheCompanyofBiologistsLtd
doi:10.1242/jcs.131318
Summary
Autophagy is a bulk protein-degradation process that is regulated by many factors. In this study, we quantitatively assessed the
contribution of each essential yeast gene to autophagy. Of the contributing factors that we identified, we focused on the TRAPPIII
complex, which was recently shown to act as a guanine-nucleotide exchange factor for the Rab small GTPase Ypt1. Autophagy is
defective in the TRAPPIII mutant under nutrient-rich conditions (Cvt pathway), but starvation-induced autophagy is only partially
affected. Here, we show that TRAPPIII functions at the Golgi complex to receive general retrograde vesicle traffic from early
e
c endosomes.CargoproteinsinthisTRAPPIII-dependentpathwayincludeAtg9,atransmembraneproteinthatisessentialforautophagy,
n andSnc1,aSNAREunrelatedtoautophagy.Whencellswerestarved,furtherdisruptionofvesiclemovementfromlateendosomesto
e
ci the Golgi caused defects in Atg9 trafficking and autophagy. Thus, TRAPPIII-dependent sorting pathways provide Atg9 reservoirs for
S
pre-autophagosomal structure and phagophore assembly sites under nutrient-rich conditions, whereas the late endosome-to-Golgi
ell pathway is added to these reservoirs when nutrients are limited. This clarification of the role of TRAPPIII elucidates how general
C membrane traffic contributes to autophagy.
f
o
al Keywords:Autophagy,TRAPP,Golgi-associatedretrogradeprotein,Sortingnexin,Retromer,Atg9,Snc1
n
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Introduction formation, and other small structures called peripheral reservoirs
Autophagy is an evolutionarily conserved intracellular degradation (Reggiori et al., 2004a). Mutation of Tlg2 or COG complexes
systemthatcontributestoawiderangeofphysiologicalphenomena inhibitsthisrecycling,suggestingthatthesefactorsareinvolvedin
(Mizushima and Komatsu, 2011). However, the formation of the Atg9 dynamics (Ohashi and Munro, 2010; Yen et al., 2010). In
autophagosome,asphericalmembranestructurethatsequestersthe mammalian cells, Atg9 is transiently associated with some
degradation substrates, remains poorly understood (Yoshimori and endosomes (Kageyama et al., 2011; Orsi et al., 2012). A recent
Noda,2008).Theautophagosomeissurroundedbytwomembrane study characterized Atg9-containing vesicles with respect to
layers, suggesting that it forms by membrane dynamics that differ physical characteristics such as their size and the number of
from the mechanisms generally responsible for vesicle budding. Atg9moleculescontainedineachvesicle(Yamamotoetal.,2012);
Formation of the autophagosome is mediated by the Atg proteins, however, the identity and significance of this vesicular-transport
whichincludetwoubiquitin-likeproteins(Nakatogawaetal.,2009). pathwayhavenotbeenclearlyelucidated.
InadditiontotheAtgproteins,numerousotherproteinsareinvolved, In this study, we screened a mutant collection containing
either directly or indirectly, in autophagy. However, it remains knockdownsofallessentialgenesinSaccharomycescerevisiae,by
unclear how other membrane-trafficking pathways are related to measuringtheautophagiccompetencyofeachmutant.Amongthe
autophagy(LongattiandTooze,2009).Severalyeastmutationsthat factors that we identified as contributing significantly to
cause defects in intracellular vesicular trafficking also affect autophagy,wefocusedonTRAPPIII,whichhasbeenreportedto
autophagy. For example, mutation of Tlg2, a t-SNARE localized be specifically involved in autophagy at the PAS, where
inendosomesandtheGolgi,disruptsautophagy,butdoesnotabolish autophagosome formation takes place (Lynch-Day et al., 2010).
it(Abeliovichetal.,1999).Similarly,earlyandlatesecmutants,as In contrast to the previous report, our results demonstrated that
wellasmutantsintheconservedoligomericGolgi(COG)complex, TRAPPIIIplaysaroleingeneralvesiculartraffickingattheGolgi,
exhibit defective autophagy (Geng et al., 2010; Hamasaki et al., rather than in an autophagy-specific process. These findings
2003;Ishiharaetal.,2001;Reggiorietal.,2004b;Yenetal.,2010). explainhowgeneralvesiculartrafficcontributestoautophagy.
Atg9 is a multiple-transmembrane protein that is essential for
autophagosomeformation(Nodaetal.,2000;Youngetal.,2006); Results
it is recycled between pre-autophagosomal structure/phagophore IntheyeastSaccharomycescerevisiae,analkalinephosphataseassay
assembly sites (PAS), which are involved in autophagosome is widely used to quantitatively assess autophagy (Noda and
4964 Journal of Cell Science 126 (21)
Klionsky, 2008; Noda et al., 1995). To obtain a comprehensive Atg9 is recycled between PAS and as-yet-unidentified
understandingofthemechanismsunderlyingregulationofautophagy, peripheral reservoirs, previously characterized as multiple
weextendedthealkalinephosphataseassaysystembyadaptingthe punctate compartments distributed throughout the cytosol (He
procedure to 96-well microtiter plates, enabling large-scale andKlionsky,2007;Nodaetal.,2000).AlthoughAtg9recycling
quantitativeestimationofautophagiccompetencyandtheeffectsof has previously been reported to be independent of TRAPPIII
everynon-essentialgenemutationinyeast(S.K.etal.,unpublished (Lynch-Dayetal.,2010),were-examinedthisissueusingamore
results).Here,weappliedthissystemtotheessentialgeneknockdown elaborate experimental system that exploits ubiquitylation-
collection,includingabout900mutants,inwhichthemRNAforeach mediated degradation of the IAA protein induced by addition
gene is destabilized by insertion of a marker gene into the 39- of auxin (NAA) to the medium (Nishimura et al., 2009). In the
untranslatedregion(DAmPmethod)(Schuldineretal.,2005).From presenceofauxin,Trs85taggedwithIAAisnotdetectedbecause
thiscollection,weidentifiedagroupofgenes,includingTRS20and itisubiquitylatedanddegraded,whereasintheabsenceofauxin,
BET5, whose knockdown resulted in markedly low autophagic TRS85-IAAisstablyexpressedfromitsownpromoterandisnot
activity (Fig.1A,B). Trs20 and Bet5 are core subunits of TRAPP degraded (Fig. 2A). Defect in API processing observed in the
complexes, which function at tethering step in several vesicular- absence of TRS85 due to degradation was recovered after
transport pathways (Barrowman et al., 2010). The three TRAPP removal of auxin (Fig. 2B). We observed mCherry-Atg8 dot
formation in these conditions, and found that it is severely
complexes–TRAPPI,TRAPPIIandTRAPPIII–havebeenreported
defectedinthepresenceofauxin,butrecoveredafterremovalof
tofunctioninendoplasmicreticulum(ER)-to-Golgitransport,intra-
auxin (Fig. 2C). Consistent with the results of a previous paper
Golgitransportandautophagy,respectively(Lynch-Dayetal.,2010).
(Lynch-Dayetal.,2010),punctaofAtg9-36GFPweredispersed
Each TRAPP complex serves as a guanine-nucleotide exchange
throughthecytoplasmevenintheabsenceofTrs85,apatternthat
factorfortheRabGTPaseYpt1(Lynch-Dayetal.,2010),whichwas
wasindistinguishablefromthatobservedinthepresenceofTrs85
alsohighlyrankedasacandidategeneinourscreen(Fig.1A).The
(Fig. 2D, left top and bottom panels). When we knocked out
only subunit that is specific to TRAPPIII, Trs85, was also highly
ATG1,aproteinkinaserequiredformovementofAtg9fromthe
rankedinourscreenofmutationsofnonessentialgenes(S.K.etal.,
PAS to peripheral pools, Atg9-36GFP accumulated at the PAS,
e unpublishedresults).Therefore,basedontheresultsofourunbiased
c which was observed as a bright punctate signal (Reggiori et al.,
n genome-wide screen, we focused on TRAPPIII in our subsequent
e 2004a)(Fig. 2D,rightbottompanel).Bycontrast,intheabsence
ci investigations. ofTrs85inatg1mutants,Atg9-36GFPwasdispersedthroughout
S
thecells(Fig. 2D,righttoppanel).Thus,Atg9traffickingtothe
ell PAS, and resultant PAS formation, is dependent on TRAPPIII.
C
Recently, TRAPPIII was reported to be associated with the
of vesiclewhereAtg9resides(Kakutaetal.,2012).Consistentwith
al this, subsets of Trs85-36mCherry-positive puncta and Atg9-
n 36GFP-positive puncta were also double-positive (12.2% and
r
u 5.23%, respectively, in over 300 cells) (supplementary material
o
J Fig.S1A).Furthermore,subsetsofGFP-Ypt1positivepunctaand
apartofAtg9-36mCherrywerealsodoublepositive(10.8%and
2.73%, respectively, in over 400 cells) (supplementary material
Fig. S1B). Therefore, at least some Atg9-positive puncta
correlate with the vesicles in which TRAPPIII and/or Ypt1
resides.
In contrast to the situation in nutrient-rich conditions,
TRAPPIII (trs85) and atg1 double-mutant cells did not exhibit
defective Atg9 transport to the PAS under starvation conditions
(Fig. 3A,B). On the basis of this observation, we hypothesized
that under starvation conditions, another pathway bypasses the
TRAPPIII-dependent pathway for transportto the PAS. The Cvt
pathway is a selective autophagic process in which API is
transportedtothevacuoleandprocessedtoitsmatureform(Scott
et al., 1996). TRAPPIII (trs85) null mutants did not exhibit
defects in the Cvt pathway under starvation conditions, whereas
they were severely defective under nutrient-rich conditions,
consistent with the existence of a starvation-specific bypass
pathway (Meiling-Wesse et al., 2005; Nazarko et al., 2005).
Inanefforttoidentifythe bypasspathway,wefirstexamined
Fig.1. Genome-wideexaminationofautophagyinacollectionof
Retromer, a protein complex that functions in retrograde
knockdownmutantsofessentialgenes.(A)Autophagicactivitiesofa
transport from the late endosomes to the Golgi (Seaman, 2005).
collectionofyeastmutantsinwhichexpressionofessentialgeneswas
To date, Retromer has not been considered to be crucial for
knockeddown.Cellswereassayedforautophagicactivityafter4hoursof
autophagy(Kametakaetal.,1998).Atg9traffickingisaffectedin
nitrogenstarvation.Relativeautophagicactivitiesofeachmutantcompared
withthewild-typestrainareplotted.Notethattheefficiencyofexpression several double mutants in endosomal trafficking pathways,
knockdowndifferedbetweenthestrains.(B)Detailedresultsforthe although the precise role of these pathways is still ambiguous
representativeTRAPPmutantsindicatedbyarrowsinA. (Ohashi and Munro, 2010). Although mutation in an essential
TRAPPIII and autophagy 4965
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Fig.2. Atg9movementdependsonTRAPPIII.(A)ConditionalexpressionofTrs85wasdetectedbyimmunoblottingforHA.InKNY74cells,which
al expresstheauxinreceptorgeneOsTIR1,theC-terminusofgenomicTRS85wastaggedwithIAAand36HA,makingtheproteinatargetofubiquitylationinthe
n
r presenceofauxin(NAA).WhenNAAwasremovedfromthemedium,Trs85-IAA-36HAwasexpressedatnormallevels.(B)Lysatesofwild-type,trs85D,Trs85-
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o IAA-36HAcellsbeforeandafterNAAwash-outwerepreparedbythealkalinelysismethodandsubjectedtoSDS-PAGEfollowedbyimmunoblottingfor
J API.Lysateequivalentto0.2OD unitswasloadedtoeachlane.Lane1,wild-type;lane2,trs85D;lane3,Trs85-IAA-36HA(-NAA2h);lane4,Trs85-IAA-
600
36HA(200mMNAA).Upperandlowerbandsrepresentpro-andmature-formofAPI,respectively.(C)KNY74cellsharboringpRS314-26mCherry-Atg8
wereincubatedinthepresenceof200mMNAAtodestabilizetheTrs85-IAA-36HAforanovernightinSDcontaining0.5%casaminoacids(SCD)medium.The
cellswereobservedattheindicatedtimepointsaftertheNAAwash-outusingLeicaDIM6000Bmicroscope.26mCherry-Atg8-positivepunctawerecounted
(n.1000cells)andthegraphshowsthenumberofcellsforeachcondition(mean6s.d.).(D)TRS85-IAA-36HA(KNY74)andTRS85-IAA-36HAatg1D
(KNY76)cellsexpressingAtg9-36GFPfromthenativepromoterwereculturedinYPDinthepresenceof200mMNAA.Imageswereobtained0and2hours
afterNAAwasremoved,usingaLeicaDIM6000Bconventionalfluorescencemicroscope.
subunit of Retromer, Vps17, did not affect the distribution of trs85 atg1 triple mutant, we further observed that Atg9 failed to
Atg9-36GFP under nutrient-rich or starvation conditions (not reach the PAS: Atg9-36GFP was scarcely associated with API
shown),acombinationofvps17andaTRAPPIIImutation(trs85) puncta, which represent the PAS, in sharp contrast to the
increased the number of Atg9 puncta, even under starvation significant overlap between Atg9-36GFP and API puncta in the
conditions (Fig. 3A,B). This observation suggests that a atg1 single mutant (Fig. 4). This increase was not due to
Retromer-dependent process bypasses the TRAPPIII-dependent elevation of the amount of Atg9-36GFP protein because it was
pathway when cells are starved. notchangedbythesemutations(supplementarymaterialFig.S2).
On the basis of the possibility that the TRAPPIII-derived Consistentwiththeseresults,weobservedamarkedreduction
defect is bypassed by a Retromer-dependent pathway, we in API processing after 1 hour of starvation in the vps17 trs85
hypothesized that TRAPPIII has some unknown connection to double mutant, relative to each single mutant (Fig. 5A–C).
endosome-to-Golgi trafficking. Therefore, we asked whether Likewise, the vps51 trs85 double mutant exhibited a severe
other retrograde endosome-to-Golgi transport processes play defectafter3 hoursofstarvation(Fig. 5A–C),althoughmutation
bypass roles similar to that of the Retromer-dependent pathway. of the GARP subunit Vps51 was previously shown to only
Golgi-associated retrograde protein (GARP) is a complex that slightly disrupt API processing (Reggiori et al., 2003). By
functions as a tethering factor in endosome-to-Golgi transport, contrast, combination of the GARP mutation (vps51) with a
andVps51isitsessentialsubunit(BonifacinoandHierro,2010). Retromermutation(vps17)didnotresultinamarkeddefecteven
Similar to the case of Retromer, the vps51 trs85 double mutant under starvation conditions (Fig. 5B,C). These observations are
exhibited a severe defect in Atg9 trafficking under starvation consistent with a model in which Retromer and GARP function
conditions in the atg1 background (Fig. 3A,B). In this vps51 inthesamebypasspathwayfromthelateendosometotheGolgi.
4966 Journal of Cell Science 126 (21)
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of Fig.3. Retromer-andGARP-dependentbypasspathwaysforAtg9traffickingunderstarvation.(A)Wild-type(KNY93),atg1D(KNY94),atg1D
al trs85D(KNY80),atg1Dtrs85Dvps17D(KNY105)andatg1Dtrs85Dvps51D(KNY280)cellsexpressingAtg9-36GFPwereculturedat26˚CinYPDand
n subjectedtonitrogenstarvationfor2hours.ImageswereobtainedusinganOlympusIX71microscopeequippedwithaspinning-diskconfocalsystem.
ur (B)Atg9-36GFP-positivepunctaineachstraindescribedinAwerecountedusingG-countsoftware;means6s.d.areshown(n.100).Representativeresults
o fromtwoindependentexperimentsareshown.
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To further understandthe relationship between TRAPPIII and secretory pathway (Lewis et al., 2000). Compared with wild-
the other endosomal pathways, we next examined the sorting type cells, the TRAPPIII (trs85) mutant exhibited altered
nexin complex, which functions in a retrograde pathway that is distribution of GFP-Snc1 (Fig. 6A,B). Furthermore, when the
distinct from the Retromer-mediated process (Hettema et al., TRAPPIII (trs85) mutationwas combinedwitha GARP (vps51)
2003). Mutation of the sorting nexin subunit Atg24 (also called mutation, marked changes in the localization of GFP-Snc1 were
Snx4) disrupts API processing under nutrient-rich conditions observed (Fig. 6A,B): no GFP signals were detected at the
(Nice et al., 2002). When the sorting nexin mutation atg24 was plasma membrane, and most signals were dispersed throughout
introducedintotheTRAPPIII(trs85)mutant,thephenotypewas thecytoplasm,implyingthattheGFP-Snc1proteinwaspackaged
similartothat ofeachsingle mutant,unlikethe combinations of in small transport vesicles (Fig. 6A,B). Ric1 is a guanine-
trs85with mutations inGARP and Retromer subunits (Fig. 5A– nucleotide exchange factor for Ypt6 that functions in a GARP-
C).Bycontrast,combinationofatg24withamutationinaGARP dependent pathway (Siniossoglou et al., 2000). Consistent with
(vps51) or Retromer subunit (vps17) exacerbated the API this, the phenotype of the ric1 trs85 double mutant was quite
processing defects relative to each single mutant (Fig. 5B,C). similartothatofthevps51trs85mutant(supplementarymaterial
This result indicates that TRAPPIII functions in the same Fig. S3A). We also noticed that GARP (vps51) and TRAPPIII
pathway as sorting nexin, and leads to the novel idea that (trs85) double mutant, and ric1 and trs85 double mutant,
TRAPPIII is involved in endosome-to-Golgi retrograde exhibited severe growth defects, growing only at lower
trafficking. temperatures and at a slower overall rate (Fig. 6C and not
On the basis of this finding, we asked whether TRAPPIII shown). Furthermore, combination of the TRAPPIII (trs85) and
functionmightnotbespecifictoautophagy,butratherassociated Retromer (vps17) mutations resulted in a phenotype that was
with general vesicular traffic. To answer this question, we milderoverallthanthatofvps51trs85,butstilladditive,whereas
assessedthelocalizationofageneralcargo,GFP-taggedSnc1,a the TRAPPIII (trs85) and sorting nexin (atg24) double mutant
v-SNARE protein involved in the fusion of secretory vesicles to did not exhibit an additive growth defect (Fig. 6C). Next, we
the plasma membrane. Snc1 recycles between the plasma investigated another cargo of the endosome-to-Golgi pathway,
membrane, endosomes, and the Golgi complex by a Vps10, which is a receptor for the vacuolar protease
combination of endocytosis, retrograde transport and the carboxypeptidase Y. Vps10 mostly localized to the Golgi and
TRAPPIII and autophagy 4967
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Fig.4. Atg9movementtothePASisseverelyaffectedintrs85vps51cells.atg1D(KNY310)andatg1Dtrs85Dvps51D(KNY279)cellsharboringgenomic
of Atg9-3xGFPandAPI-mStrawberrywereculturedat26˚CinYPDandshiftedtonitrogenstarvationfor2hours.Imageswereobtainedattheindicatedtime
pointsusinganOlympusIX71microscopeequippedwithaspinning-diskconfocalsystem.ThegraphshowsthequantificationofAtg9-3xGFPandAPI-
al
n mStrawberrycolocalization.Foreachstrain,90–250API-mStrawberry-positivepunctawereexamined.Scalebars:5mm.
r
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endosome,andexhibitedatypicalpatchypattern(supplementary only 3.7% of GFP-Ypt1-positive dots were positive for
material Fig. S3B) (Shi et al., 2011). However, the pattern of 26mCherry-Atg8. Furthermore, a recent report showed that
Vps10 was more dispersed in the trs85 vps51 double mutant, Atg11 also acts together with Ypt1 and TRAPPIII in autophagy
appearingasnumeroussmallparticles,andwasalsodispersedto (Lipatovaet.al,2012).However,inacountofmorethan500cells,
some extent in each single mutant (supplementary material Fig. only 0.95% of GFP-Atg11-positive puncta were also positive for
S3B). Taken together, these results indicate that TRAPPIII is Sec7-mStrawberry (supplementary material Fig. S4C). Together,
involved in general retrograde transport from the early these results suggest that association of TRAPPIII and Ypt1 to
endosomes to the Golgi. PASislimitedtoasmallpopulation.
Because other TRAPP family members, TRAPPI and
TRAPPII, act in tethering step at the destinations of their Discussion
respectivepathways(Barrowmanetal.,2010),wepredictedthat In this study, we revealed an important novel feature of
TRAPPIII would also function at its destination, the Golgi. To membrane trafficking, namely, a role for TRAPPIII in
investigate this idea, we determined whether Trs85-GFP endosome-to-Golgi retrograde transport. The identity of the
expressed at endogenous levels would colocalize with the tetheringfactoratthisstephasbeena‘missingpiece’inthestudy
Golgi marker Sec7-mStrawberry in wild-type cells. In cells of membrane traffic. We also reinterpreted the observation that
under starvation conditions, 80.9% of Trs85-GFP signals were TRAPPIII-dependentAtg9movementiscrucialforautophagy,in
also positive for Sec7-mStrawberry (Fig. 7A), whereas only termsofthenutrientresponse.Onthebasisofthesefindings,we
0.58% of Trs85-GFP signals overlapped with API-mStrawberry, propose a model (below) describing how general membrane
whichrepresentsthePASinwild-typecells(Fig. 7B);morethan traffic is involved in the regulation of autophagy.
200 cellswere counted for eachquantification of colocalization. Combinations of mutations in TRAPPIII and GARP subunits
Together,theseresultssupportaroleforTRAPPIIIintetheringto producedadditivedefects,whereascombinationsofmutationsin
the Golgi retrograde vesicles that originate from endosomes. TRAPPIII and sorting nexin subunits did not result in more
Accordingly,weinvestigatedthelocalizationofYpt1inrelation severe phenotypes. These findings indicate that TRAPPIII
totheGolgiandAtg8inwild-typecells(supplementarymaterial receives vesicles from early endosomes at the Golgi (Fig. 8).
Fig.S4A,B).Inacountofmorethan60cells,77.7%ofGFP-Ypt1- Our results showing that TRAPPIII functions in retrograde
positive dots were also positive for Sec7-mStrawberry, whereas transport from endosomes to the Golgi are consistent with the
4968 Journal of Cell Science 126 (21)
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S
combiningmutationsthataffect
ell TRAPPIIIandlateendosome-to-Golgi
C transport.(A)Summaryofgenesand
f proteinsinvestigatedintheseexperiments.
o
(B)Eachstrain(SEY6210;NTY41;KNY92,
al KNY125,KNY189,KNY191,KNY151,
n
r KNY216,KNY217,KNY218,KNY222or
u
o KNY223)wasgrownat26˚CinYPD,
J transferredtomediumlackingnitrogen,and
collectedattheindicatedtimepoints.Cell
lysateswereanalyzedbyimmunoblotting
withanti-APIantibody.Whitearrowhead,
pro-API;blackarrowhead,mature-API.
(C)Bandintensitieswerecalculatedfor
matureAPIrelativetototalAPI(matureAPI
+pro-API)inB.Representativeresultsfrom
twoindependentexperimentsareshown.
previously reported roles of TRAPPI and TRAPPII in ER-to- structures (8–12%) colocalize with Golgi and endosomal
Golgiandintra-Golgitrafficking,respectively.Thus,allTRAPP markers (Mari et al., 2010). The other labeled structures were
familymemberscannowbeconsideredtofunctionattheGolgi. probably a mixture of vesicles providing anterograde and
Furthermore,ourresultssuggestthatYpt1functionsattheGolgi retrograde transport between the Golgi and endosomes. During
together with all members of the TRAPP family. autophagosome formation, some Atg9 will depart from these
Thesefindingsprovideimportantinsightsintothedynamicsof pathwaysandarriveatthePAS.InTRAPPIIImutantcells,Atg9
Atg9(Fig. 8).Undernutrient-richconditions,earlyendosome-to- wastrappedinthevesicleanddidnotreachthePAS.Aslongas
Golgi transport is crucial for the Cvt pathway. Because COG- autophagy proceeds normally, Atg9 is recycled back to the
dependent Golgi function and exit from the Golgi are important peripheral pool via the PAS and the autophagosome and/or
for autophagy (van der Vaart et al., 2010; Yen et al., 2010), vacuoles, but in mutant cells (e.g. atg1), it accumulates
shuttlingbetweentheGolgiandearlyendosomesalsoappearsto abnormally in the PAS.
be crucial. The essential cargo molecule for autophagy is Atg9, Another finding of this study is that the TRAPPIII-dependent
although additional transmembrane cargo proteins, such as pathway is bypassed under starvation conditions (Fig. 8).
Atg27, could also participate in this process. Atg9-GFP-positive Previously, the existence of this alternative pathway might
puncta in peripheral reservoirs exhibit marked movement have obscured the role of TRAPPIII in this process. Here, we
(Sekito et al., 2009), and only a small minority of these proposeamodelinwhichTRAPPIIIandGARPfunctionastwo
TRAPPIII and autophagy 4969
Fig.6. Snc1traffickingandgrowthare
disruptedbymutationsaffecting
TRAPPIIIandlateendosome-to-Golgi
transport.(A)Strains(KNY201,KNY204,
KNY205andKNY225)expressingGFP-
Snc1wereculturedat26˚CinYPD,and
GFP-derivedsignalswereobservedusingan
OlympusIX71microscopeequippedwitha
spinning-diskconfocalsystem.(B)Based
onthedistributionofGFP-Snc1inA,the
cellswerecategorizedintofourgroups,and
therelativesizeofeachpopulationwas
determined(n.200cells).(1)Diffuse:GFP
signalswerediffuselylocatedthroughout
thecell.(2)Internal:GFPsignalswere
observedasinternalpuncta.(3)Polar:in
additiontointernalpuncta,GFPsignals
wereobservedontheplasmamembraneofa
bud.(4)Nonpolar:inadditiontoabud,GFP
signalswereobservedontheplasma
membraneofthemothercell.
Representativeresultsfromtwo
independentexperimentsareshown.
(C)Seriallydilutedcellsfromeachstrain
(SEY6210;NTY41;KNY92,KNY125,
KNY189,KNY191,KNY151,KNY217and
e
c KNY218)werespottedontoYPDplatesand
n incubatedfor3daysattheindicated
e
ci temperatures.
S
ell
independentcomplexes attheGolgi.Althoughthevps51mutant must represent an alternative, rather than primary, pathway in
C
exhibited defective API processing under nutrient-rich autophagy. However, Snc1 recycling mainly uses the GARP-
f
o
conditions, this defect was not as pronounced as that of the dependent pathway, and in this case the TRAPPIII-dependent
al trs85 mutant, and autophagic activity was not affected under pathway appears to serve as the backup. In general, starvation
n
r starvation conditions. Therefore, the GARP-dependent pathway alters vesicular trafficking, and the early endosome-to-late
u
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Fig.7. Trs85localizationattheGolgicomplex.(A)Wild-
typecells(KNY306)harboringchromosomallyintegrated
TRS85-GFPandSEC7-mStrawberrywereculturedinYPD
andshiftedtomediumlackingnitrogenfor3hours.Images
wereobtainedattheindicatedtimepointsusinganOlympus
IX71microscopeequippedwithaspinning-diskconfocal
system.(B)Wild-typecells(KNY305)harboring
chromosomallyintegratedTrs85-GFPandAPI-
mStrawberrywereculturedinYPDandshiftedtomedium
lackingnitrogenfor3hours.Imageswereobtainedatthe
indicatedtimepointsusinganOlympusIX71microscope
equippedwithaspinning-diskconfocalsystem.Scalebars:
5mm.
4970 Journal of Cell Science 126 (21)
proposed in those studies. Future analysis of TRAPP-dependent
Atg9 trafficking inmammalian cells will determine whether our
model is universally applicable.
In preparing for sudden demand for autophagy, pooling the
transmembrane protein Atg9 as a cargo of vesicular transport
may be more favorable than statically storing the protein in a
specific organelle, such as the Golgi. How Atg9 is transferred
from these reservoirs to the PAS is still unclear. Our model and
those proposed in other studies predict that Atg9 is delivered to
thePASviatheGolgithroughacanonicalmechanismsuchasthe
secretory pathway (Geng et al., 2010; Nair et al., 2011). This
important issue should be addressed in future studies.
MaterialsandMethods
Growthconditionsandmedia
Cellswereincubatedat30˚CunlessotherwiseindicatedinYPD(1%Bactoyeast
extract,2%Bactopeptoneand2%dextrose) orSD(0.17%yeastnitrogenbase
withoutaminoacidsorammoniumsulfate,0.5%ammoniumsulfate,2%dextrose,
andappropriateaminoacids)untilanOD valueof0.8–1.2wasreached.For
600
starvationexperiments,cellswerewashedindeionizedwater,transferredtoSD-N
(0.17% yeast nitrogen base without amino acids or ammonium sulfate, 2%
dextrose),andincubatedattheindicatedtemperature.Forauxin-baseddegradation
of Trs85, cells were grown overnight in YPD containing 200mM 1-
naphthaleneaceticacid(NAA;SigmaN0640)andwashedtwicewithfreshYPD
toremovetheNAA.ThecellswerethenresuspendedinYPDatanOD valueof
600
0.5,andincubatedat30˚Cfortheindicatedtimes.
e
Fig.8. RoleofTRAPPIIIinmembranetraffickingandautophagy.Model
c
n ofmembranetraffickingandAtg9dynamics.TRAPPIIIfunctionsattheGolgi Geneticprocedures
e YeaststrainsusedinthisstudyarelistedinTable1.PCRwasusedtocreateand
ci inthesamepathwayassortingnexin,butinadifferentpathwaytoGARPand confirmallgenedisruptions,taggedconstructsandothermodifications(Goldstein
S Retromer.Undernutrient-richconditions,TRAPPIIIplaysacentralrolein etal.,1999;Jankeetal.,2004;Longtineetal.,1998;Nakatogawaetal.,2012).The
ell Acotngs9timtuoteveamsehnutt.tlTinhgisppaaththwwaayy.UannddeGrosltgair-vtoa-tieonndocsoonmdietiaonntse,rtohgeraGdAeRtraPn-sport PSyHsOte8ml)oc(uSschinultdhienekrnoectkaolu.,tc2o0l0le5c)tiwonasanredptlhaeceDdAwmiPthhpahpolo8iDd6c0olluescitnigona(OsypnetnheBtiioc
C
dependentpathwayactsasabypassroute.Thesepathwaysprovideareservoir geneticarraymethod(TongandBoone,2007).Briefly,theTNY509parentalstrain
of ofAtg9,andsomeAtg9isrelocalizedatthePAS,whereitcontributesto harboringpho8D60(NodaandKlionsky,2008;TongandBoone,2007)wascrossed
witheachmutantstrainonYPDinarectangularplate(OmniTray,Nunc)witha96-
al autophagy.Solidlinesrepresentpathwaysexperimentallyexaminedinthis pinnedreplicator(VP408,V&PScientific).Haploidcellscontainingthepho8D60
n study,anddashedlinesreflectotherpublishedresults. alleleandeachmutantallelewereselectedusingauxotrophicanddrug-resistance
r
u markersandsequentialreplicaplating(TongandBoone,2007).ThemStrawberry
Jo endosomepathwayisenhancedbystarvation,possiblyduetothe sequencewasPCR-amplifiedfrompmStrawberry(Shuetal.,2006)andusedto
replacetheyeGFPsequenceofpYM25togeneratepKN14;theresultingplasmid
need for increased degradation in the vacuole (Hamasaki et al.,
was used for C-terminal tagging with mStrawberry. A partially digested PvuII
2005; Jones et al., 2012). As a result, some Atg9 in early fragment of pGFP-Snc1 (306) (Lewis et al., 2000) was subcloned into pRS305
endosomes will be relocated to late endosomes, and the late digestedwithPvuIItogeneratepKN24.Theresultingplasmidwaslinearizedwith
EcoRVandintegratedatthechromosomalLEU2locus.ABamHI-SacIfragment
endosome-to-Golgi pathway becomes another reservoir of Atg9.
encoding36GFPfromthepAtg9-36GFP(306)plasmid(Gengetal.,2010)was
Our model also explains why Tlg2, a t-SNARE localized on the subcloned into pRS303 digested with the same enzymes to generate pKN15
Golgiandearlyendosomes,isrequiredonlyfortheCvtpathway (pRS303-36GFP). SequenceencodingtheC-terminal region ofATG9wasPCR
amplifiedusingprimers59-CGGGATCCCCTCTTCCGACGTCAGAC-39and59-
(Abeliovich et al., 1999). When mutations affecting vesicles at
CCGCTCGAGGCACCATTTCTGGTCACATAC-39.Theamplifiedfragmentwas
theirorigin(Retromer,sortingnexin)anddestination(TRAPPIII, digestedwithBamHIandXhoI,andthensubclonedintopKN15digestedwiththe
GARP) were combined, the phenotypes were milder (see sameenzymestogeneratepKN17(pRS303-Atg9-36GFP).pKN17waslinearized
Fig. 5B,C; vps51 trs85 and atg24 vps51 double mutants) than with BglII and integrated at the ATG9 locus. pNHK53 and pMK43 (National
BioResource Project, Japan) harbored OsTIR1 and AtIAA17, respectively
that of the trs85 vps51 double mutant. This implies that the
(Nishimuraetal.,2009).pNHK53wascutwithStuIandintegratedattheURA3
apparatuses at the origin and destination do not need to strictly locusofawild-typestrain.AtIAA17inpMK43wasPCR-amplified,cutwithHindIII
match. Also of note, even in the context of the strongest andXhoI,andsubclonedintopYM24digestedwithHindIIIandSalItoadd36HAto
theCterminusofIAA17.Theresultantplasmid,pKN19,wasusedasthetemplate
phenotype(i.e.thatofthetrs85vps51mutant),somelimitedAPI
forC-AIDtaggingofTRS85.pRS314-26mCherry-Atg8wasagiftfromtheOhsumi
processing still occurred. This might reflect the existence of lab.pKN38toexpressGFP-Ypt1undertheADH1promoterwasconstructedas
another bypass pathway, such as the Fab1-dependent retrograde follows.TheYPT1genewasamplifiedfromthewild-typegenomicDNAbyPCR
withtheprimerpairof59-GGGGATCCAATATGAATAGCGAGTACG-39GG
pathway (Efe et al., 2007). Alternatively, even without the
GA59GG GAT CCA ATA TGA ATA GCG AGT ACG-3G, and digested with
reservoirsources,Atg9expressedduringstarvationcouldsupport BamHIandXhoI.TheresultingfragmentwasintroducedintopBP73-Adigested
a small amount of autophagic activity. withthesameenzymestogeneratepKN37,whichhasaGFP-YPT1fusiongene
undertheADH1promoter.TheplasmidwasthendigestedwithSacIandXhoIand
Our model mightappear toconflict with previousreports that
thefragmentcontainingGFP-YPT1 withtheADH-promoterwassubclonedinto
TRAPPIII functions at the PAS in yeast and in forming pRS304 digested with the same enzymes. The resulting plasmid pKN38 was
autophagosome in mammalian cells (Lynch-Day et al., 2010; linearizedattheuniqueEcoRIsitewithintheYPT1geneandintegratedattheYPT1
Kakuta et al., 2012; Lipatova et al., 2012; Huang et al., 2011; locusofawild-typestrainharboringchromosomallytaggedSec7-mStrawberry.
Wangetal.,2013).Ourmodeldoesnotnecessarilyexcludethese
Large-scaleALPassay
possibilities,butourresultssuggestthatthedirectcontributionof
YPDplateswereinoculatedwithcellsfromeachpoolusinga96-pinreplicatorand
TRAPPIII to autophagosome formation is not as large as incubated for 16–24hours at 30˚C. Subsequently, the cells were suspended in
TRAPPIII and autophagy 4971
Table 1. Strains used in this study
Strain Genotype Reference
Y3656 MATacan1D::MFA1pr-HIS3-MFa1pr-LEU2ura3Dleu2Dhis3Dmet15Dlys2D (TongandBoone,2007)
TNY509 Y3656;pho8::pho8D60:natNT2 Thisstudy
SKY001 TNY509pep4D::kanMX6 Thisstudy
SEY6210 MATaura3-52leu2-3,112his3D200trp1-D901lys2-801suc2-D9 (Robinsonetal.,1988)
SEY6210.1 MATaura3-52leu2-3,112his3D200trp1-D901lys2-801suc2-D9 (Robinsonetal.,1988)
NTY41 SEY6210;atg1D::kanMX6 Taguchietal.,submitted
KNY67 SEY6210;OsTIR1-9xmyc:URA3atg9::ATG9-3xGFP:HIS3 Thisstudy
KNY74 KNY67;trs85::TRS85-AtIAA17-36HA:hphNT1 Thisstudy
KNY76 KNY67;atg1D::kanMX6trs85::TRS85-AtIAA17-36HA:hphNT1 Thisstudy
KNY93 SEY6210;atg9::ATG9-36GFP:HIS3 Thisstudy
KNY94 SEY6210;atg1D::kanMX6atg9::ATG9-36GFP:HIS3 Thisstudy
KNY95 SEY6210;trs85D::URA3MXatg9::ATG9-36GFP:HIS3 Thisstudy
KNY80 SEY6210;atg1D::kanMX6MX6trs85D::CaURA3atg9::ATG9-36GFP:HIS3 Thisstudy
KNY103 SEY6210;vps17D::TRP1atg9::ATG9-36GFP:HIS3 Thisstudy
KNY104 SEY6210;trs85D::CaURA3vps17D::TRP1atg9::ATG9-36GFP:HIS3 Thisstudy
KNY105 SEY6210;atg1D::kanMX6trs85D::CaURA3vps17D::TRP1atg9::ATG9-36GFP:HIS3 Thisstudy
KNY279 SEY6210;atg1D::kanMX6trs85D::CaURA3vps51D::natNT2atg9::ATG9-36GFP:HIS3 Thisstudy
ape1::APE1-mStrawberry:hphNT1
KNY92 SEY6210;trs85D::URA3MX Thisstudy
KNY125 SEY6210;vps17D::TRP1 Thisstudy
KNY189 SEY6210;atg24D::zeoNT3 Thisstudy
KNY191 SEY6210;vps51D::natNT2 Thisstudy
KNY151 SEY6210;trs85D::URA3MXvps17D::TRP1 Thisstudy
KNY217 SEY6210;atg24D::zeoNT3trs85D::URA3MX Thisstudy
KNY218 SEY6210;trs85D::URA3MXvps51D::natNT2 Thisstudy
e KNY216 SEY6210;atg24D::zeoNT3vps51D::natNT2 Thisstudy
c KNY222 SEY6210;vps17D::TRP1vps51D::natNT2 Thisstudy
n
e KNY223 SEY6210;atg24D::zeoNT3vps17D::TRP1 Thisstudy
ci KNY201 SEY6210;leu2::GFP-SNC1:LEU2 Thisstudy
S KNY204 SEY6210;vps51D::natNT2leu2::GFP-SNC1:LEU2 Thisstudy
ell KKNNYY220255 SSEEYY66221100;;ttrrss8855DD::::CCaaUURRAA33lvepus25:1:DG:F:nPa-tSNNTC21l:eLuE2U::2GFP-SNC1:LEU2 TThhiissssttuuddyy
C
KNY305 SEY6210;trs85::TRS85-GFP:HIS3MX6sec7::SEC7-mStrawberry:hphNT1 Thisstudy
of KNY306 SEY6210;trs85::TRS85-GFP:HIS3MX6ape1::APE1-mStrawberry:hphNT1 Thisstudy
KNY310 SEY6210;atg1D::kanMX6atg9::ATG9-3xGFP:HIS3ape1::APE1-mStrawberry:hphNT1 Thisstudy
al KNY345 SEY6210;atg9::ATG9-3xGFP:HIS3sec7::SEC7-mStrawberry:hphNT1 Thisstudy
n
r KNY351 SEY6210;ric1::kanMX6snc1::GFP-SNC1:LEU2 Thisstudy
u
KNY352 SEY6210;ric1::kanMX6trs85::CaURA3snc1::GFP-SNC1:LEU2 Thisstudy
o
J KNY353 SEY6210;vps10::VPS10-GFP:His3MX6 Thisstudy
KNY354 SEY6210;trs85::CaURA3vps10::VPS10-GFP:His3MX6 Thisstudy
KNY355 SEY6210;vps51::natNT2vps10::VPS10-GFP:His3MX6 Thisstudy
KNY356 SEY6210;trs85::CaURA3vps51::natNTSvps10::VPS10-GFP:His3MX6 Thisstudy
KNY357 SEY6210;atg9::ATG9-3xmCherry:hphNT1ypt1::PADH-GFP-YPT1:TRP1 Thisstudy
KNY358 SEY6210;atg9::ATG9-3xGFP:HisMX6trs85::TRS85-3xmCherry:hphNT1 Thisstudy
KNY359 SEY6210;sec7::SEC7-mStrawberry:hphNT1ypt1::PADH-GFP-YPT1:TRP1 Thisstudy
KNY360 SEY6210;ypt1::PADH-GFP-YPT1:TRP1pRS314-2xmCherry-Atg8 Thisstudy
KNY361 SEY6210;atg11::PADH-GFP-Atg11:natNT2sec7::SEC7-mStrawberry:hphNT1 Thisstudy
200mlofSD-Nmediumin96-wellplatesandincubatedfor4hoursat30˚C.The objective lens, Leica Microsystems) was used to observe the cells in
plates were centrifuged, and the supernatant was discarded. To each well was supplementarymaterialFig.S2.
added50mlofice-coldlysisbuffer(10mMTris-HClatpH9.0,10mMMgSO
4
and 10mM ZnSO,) and ,10ml of 0.6 mm zirconia/silica beads (Biomedical
4 Proteinextractionandimmunoblotting
Science).Theplatesweresealedwithparafilmandmixedvigorouslyonarotary
Yeastcellswereharvested,suspendedin100mlof0.2MNaOHand1%(v/v)2-
shakerat2500rpmfor10minutesat4˚C.Afterabriefcentrifugation,150mlof
mercaptoethanol, and incubated on ice for 10minutes. One milliliter of chilled
ice-coldlysisbufferwasadded,andtheplateswerecentrifugedfor15minutesat
acetone was added to the suspension, and the samples were incubated for
490 g at 4˚C. Protein levels were determined from 50 ml aliquots of the
10minutes on ice. After centrifugation at 16,000 g for 10minutes, cell pellets
supernatants using the bicinchoninic acid method, and enzymatic activity was
werewashedoncewithchilledacetoneanddissolvedinLaemmlisamplebuffer
measuredin50mlaliquotsasdescribedinapreviousreport(NodaandKlionsky,
usingawater-bathsonicatorandboiledfor5minutes.Aliquots(0.2OD units)
2008)withslightmodifications. 600
wereresolvedusingSDS-PAGE,andproteinsweredetectedwiththeappropriate
antibodies[anti-aminopeptidaseI(Nodaetal.,2000),anti-HA(12CA5),anti-Atg9
Microscopy (a gift from the Ohsumi lab)]. Band intensities were measured using ImageJ
Cells were observed Leica AF6500 fluorescent imaging system mounted on a software. Lysates to detect Atg9 were prepared by glass bead lysis. Cells were
DIM6000Bmicroscope(HCXPLAPO636/1.40–0.60oil-immersionobjective suspendedinlysisbuffer(20mMPIPES,pH6.8,200mMsorbitol,5mMEDTA)
lens, mercury lamp) under the control of LAS-AF software (Leica containing Complete protease inhibitor cocktail (Roche) and 4mM PMSF, and
Microsystems) or on a Yokogawa CSU-X spinning-disk confocal system disruptedbysixroundsofvortexingfor30secondswith30secondintervalson
(Yokogawa Electric Corp., Japan) mounted on an Olympus IX71 microscope ice.TritonX-100wasaddedtothelysateatthefinalconcentrationof0.5%.The
(1006NA 1.4 PlanApo objective lens) equipped with an Andor iXon CCD lysates were incubated on ice for 5minutes and centrifuged at 5000 g for
camera (Andor Technology, UK) under the control of the Andor IQ software. 10minutes at 4˚C. The supernatants were incubated at 65˚C for 10minutes in
ImageswereprocessedusingAdobePhotoshop.Atg9punctawerecountedusing Laemmli sample buffer before performing SDS-PAGE followed by
the G-Count software (G-Angstrom, Japan). Leica TCS SP8 confocal system immunoblotting with anti-Atg9 (a gift from the Ohsumi lab) and anti-PGK (A-
mountedon aDMI 6000CS microscope (HCXPL APO 1006/1.4 Oil STED 6457,MolecularProbes).
4972 Journal of Cell Science 126 (21)
Acknowledgements Lewis,M.J.,Nichols,B.J.,Prescianotto-Baschong,C.,Riezman,H.andPelham,
TheauthorswouldliketothankDrDanielJ.Klionsky(Universityof H. R. (2000). Specific retrieval of the exocytic SNARE Snc1p from early yeast
endosomes.Mol.Biol.Cell11,23-38.
Michigan),DrScottD.Emr(CornellUniversity),DrCharlesBoone
Lipatova, Z., Belogortseva, N., Zhang, X. Q., Kim, J., Taussig, D. and Segev,
(University of Toronto), Dr Yoshinori Ohsumi, Dr Hayashi N.(2012).RegulationofselectiveautophagyonsetbyaYpt/RabGTPasemodule.
Yamamoto (Tokyo Institute of Technology), and Dr Roger Tsien Proc.Natl.Acad.Sci.USA109,6981-6986.
(University of California, San Diego) for various strains and Longatti, A. and Tooze, S. A. (2009). Vesicular trafficking and autophagosome
formation.CellDeathDiffer.16,956-965.
plasmids; the National BioResource Project Yeast (Japan) for
Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A.,
plasmids;and MrYuuma Itofortechnical assistance. Brachat, A., Philippsen, P. and Pringle, J. R. (1998). Additional modules for
versatile and economical PCR-based gene deletion and modification in
Saccharomycescerevisiae.Yeast14,953-961.
Authorcontributions
Lynch-Day, M. A., Bhandari, D., Menon, S., Huang, J., Cai, H., Bartholomew,
K.S.N. did most of the experiments. S.K. established the novel C.R.,Brumell,J.H.,Ferro-Novick,S.andKlionsky,D.J.(2010).Trs85directsa
autophagy screening method and performed the genome-wide Ypt1GEF,TRAPPIII,tothephagophoretopromoteautophagy.Proc.Natl.Acad.
screening. T.Y. contributed valuable discussion. T.N. designed the Sci.USA107,7811-7816.
Mari, M., Griffith, J., Rieter, E., Krishnappa, L., Klionsky, D. J. and Reggiori,
studyandwrote themanuscript.
F. (2010). An Atg9-containing compartment that functions in the early steps of
autophagosomebiogenesis.J.CellBiol.190,1005-1022.
Funding Meiling-Wesse, K., Epple, U. D., Krick, R., Barth, H., Appelles, A., Voss, C.,
ThisworkwassupportedinpartbytheSpecialCoordinationFunds Eskelinen,E.L.andThumm,M.(2005).Trs85(Gsg1),acomponentoftheTRAPP
complexes,isrequiredfortheorganizationofthepreautophagosomalstructureduring
for Promoting Science and Technology from the Ministry of
selectiveautophagyviatheCvtpathway.J.Biol.Chem.280,33669-33678.
Education, Culture, Sports, Science and Technology (MEXT) of Mizushima,N.andKomatsu,M.(2011).Autophagy:renovationofcellsandtissues.
Japan;and byagrant from theCell Science Research Foundation. Cell147,728-741.
Nair,U.,Jotwani,A.,Geng,J.,Gammoh,N.,Richerson,D.,Yen,W.L.,Griffith,J.,
Nag, S., Wang, K., Moss, T. et al. (2011). SNARE proteins are required for
Supplementarymaterialavailableonlineat macroautophagy.Cell146,290-302.
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.131318/-/DC1 Nakatogawa, H., Suzuki, K., Kamada, Y. and Ohsumi, Y. (2009). Dynamics and
diversityinautophagymechanisms:lessonsfromyeast.Nat.Rev.Mol.CellBiol.10,
458-467.
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