The Journal of Cell Biology

JCB

C

Introduction Thus, twoproperties,Ca John V.Kilmartin and geneticinteractions with spindle polebody duplication and anessentialfunctioninbuddingyeast Sfi1p hasconserved -bindingsites changes inCa attached totheMTOC,whichcancontractinresponse the otherisasconstituentsofcontractilefiberswithinand 1981; Middendorpetal.,2000;Salisbury2002),and lished. OneisintheduplicationofMTOC(Byers, suggesting multipleroles.Twoclearroleshavebeenestab- different partsoftheMTOC(Levyetal.,1996),again organizing centers(MTOCs),andareoftenlocatedwithin ticular, centrinsareubiquitouscomponentsofmicrotubule- 1998; Arakietal.,2001;Pulvermuller2002).In processes (SchiebelandBornens,1995;Sullivanetal., calmodulin, haverolesinanumberofdifferentcellular areconservedcalmodulin-likeproteinsand,like Medical Research Council(MRC) ofMolecular Laboratory Biology, CambridgeCB22QH,UK on theCa moneme alsohaselasticpropertiesthatarenotdependent centrin (Maciejewskietal.,1999).Inaddition,thespas- 1975), hashomologytoEF-handdomainproteinssuch as 1999). Itsprincipleconstituent,spasmin(Amosetal., moneme (Weis-FoghandAmos,1972;Moriyamaetal., changes inCa related fibrousstructurethatalsocontractsinresponseto sensitive mutantsin body (SPB),where Cdc31pisalsolocalized. Temperature- these, Sfi1p,localizestothehalf-bridgeofspindlepole experiments toisolateCdc31p-bindingproteins.Oneof single Zdomainofprotein a with A, andusedinpull-down  Cdc31p; centrin Key words: SPB;duplication; Sfi1p; Fax: 44-1223-412142.email:[email protected] Biology, HillsRd.,CambridgeCB2 2QH,UK.Tel.:44-1223-402242. J.V. Kilmartin,MRCLaboratory ofMolecular to correspondence Address http://www.jcb.org/cgi/doi/10.1083/jcb.200307064 The Journal ofCellBiology TheRockefeller University Press, 0021-9525 Article

cerevisiae crotubule-organizing centers. The entrins arecalmodulin-likeproteinspresentinmi-

2

concentration(Weis-FoghandAmos,1972).

2

2

centrin,Cdc31p,was functionallytagged concentration(Salisburyetal.,1984).A concentrationisthevorticellidspas- SFI1 , Volume 162, Number 7,September 29,20031211–1221 show adefectinSPBduplication 2 -dependent contractionand cdc31-1 /2003/09/1211/11 $8.00 . Sfi1pcontains Saccharomyces par- to individual Sfi1repeatsina1:1ratio, soasinglemolecule localizes closetothecentrioleregion.Cdc31pbindsdirectly to theSPB, andinseveral humanproteins,oneofwhich sensitive mutantsarrestwith asinglelargeSPB(Byers, elastic propertiesofthehalf-bridge. Kilmartin, 1999).Theretractionprocessmayrelyonpossible SPB componentsandcompletingassembly(Adams plaque tonucleoplasm,therebyallowingassemblyofnuclear the newlyassembledduplicationplaque.Thisexposes followed byretractionofthehalf-bridgeacrossface nuclear lipidbilayersatthedistalendofhalf-bridge, transit processoccursbyfusionofthecytoplasmicand spindle canbeformed(AdamsandKilmartin,1999).This the twolipidbilayersofnuclearenvelopesothata bridge allowsthetransitofpartlyassembledSPBacross cytoplasmic sideofthehalf-bridge;andsecond,half- duplication. First,thenewSPBstartstoassembleondistal 1999). Thehalf-bridgeplaysanimportantroleinSPB Goetsch, 1974;AdamsandKilmartin,1999;O’Tooleetal., of theenvelopeattachedtoonesideSPB(Byersand envelope, andthehalf-bridgeisaspecializedrectangulararea dle polebody(SPB).TheSPBisembeddedinthenuclear localized (Spangetal.,1993)tothehalf-bridgeofspin- trin-containing fibrousstructures. motif isconserved. in therepeatregion,showing thatthiscentrin-binding human proteincontainingSfi1 repeatsalsobindscentrin of Sfi1pbindsmultiplemoleculesCdc31p. The centrosomal Schizosaccharomyces pombe multiple internalrepeatsthatarealsopresentina NH ZCdc31p, Cdc31pcontainingasingle ZdomainofproteinAatthe Abbreviations usedinthispaper:prA, proteinA;SPB,spindlepolebody; Ca Cdc31p isessentialforSPB duplication:temperature- Budding yeasthasonecentrin,Cdc31p,someofwhichis 2 2 terminus. -independent elasticity,maybecharacteristicofcen- ,which alsolocalizes 1211 The Journal of Cell Biology 1212 ever, shortNH of Cdc31pwithproteinsotherthanKar1parelessCa 2001). Thiswasbecausethereisevidencethatinteractions (seeMaterials andmethods). proteosome components, ribosomalproteins,keratin, ornonspecific spectrometry. Numberedbands contained heatshockproteins, were identifiedbymatrix-assisted laserdesorption/ionizationmass ZCdc31p orSfi1p-prAafterbinding toIgG-Sepharose. Figure 1. longer NH proach wasappliedtoCdc31p,butallCOOH-terminaland (Grandi etal.,1993;KnopandSchiebel,1997).Thisap- lowed bypurificationofthecomplexonanIgGcolumn plex istaggingoneoftheproteinswithproteinA(prA),fol- A powerfulwayofidentifyinginteractingproteinsinacom- A complexcontainingCdc31pandSfi1p ity ofthehalf-bridgeSPB. Sfi1p suggestsamechanismforeithercontractionorelastic- role inSPBduplication.Therepeateddomainstructureof conserved centrin-bindingsites,andwhichhasanessential protein, Sfi1p,whichbindsCdc31pdirectlyviamultiple al., 2002;Nishikawaet2003).Thispaperdescribesa al., 1995),andmorerecently,Mps3p/Nep98p(Jaspersenet been identified;Kar1p(BigginsandRose,1994;Spanget Cdc31p-binding proteinsinthehalf-bridgehavealready 1981) andfailtoformasatellite(Wineyetal.,1991).Two Ca a pull-downexperiment(Fig.1a). and inthecaseofZCdc31p,broughtdownotherproteins or asingleZdomainofprA(ZCdc31p)werefunctional, and centrinwitha350-kDproteininthe min withthespasmoneme(Amos,1971;Amosetal.,1975), sitive (Geieretal.,1996).Inaddition,interactionsofspas- Results ciliary lattice(Klotzetal.,1997),arestableinthepresence For theseexperiments,lysiswasperformedunderlowfree 2 The JournalofCellBiology conditions(5mMEGTA;WiggeandKilmartin, SDS gelofproteinsisolatedfrom yeastcellscontaining 2 -terminal tagsfailedtorescueadeletion.How-

2

-terminal tagscontainingsingleepitope

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Volume 162,Number7,2003

Paramecium Bands 2 infra- sen- 36 amined byimmunofluorescent stainingafterarrestfor4hat published data). However,cellssynchronized inG (Ma etal.,1999), i.e.,failuretoformamitotic spindle(un- tion causesaG cyclase pathway,butitisanessentialproteinwhosedeple- characterized, itwasfoundnottobeinvolvedintheadenyl adenyl cyclase(Maetal.,1999).WhenSfi1pwaspartly the heatsensitivityassociatedwithaparticularmutationin lar interest,Sfi1p,wasinitiallyidentifiedasasuppressorof is ferrochelatase(Labbe-Bois,1990).Oneproteinofparticu- recombination (GallardoandAguilera,2001),Hem15p (Brickner andFuller,1997),Thp1pisinvolvedinmitotic of diversefunction:Vps13pisinvolvedmembranetraffic of EGTA.TheproteinsthatcopurifiedwithZCdc31pwere cdc31-1 tions with plication hadoccurredandalso tolookforgeneticinterac- in moredetail,particularto determinewhetherSPBdu- perature-sensitive mutantswerepreparedtocheckthearrest gests thatitmighthaveafunctioninSPBduplication.Tem- The localizationofSfi1ptothehalf-bridgeSPBsug- Phenotype of and Cdc31parelocatedinthesamepartofSPB. calized tothehalf-bridge(Spangetal.,1993),thusSfi1p or merelyantigenaccessibilityisnotclear.Cdc31palsolo- cates amorespecificlocalizationofSfi1ponthehalf-bridge Spc72p (AdamsandKilmartin,1999).Whetherthisindi- the bridgecomparedwithothercomponentssuch as ing SPB,andinpairedSPBswasmorecentrallylocatedon Sfi1p insingleSPBsappearedsomewhatdistaltotheexist- the half-bridge(Fig.2e).Theimmuno-EMstainingof Immuno-EM thendefinedthelocalizationmorepreciselyto was confirmedbyimmunofluorescence(Fig.2,candd). a andb).ThissuggestslocalizationtotheSPBregion,which or twospotsthatwerecoincidentwithnuclearDNA(Fig.2, Sfi1p wastaggedwithGFPandfoundtolocalizeone Localization ofSfi1p-GFP dle (Maetal.,1999).Thisissimilartothephenotypeof gests thatCdc31pispresentinamolarratio Cdc31p (thebandsweretoofainttoscanaccurately)sug- tensities oftheCoomassie-stainedbandsforSfi1p-prAand Sfi1p andCdc31p.Inaddition,inspectionoftherelativein- difficult todetect. present inFig.1b,theseotherSPBcomponentswouldbe Spc110p (Elliottetal.,1999).Atthelowlevelsofprotein and Kilmartin,1996),whicharepresentinacomplexwith with otherSPBcomponentssuchasSpc42p(Donaldson whether thisreflectsadirectinteractionwithSpc110por Kilmartin, 1990;Kilmartinetal.,1993).Itisnotclear gether withanotherSPBcomponent,Spc110p(Routand pull-down wasnotasclean,butitdidisolateCdc31pto- experiment underthesamelysisconditions(Fig.1b).This Cdc31p wasreciprocal,Sfi1p-prAusedinapull-down the SPB. These experimentsshowareciprocalinteractionbetween To confirmthattheinteractionbetweenSfi1pand C, andshowedaverysimilar phenotype tothedepletion (Byers,1981),andindicatesapossiblefunctionat CDC31 2 sfi1-3 /M arrestwithfailuretoformamitoticspin- . Twoalleles, and sfi1-7 sfi1-3 and sfi1-7 1. , wereex- 1 with The Journal of Cell Biology cell cycle.Thisbehaviorisvery similartothatofdifferent At 1.5hafterrelease, At ters from2.5to5h(Fig.3b).However, a). Allofthecellsthenarrestedwithsinglemicrotubuleas- peared toshownormalseparatingorseparatedDNA(Fig. 3 of thecellshadspindles,andpost-anaphasespindlesap- the firstcellcycle,and cdc31 sfi1-7 Figure 2. (Fig. 3c).Theseresultsshowthat arrested withsinglemicrotubuleastersbetween1.5and5 h 36 of thecellsexamined),despitebuddingatsametimeas of of asynchronous cellsafter4hat36 SPB duplication hadoccurred.TheSPB-containing regions ers, 1981;Vallenetal.,1994). ters inthefirstcellcycle,and somearrestinthesecond(By- bridge. Bars:(a–d)2 (e) Immuno-EMofGFP-Sfi1pshowingstaininglocalizedtothehalf- anti-GFP (c)andanti-90-kD(Spc98p)mAbstolocalizeSPBs(d). bright spotsaremitochondria.(candd)Immunofluorescencewith with nuclei.Inb,nucleiarethelargerareasofstaining;small DAPI fluorescenceforDNA(b)showoneortwoGFPspotscoincident of unfixedcellscontainingSfi1p-GFP.GFPfluorescence(a)and -factor (10 -factor Both alleleswereexaminedby EMtodeterminewhether C showedinterestingdifferences between thetwoalleles. , didnotpassthroughmitosisat1.5h,butremained alleles,wheresomearrestwith singlemicrotubuleas- Localization of g/ml for150minat23 m; (e)0.1 sfi1-7 S. cerevisiae sfi1-7 cellspassedthroughmitosis:78% cellsarrestduringthesecond m. Sfi1p. sfi1-3 C andsynchronized (aandb)Fluorescence C) andreleasedat cellsarrestduring sfi1-3

cells(95%

plaque diameterincreasingfrom0.10 section. TherewasaclearincreaseinSPBsize,thecentral difficult torecognizeunlessitisalmostperpendicularthe (Fig. 3e),butthisisanunderestimate,asthehalf-bridge is About onethirdofthesingleSPBshadclearhalf-bridges only singleSPBs(Fig.3,d–g).Theoneexceptionwasan rial sectioning.Inatotalof50cellsfrombothalleles,49had cells between2.5and5hafterreleasewereexaminedbyse- to 0.16 cdc31-1 The similarity between thephenotypesof and Genetic interactionsbetween tive temperature. large SPBineitherthefirstor secondcellcycleattherestric- Vallen etal.,1994).Alloftheseallelesarrestwithasingle show strikingsimilaritiesto cdc31-1 sfi1-7 Figure 3. similar tothephenotypeof bled SPBstillonthecytoplasmicsideofhalf-bridge, smaller. 36 and DAPIforDNA.(d)EMof (c) (d-g) 0.1

-factor werereleasedat36

In conclusion,thephenotypesofboth sfi1-3

C, (f)

cdc31-1

cellwithonenormalSPBandsmallpartlyassem-

suggestthatthere mightbegeneticinteractions be- (Byers,1981),althoughtheincreaseinSPBsizeis at1.5h,stainedwithanti-Tub4pforSPBsoranti-tubulin

sfi1-7

m.

Phenotype of

0.08

after4h,and(g)

Yeast spindlepolebodyduplication| m (Fig.3,e–g).Thisisagainsimilarto sfi1 C. (a) alleles. sfi1-3 sfi1-3 mps2-1 sfi1-7 at23 cdc31 CellssynchronizedinG sfi1-3 after2.5h.Bars:(a-c)2 at1.5h,(b) (Wineyetal.,1991). alleles(Byers,1981; C, (e) , 0.02 sfi1-7 sfi1-7 sfi1-3 sfi1-3 Kilmartin , sfi1-7 after5hat m (Fig.3d) , and sfi1-7 at2.5h, 1 sfi1-7 with , and 1213 m; The Journal of Cell Biology 1214 between the There isonly24.5%sequenceidentity(Cliftenetal.,2003) further. Allexceptoneoftheexpected variable colonysizeonstreakingout,andwerenotstudied both 2- Transformation ofthesecellswith 37 Saccharomyces cerevisiae sfi1-7 cdc31-1 tween on fluorooroticacidmedium(whichselectsagainst does notgrowat30 (a) Suppressionof Figure 4. Structural featuresandconservationofSfi1repeats slowly thanthewildtype,andfailedtogrowatall30 served intheother the twospecies.However, son andKilmartin,1996)has only16.5%identitybetween worse formostSPBproteins; for example,Spc42p(Donald- org). Thisdegreeofdivergence issimilarorsometimeseven http://db.yeastgenome. (these alignmentsaredisplayedat ments showgeneticinteractionsbetween between the b). Theseexperimentsshowasyntheticgrowthinteraction 23 temperature-sensitive sporesgrewat23 30 quence AX striking internalrepeats(Fig. 5 a)containingaconsensusse- plasmid. (b)Thedoubletemperature-sensitivemutant C, whichwasdependentonthepresenceof C, andtheseallgrewextremelyslowly,producedavery C. Therewerealsosyntheticgrowthdefectsbetween m plasmidsuppressedboth The JournalofCellBiology sfi1 SFI1 allelesand Genetic interactionsbetween 7 and LLX sfi1 S. cerevisiae doubletemperature-sensitivesporesgrewat allelesand sfi1-3 3 F/LX CDC31 C andcannotlosetherescuing Saccharomyces cdc31-1 by 2 WK/R. Thisrepeat structureiscon- Sfi1pisaverydivergentprotein. CDC31 and . Overexpressionof

| S. cerevisiae cdc31-1 . Onlyonethirdoftheexpected

Volume 162,Number7,2003 Saccharomyces castelli inanoverexpressing2- sfi1-3 species,withthe possible , andbothsetsofexperi- SFI1 (Fig.4a)and SFI1 sfi1-3 cdc31-1 Sfi1phasaseriesof C, butslightlymore restoredgrowthat SFI1 and URA3 CEN-URA3-SFI1 and CDC31 sfi1-3cdc31-1 CDC31 SFI1 plasmids). CDC31 proteins sfi1-7 double (Fig.4 m . ona

at C. .

the versionwith 1242aminoacidsisanalyzed here.There- either 968or1242 aminoacids(seeMaterials andmethods); with almostidenticalsequence, butwithstopcodonsafter (Fig. 5,bandc).Thereare at leasttwoversionsofhSfi1 here, andahumanprotein (KIAA0542) calledhSfi1here ing. Thesewerean gaps betweentherepeatswere identifiedbyBLASTsearch- amino acids. clusters ofrepeatsin times thealanineisnotasprominent.Thegapbetween exception ofrepeatsaroundW304andF698,some- of theconservedtryptophans(position22)marked. The domainstructureforeachproteinisalsoshownwiththepositions potential homologuesin Figure 5. Proteins withsimilarrepeatstoSfi1pand Sequence logosofSfi1repeatsin S. pombe S. cerevisiae S. pombe protein(T40750)calledSpSfi1 , SpSfi1,andhumans,hSfi1. Sfi1pisbetween23and35

S. cerevisiae

and

The Journal of Cell Biology ogy wasdetectedbetweentheNH outside oftherepeatregionshowninFig.5,andnohomol- two forhumanSfi1.Therearenofurtherconsensusresidues within oneorganism. and humans,maybepresentinmultipleproteins sults suggestthatSfi1repeatsareconservedbetweenfungi anti-Sad1 (f)toshow localizationtotheSPBregion. Bars,2 of SpSfi1ptothe ends ofthemitoticspindle,andwith theSPBmarker GFP fluorescence(cande)withanti-tubulin (d)toshowlocalization withnuclei.(c–f)GFPfluorescence andimmunofluorescence.incident and DAPIfluorescenceforDNA (b) showoneortwoGFPspotsco- rescence ofunfixedcellscontaining SpSfi1p-GFP.GFPfluorescence(a) Figure 6. ized totheSPB.Tagged siae S. pombe Localization of proteins waslow:oneforSfi1p,none line contentwithintherepeatregionsforallofthree tophan, W,isshowndiagrammaticallyinFig.5).Thepro- three 33merrepeats(thepositionoftheconservedtryp- 35mer, afurthernine33merrepeats,one37mer,andfinally and 862thereareninecontinuous33merrepeats,one peats inhSfi1areparticularlyuniform.Betweenresidues96 No further than hSfi1,andarethuslesssimilartotheyeastproteins. ods). However,thesesequenceshavemanyfewerrepeats ing multiplecopiesofthisrepeat(seeMaterialsandmeth- man proteinsandthepartialsequenceofanothercontain- (see Materialsandmethods).Thisidentifiedtwomorehu- able, soScanProsite(Gattikeretal.,2002)wasusedinstead erogeneous insequence,BLASTsearchingwasnotsuit- also containSfi1repeats?Becausetherepeatisquitehet- domains ofthethreeproteins. two spotspercellcoincidentwiththenuclearDNA(Fig.6, Are thereotherproteinsinthesethreeorganismsthat Sfi1p,wastaggedwithGFPtoseewhetherittoolocal- Sfi1(Fig.5b),apotentialhomologueof Localization of S. cerevisiae S. pombe proteinswereidentified.Thesere- S. pombe S. pombe Sfi1andhuman Sfi1(SpSfi1). Sfi1localizedtooneor 2 - andCOOH-terminal S. pombe (aandb) Sfi1,and S. cerevi- m. Fluo- GT335 arered.Bars,5 merged images(eandh).Anti-GFP isgreenandanti-centrin2 GFP (candf),anti-centrin2(d),GT335 (g),andcorresponding shown atthebottom.Stainingof the centrosomeregionwithanti- with anti-GFP(a)andanti- Figure 7. Transfection ofHeLacellswithGFP-hSfi1. Yeast spindlepolebodyduplication| m. -tubulin (b).Anuntransfectedcell is Kilmartin Cellsstained 1215 The Journal of Cell Biology 1216 Sfi1p and(f)forhSfi1. GelswerestainedwithCoomassie blueand Sfi1p andhuman hSfi1. Figure 8. The JournalofCellBiology GST pull-downstolocalizethecentrin-binding sitesin MapsoftheGST fusionsareshown(a)for

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Volume 162,Number7,2003 in therepeatregion. positioned, similarlyspaced,andalsoalowprolinecontent tion, asinthetwoyeasts,ithadrepeatscentrally lar totherepeatnumberintwoyeasts(Fig.5).Inaddi- tween 4and10repeats,whereashSfi1had23simi- that Cdc31pfailstobindtheNH the pull-down(P)foreachoffiveconstructs.Itisclear natant fromthepull-down(S),andwashedpellet in theleftlane,followedbyfusionprotein(F),super- GST pull-downs(Fig.8b)showstherecombinantCdc31p COOH-terminal domain(fusion5).TheSDSgelofthe three domainscontainingtherepeats(fusions2–4),and centrin 2binding toGSTfusionsofhSfi1. 1–3 repeats.(g)Cdc31p bindingtoGSTfusionsof hSfi1.(h)Human concentration. (dande)Cdc31pbinding toSfi1pfusionscontaining Sfi1p. (c)BindingofCdc31ptofusion 2withincreasingCdc31p ml buffer).(b)GSTpull-downsfor Cdc31pbindingtofusions1–5in pull-down, andPthepellet(3- pull-downs, Fisthefusionprotein, Sisthesupernatantafter human centrin2(firstlaneinh). In eachsetofthreelanesforthe show therecombinantCdc31p(first laneinb)andtherecombinant Sfi1p and repeats, hSfi1seemedthemosthomologousto cerevisiae (Hagan andYanagida,1995).Thus, coincident withSad1(Fig.6,eandf),amarkerfortheSPB to theendsofmitoticspindle(Fig.6,candd)were a andb).Doublelabelingshowedthatthesespotslocalized downs usingproteinsexpressedin Cdc31p bindsdirectlytoSfi1p.ThiswastestedbyGSTpull- The prApull-downexperimentsshowninFig.1suggestthe Binding ofCdc31ptoSfi1p number isapproximateforfusion3becauseofproteolysis). tween 3and4molofCdc31pperfusionprotein(this Cdc31p tofusionis Coomassie-stained bandsshowthatthemolarratioof repeats (Fig.8b).Inaddition,therelativeintensitiesof domains, butdoesbindtothethreefusionscontaining hSfi1 localizesclosetothecentriole. oles (Bobinnecetal.,1998).Theseresultsindicatethat 1992), againstglutamylatedtubulinandamarkerforcentri- incident (Fig.7,f–h)withanmAb,GT335(Wolffetal., most completelycoincident(Fig.7,c–e)withanmAb, at theCOOHterminusofeitherform.Thestainingwasal- the experiments.NostainingwasseenwhenGFPplaced higher forthe1242aminoacidform,sothiswasusedall forms ofhSfi1,butthetransfectionefficiencywasmuch sults werefoundforboththe1242and968aminoacid centrosomal anti-GFP consistingofoneortwodotswithinclosetothe transfected intoHeLacells.Afaintsignalwasdetectedwith domains (Fig.8a);theNH domains region. Accordingly,Sfi1pwasinitiallydividedintofive the repeats,itseemedlikelythatCdc31pwouldbindtothis Cdc31p isconservedandtheonlypartofSfi1p Cetn2 (Hartetal.,2001),againstcentrin2,andalmostco- hSfi1 wastaggedattheNH Of thefourhumanproteinsidentifiedwithpotentialSfi1 Sfi1p,localizestotheSPBregion. S. pombe -tubulin staining(Fig.7,aandb).Similarre- Sfi1.Theotherhumanproteinshadbe- 1. Scanningofthegelsshowedbe- l beadswashed4–5timeswith0.5 2 2 -terminal domain(fusion1), terminuswithGFPandwas Escherichia coli 2 - andCOOH-terminal S. pombe Sfi1,like S. cerevisiae . Because S. The Journal of Cell Biology these repeats. also bindsCdc31paswellmammaliancentrinsdirectly at man homologueofSfi1p,hSfi1containingsimilarrepeats, Cdc31p directlythroughaseriesofconservedrepeats.Ahu- creased (unpublisheddata). EGTA, thoughthebindingofcentrin2wasslightlyde- Again, thebindingofCdc31pwasunaffectedby5mM tions betweenthedifferentcentrins(unpublisheddata). binding tofusions6–9,butnotfusion10,withslightvaria- 1997) andmousecentrin4(Gavetetal.,2003)gavesimilar trins 1(Erraboluetal.,1994)and3(Middendorp did notbindeitherprotein(Fig.8,gandh).Humancen- the COOHterminus,containingnodetectableSfi1repeats, g) andhumancentrin2(Fig.8h),whereasfusion10from fusions containingSfi1repeatsboundbothCdc31p(Fig.8 Cdc31p andhumancentrin2(LeeHuang,1993).The also preparedforhSfi1(Fig.8f)andtestedbindingto (see Fig.5candMaterialsmethods),sofusionswere that mostoftheSfi1repeatsinSfi1pcanbindCdc31p. mM EGTA(unpublisheddata).Together,thesedatasuggest sensitive becauseitwasunaffectedbyremovalofCa shows thatthe Sfi1 repeatsarealsopresentin twopreviously et al.,2002;Nishikawa etal.,2003),andSfi1p. Thispaper Rose, 1994;Spangetal.,1995), Mps3p/Nep98p(Jaspersen ing proteinsinthehalf-bridge tothree;Kar1p(Bigginsand the protein.Thisnowbrings thenumberofCdc31p-bind- consist ofaseries17novel repeats inthecentralregionof Cdc31p bindsdirectlytomultiple bindingsitesinSfi1pthat show geneticinteractions,suggesting theyinteractinvivo. an essentialfunctioninSPBduplication. 1993), Sfi1plocalizestothehalf-bridgeofSPBandhas centrin, Cdc31p.LikeCdc31p(Byers,1981;Spangetal., down usingafunctionaltaggedversionofthebuddingyeast This paperdescribesaprotein,Sfi1p,isolatedfrompull- Discussion ing ofCdc31ptotheGSTfusionsdidnotappearbeCa conditions oftheyeastlysatepull-down(Fig.1a),bind- of therepeatsisinactive.AsexpectedfromlowfreeCa tration wasincreased(unpublisheddata),suggestingthatone two repeatsneverroseabove1.1whentheCdc31pconcen- with thepredictedthreerepeats.Theratiofor4ccontaining concentration wasincreased(unpublisheddata),compared ratio of1.1inFig.8d,whichroseto2.5whentheCdc31p repeats, 2aunfortunatelysufferedfromproteolysis,2cgavea concentration (unpublisheddata).Forthefusionswith2to3 which didnotriseabove1.1overa10-foldrangeofCdc31p repeats (2b,3a,4a,and4b)showedmolarratioscloseto1, 3b theaffinitywaslow.Theotherfusionscontainingsingle positive forCdc31pbinding(Fig.8,dande),although element, soitwasplacedcentrally.Allofthesefusionswere was assumedthattheconservedtryptophancritical ined. Indesigningthefusionsforindividualrepeats,it for saturationfusion2,wherethemolarratioroseto Increasing theCdc31pconcentrationshowedsomeevidence 5.5 comparedwiththesixpredictedrepeats(Fig.8c). These resultsshowthatSfi1pbindsmultiplemoleculesof Sfi1 repeatshavebeenfoundinseveralhumanproteins Next, individualandsmallergroupsofrepeatswereexam- SFI1 and 2 CDC31 with5 2 2 - helical conformationbetweentheNH 2003). MyosinIQdomainsarethoughttobepresentina residues intheIQGAP-relatedproteinIqg1p(Terraketal., ple IQdomainsarespacedmorewidely,forexample,28–32 shorter distanceapart(23–26residues),thoughothermulti- multiple continuouscopies.Theyarespacedataslightly yeast uncharacterized proteins,onefromtheevolutionallydistant the centrins,givencharacteristic largerNH may alsobecompatiblewith directconnectionsbetween adjacent Sfi1repeats(usually between23and33residues) formation liketheIQrepeats.Thewiderspacingbetween generally lackprolines,andthusmightadoptahelicalcon- domain (Houdusseetal.,1996). first boundcalmodulinmaybedirectlyincontactwiththat may regulatethefunctionofmotordomainbecause mational changesinthealreadyboundcalmodulin.This tional myosins.Here,additionofCa striated flagellar roots(Salisburyetal.,1984), whichrap- organelles such asthespasmoneme(Amoset al.,1975)and relevance tootherEF-hand domain orcentrin-containing charya etal.,1993).Thistype ofstructuremayhavesome domains ofcentrinscompared withcalmodulin(Bhatta- the Ca tween thecalmodulinmolecules,whichcanexplainsomeof cent IQdomainsiscompatiblewithdirectconnectionsbe- Cohen, 1996).Thespacingof23–26residuesbetweenadja- ulin-like lightchainsbound(Xieetal.,1994;Houdusseand is basedonthestructureofscallopmyosinwithtwocalmod- nal lobesofcalmodulin(Houdusseetal.,1996);thismodel mains showCa Mooseker, 1992).LiketheSfi1repeats,myosinIQdo- neck regionofunconventionalmyosins(Cheneyand the centrin-binding Sfi1repeatsmightbetheIQdomainsin binding sites(HoeflichandIkura,2002). is verysimilartothelargevariationsbetweencalmodulin- from Sfi1repeats.Thisheterogeneityinbindingsitemotifs and Rose,1994;Spangetal.,1995),butappearsdifferent Cdc31p-binding siteinKar1phasbeenidentified(Biggins (Jaspersen etal.,2002;Nishikawa2003).The 1995), Kic1p(Sullivanetal.,1998),andMps3p/Nep98p Cdc31p, Kar1p(BigginsandRose,1994;Spangetal., present inthethreeotherproteinsthatdirectlybindto teins isolatedintheCdc31ppull-down(Fig.1),andarenot peats. Sfi1repeatsareapparentlyabsentfromtheotherpro- two repeatsmayalsoaffectthefunctionofrestre- the firstrepeat.Theseresultssuggestthatdisablingoneor (F208C) intheconsensusphenylalanineatposition19 terials andmethods),indeed, have mutationswithinthefirstandsecondrepeats(seeMa- 5) ofSfi1repeats.Bothtemperature-sensitiveallelesSfi1p WD repeatslacktheconsensusleucineatposition14(Fig. WD repeats(thatwouldbeatposition16inFig.5).Also, the conservedasparticacid(Garcia-Higueraetal.,1998)in et al.,1994),butareclearlydistinctbecauseSfi1repeatslack teins localizetothespindlepole. tein, hSfi1,bindscentrinattherepeats,andallthreepro- The Sfi1repeatregionsoftheproteinsdescribedhere The mostanalogouscalmodulin-bindingdomaintothe Sfi1 repeatshavesomeresemblancetoWD(Neer S. pombe 2 -dependent regulationoftheactivityunconven- , andanotherfromhumans.Thehumanpro- Yeast spindlepolebodyduplication| 2 -independent bindingandarepresentin sfi1-3 2 2 - andCOOH-termi- couldcauseconfor- hasamutation Kilmartin 2 -terminal 1217 The Journal of Cell Biology 1218 at leastspasminwithsuchaproteinislikelytobeCa min orcentrintotheSfi1p-likeprotein.Theassociationof gels oftheorganelle,duetohighmolarratiospas- cause itwouldbefaintlystainedinCoomassie-stainedSDS teractions. Suchaproteinwouldbedifficulttoidentifybe- be madelongerbyend-to-endorstaggeredside-to-sidein- filament ofspasminorcentrin.Thesefilamentscouldthen could formatemplateforwhatwouldeffectivelybeshort ple Sfi1repeatswerepresentintheseorganelles,thenit proteins orcalmodulin.However,ifaproteinwithmulti- given thehomologyoftheseproteinstoEF-handdomain Schiebel andBornens,1995).Thisnowseemsunlikely, filamentspresentintheseorganelles(Amos,1975; 2–7-nm originally thoughtthattheythemselvesmightformthe the mainproteincomponentsoftheseorganelles,soitwas EGTA (Amos,1971).AdditionofCa becausespasmonemesareglycerinatedin4mM independent All S. cerevisiae Materials andmethods idly contractonadditionofCa is elasticity,whichnotdependentontheCa the caseofspasmoneme(Weis-FoghandAmos,1972), additional featureofcentrin-bindingfilaments,atleastin pand andcontractanSfi1p-Cdc31pfilament.However, this pointinthecellcycle,whichmightbenecessarytoex- Ca conformational changes,inthesamewaythatadditionof trin alreadyboundtoanSfi1p-likeproteincouldcause isogenic diploidK842 (Nasmythetal.,1990),andyeast vectorsusedwere 1999). Atpresent,thereisnoevidenceforCa the half-bridgeduringSPBinsertion(AdamsandKilmartin, may playaroleintheproposedextensionandretractionof tant structuralelementsofthehalf-bridge.Inaddition,they Clearly, ifSfi1pdoesformfilaments,thesemightbeimpor- nas, thusleadingtoshortening. change wouldbeequivalenttofoldingindividualconcerti- the concertinaswerestretchedout,conformational flexible drinkingstraw.Theextendedformwouldbewhen simple analogymightbetheconcertinapartofachild’s changes couldleadtoarapidshorteningoffilament.A bound toanSfi1p-likeprotein,similarconformational the gate(Schumacheretal.,2001).Incaseofcentrin test anyelasticpropertiesorCa sions oftheserecombinantproteins, itshouldbepossibleto crystallographic investigation. Inaddition,usinglongerver- recombinant proteinsdescribedhere,shouldbeamenableto is aninterestingstructuralproblem,andusingsomeofthe driven byreassociationofCdc31p. would bereleasedinsomewayandretraction some oftheCdc31p.Aftercompletionassembly,tension ing ofSfi1repeats,withpossibleconcomitantdissociation be stretchedduringduplicationplaqueassemblybyunfold- like repeats(Lietal.,2000).Possibly,Sfi1p-Cdc31pmight ple oftitin,thestretchingoccursbyunfoldingtandemIg- (Tatham andShewry,2000),inthewell-studiedexam- tration. Elasticproteinsgenerallyhavemultiplerepeats What wouldtheroleofSfi1pbeinSPBduplication? Clearly, themultiplebindingofcentrintoSfi1repeats S. cerevisiae 2 The JournalofCellBiology tocalmodulinalreadyboundaK strains strainswereprepared inK699(W303a

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Volume 162,Number7,2003 2 2 -dependent contraction. . Spasminorcentrinare 2 tospasminorcen- ) backgroundorthe channelopens 2 2 pulsesat concen- 2 - in pull-downswith functionallyunrelatedprAfusions. of theseproteinsare probablynonspecificbecausethey havebeenfound formation onthese proteinscanbefoundathttp://db.yeastgenome.org. All gation factorandRps18apRpl23ap areribosomesubunits.Furtherin- components ofachromatinremodeling complex;Tef2pistranslationelon- Sse1p, Hsp60p,andScj1pareheat shock proteins;Rvb1pandRvb2pare and Rpn2pareproteosomecomponents; Ssa1p,Ssa2p,Ssb2p,Ydj1p, 61%; band12,Rps18ap:9,50%;and band13,Rpl23ap:10,42%.Rpn1p 45%; band11,32peptidesfromhuman keratintypeIandII;Cdc31p:10, 27%; band9,Ydj1p:17,40%and Tef2p: 12,32%;band10,Scj1p:18, 31%; band7,Hsp60p:25,55%;8,Rvb1p:12,35%andRvb2p:10, 13, 15%;band5,Sse1p:8,6,Ssa2p:29%andSsb2p: 28, 29%;band3,Rpn1p:24,25%;Spc110p:17,18%;4,Rpn2p: 52%. IntheSfi1p-prApulldown,bandsidentifiedwere:Sfi1p-prA: 12, 34%;band2,Ydj1p:9,29%;Hem15p:13,32%andZCdc31p: 9, 21%; band1,Ssa1p:8,18%;Ssa2p:10,24%andSsb2p:22%;Thp1p: Vps13p: 26,11%;Sfi1p(topband):29,33%;(bottom19, these peptideswereasfollows:intheCdc31ppull-downgel(Fig.1a), numbers ofpeptidesidentifiedandthesequencepercentagecovered by missed trypticcleavage.Allidentificationswerethetopmatch,and between 40–60ppm,allowingformethionineoxidationandupone base wassearchedusingtheMascotsearchengine(MatrixScience,Ltd.), the entireNationalCenterforBiotechnologyInformation(NCBI)data- identified bymassspectrometry(WiggeandKilmartin,2001),exceptthat plied toasingleSDSgellane.Proteinsinbands(Fig.1)were of yeastcellsharvestedat5 Complexes wereisolatedasbefore(WiggeandKilmartin,2001)from10l Isolation ofcomplexesandmassspectrometry dure (Boekeetal.,1987).Ura type genewiththetemperature-sensitiveallelebypopin-popoutproce- the Plasmids conferringatemperature-sensitivephenotypeweretransferredto ature-sensitive colonieswereselected,andtheplasmidwasrecovered. URA3 orotic acid(FOA)medium(Boekeetal.,1984)toremovethewild-type and 1mMdCTPdTTP.Transformantswerereplicaplatedontofluoro- N218D andD242A. used tomake ture-sensitive mutation.Theaminoacidchangesfoundintheplasmid to showrescueandbackcrossedthepresenceofasingletempera- nally, thetemperature-sensitivemutantstrainsweretransformedwith plasmid atthewild-typelocusanditsremovalafterFOAtreatment.Fi- ture-sensitive alleleswerecheckedbyPCRtoconfirmthepresenceof sensitive colonies.Boththepopinparentalstrainandpopouttempera- replica platedontowarmandambientYEPDplatestoselecttemperature- PCR (Akadaetal.,2000)with7.5mMMgCl presumed initiatormethionine.ThePCRconditionswerethoseforcolony same StyIsite.AllbasepairnumbersstartfromtheAofcodon 2820–2800, andtheplasmidwasgappedbetweenMscI(748) AgeI (242)andStyI(2676)sites;for PCR were97–119and2820–2800,theplasmidwasgappedbetween methionine inCdc31pwiththesequence:MGSVDNKF orientation intotheNcoIsiteplacedin M74186) betweenNcoIandBspLUIIIsiteswasinsertedinthecorrect prA wasamplifiedfrompEZZ18(GenBank/EMBL/DDBJaccessionno. cause thisconstructwasabletorescueadeletion.AsingleZdomainof change S2G,anddidnotappeartoaffectthefunctionofproteinbe- site byPCRattheinitiatormethionineof (Vallen etal.,1994),wasverifiedbyPCRsequencing. crossed toK699fivetimes,andthesequencechangeinthisallele,A48T the pRSseries(SikorskiandHieter,1989).The procedure (Muhlradetal.,1992).For sensitive mutantswerepreparedbyslightmodificationsofthegaprepair ences andwere:L96P,F278L,D541G,I623V,G935E..Temperature- lished sequence(Maetal.,1999);theseareprobablyduetostraindiffer- showed thesameaminoacidchangescomparedwithpreviouslypub- cells showinganapparentlynormalgrowthrate. vector pRS314wereabletocomplementadeletionof VPDYAHM inplaceoftheinitiatormethionine.Bothconstructs using NcoIandBspLUIIIsitesinsertedintothesequenceMGSYPYD- 1987). TheHA-Cdc31pconstructwaspreparedfromlinkeroligosagain corresponding tothesynthetic58-aminoacidZdomain(Nilssonetal., total of63aminoacidswiththesequencebetweenVDNKFandAQAPK The constructtomakeZCdc31pwaspreparedbyfirstplacingaNcoI SFI1 URA3 plasmid,thenontowarm(37 wasclonedbyVentPCRandgaprepair.Bothconstructs plasmidpRS306andtransformedintoK699toreplacethewild- sfi1-3 wereH207Q,F208CandY247C,for transformantswerepatchedontoFOAand 10 7 cells/ml,withmostofthesampleap- sfi1-7 C) andambientYEPDplates.Temper- sfi1-3 CDC31 , theprimerswere540–561and , theprimersformutagenic CDC31 2 , 0.2mMdATPanddGTP, . Thisreplacedtheinitiator cdc31-1 ; thiswouldcausethe allelewasback- CDC31 … AQAPKHM, a withthe sfi1-7 CEN SFI1 , The Journal of Cell Biology lin (Bobinnecetal.,1998).SecondaryantibodieswereAlexa against centrin2(Hartetal.,2001),andGT335glutamylatedtubu- GFP, mousemAbsGTU-88against with methanol/acetoneat were transfectedwithFuGENE™6(Roche).After48h,cellsfixed were fixedandstainedasbefore(WiggeKilmartin,2001). combinant PCR(Bähleretal.,1998;WiggeandKilmartin,2001),cells al., 1997). erinary andAgriculturalUniversity,Frederiksberg,Denmark;Gorodkinet residues 25–120).LogoswereobtainedfromJanGorodkin(TheRoyalVet- for twohitsintheORFidentifiedhumanBAC05092(fourrepeatsbetween tein (GenBankaccessionno. T40750; Evalue1e searching with[F/W/L]X however CAD38978isquitetryptophanrichinpartsofthisregion.Further sequence ofanother,CAD38978(10repeatsbetweenresidues152–423), tein, AAM34297(fiverepeatsbetweenresidues757–879)andthepartial man homologues,togetherwiththecompletesequenceofonehumanpro- tries, thisidentifiedonlySfi1p,it’s andcallingforatleastthreehitsintheORF.Outof R/H] homologue identified nameIPF5574)wasusedinstead.ABLASTPsearchwiththis tag attheNH were expressedasGSTfusions(Pulvermuller etal.,2002)withasingleHA 4657309), andmousecentrin4(Image 3657877).CentrinsandCdc31p 5163552), humancentrin2(Image 3836808),humancentrin3(Image Centrins wereobtainedasImage clones:humancentrin1(Image GST pull-downs Ziess MicroImaging,Inc.)withthe63 (Radiance 2100;Bio-RadLaboratories)onamicroscope(Axiophot;Carl Princeton Instruments),orforFig.7(c–h)only,withaconfocalmicroscope 63 MicroImaging, Inc.)withPlanNeofluar100 Light microscopywasdonewitheitheramicroscope(Axioskop;CarlZeiss Microscopy sitive toformaldehydefixationinboth kD mAbs(RoutandKilmartin,1990).Theanti-GFPstainingwasverysen- tin etal.,1993),andwerestainedwithrabbitanti-GFPpooledanti-90- (Wach etal.,1997;Wigge1998).Cellswerefixedfor2min(Kilmar- S. cerevisiae with GFPandantibodystaining Tagging of dida A BLASTPsearchwith Identification ofotherproteinswithmultipleSfi1repeats ScanProsite (Gattikeretal.,2002)usingthesequenceAX equivalent to968and1242. have homologues(AAH46305andAAH26390)thatterminateatpositions continuously overa120-kbregionofhumanchromosome22.Micealso form havinganearlierstopcodon.BothoftheseORFscouldbefollowed tion I438L,bothversionsofhSfi1areidentical,withthe968aminoacid amino acids.Apartfromtheabove31acidinsertandsubstitu- and 386,thesubstitutionsL407IP1056L.Thisproteinhas1242 YFCFRALKDNVTHAHLQQIRRNLAHQQHGVT betweenaminoacids385 length insertofthedatabasesequencehSfi1withanadditional age 5273112,BI463640)wassequencedandfoundtocontainafull- amino acids(apartfromthesubstitutionH322Y).AnImageESTclone(Im- actually tocorrespondasplicingvariant(T00322)containing968 structure (Fig.5).ThehSfi1(KIAA0542)clonewassequencedandfound 0.096) calledherehSfi1.Alloftheseproteinshaveanunusualrepeat a humanproteinof1212aminoacidsKIAA0542(BAA25468;Evalue pressed underthe same conditions,butremainedsoluble whenthemethod by thrombindigestion. GSTfusionsofSfi1pwereall insolublewhenex- ulations wereat25 as before(AdamsandKilmartin,1999). rabbit IgGandAlexa and contrastinAdobePhotoshop 2000 software.Thewholeoftheimageswerethenadjustedforbrightness ear filtertothewholeimage;confocalmicroscopeusedLasersharp Fig. 6a),interferencelineswereremovedbyapplicationoftheIPLabslin- CCD camerausedIPLabssoftware,andforfaintimages(Fig.2,a c; Human Sfi1wasinsertedintopeGFP-C1(U55763)byPCR.HeLacells S. pombe Other humanproteinsthatmightcontainSfi1repeatswereidentifiedby

NA1.4objectivesandaMicroMAXCCDcamera(RTE/CCD-1300-Y; albicans Sfi1pwastaggedattheCOOHterminusbyrecombinantPCR Sfi1wastaggedattheCOOHterminuswithGFPusingre- S. cerevisiae 2 homologue(listedathttp://genolist.pasteur.fr/CandidaDB; terminusasforHA-Cdc31p(seeabove). Allbacterialmanip- C andinductionwas for4hat25 ® 594anti–mouseIgG(MolecularProbes,Inc.). S. cerevisiae S. cerevisiae 2 W[K/R/H]X Sfi1p, 20 C, stainedwithaffinity-purifiedrabbitanti- ® Sfi1p(Evalue2e S. pombe . EMandimmuno-EMwereperformed S. pombe Sfi1pwasnotsuccessful,sothe 21–34 S. cerevisiae Apochromatobjectiveabove.The -tubulin (Sigma-Aldrich), [F/W/L]X , NA1.3orPlanApochromat Sfi1,andhumanSfi1 Aspergillus 4 ) calledhereSpSfi1,and 2 andhumancells. W[K/R/H] andcalling 14 C; GSTwasremoved ), an , mouse,andhu- S. pombe 12 800,000 en- [F/L]X ® 488anti– Candida 2 Cetn2 W[K/ pro- Can- Boeke, J.D.,F.Lacroute,andG.R.Fink.1984.Apositiveselectionformutants Byers, B.1981.Multiple rolesofthespindlepolebodiesin lifecycleof Brickner, J.H.,andR.S.Fuller.1997. Boeke, J.D.,J.Trueheart,G.Natsoulis, and G.R.Fink.1987.5-Fluorooroticacid Bobinnec, Y.,M.Moudjou,J.P.Fouquet,E.Desbruyeres,B.Edde,andBor- centrin concentrationbetween1and100 also duetoM.S.Robinsonfordiscussion. the lateDouglasKershawforcuttingserialthinsections.Thanksare anti-Sad1, KeithGull(UniversityofOxford,UK)forTAT1,andto Iain Hagan(PatersonInstituteforCancerResearch,Manchester,UK) MN) for mass spectrometry,JeffreySalisbury(TheMayoFoundation,Rochester, I amparticularlygratefultoSewYeuPeak-ChewandFaridaBegumforthe Biggins, S.,andM.D.Rose.1994.Directinteractionbetweenyeastspindlepole Bhattacharya, D.,J.Steinkotter,andM.Melkonian.1993.Molecularcloning Bähler, J.,J.-Q.Wu,M.S.Longtine,N.G.Shah,A.McKenzie,III,A.B.Steever, Amos, W.B.1975.Contractionandcalciumbindinginthevorticellidciliates. Amos, W.B.1971.Reversiblemechanochemicalcycleinthecontractionof Akada, R.,T.Murakane,andY.Nishizawa.2000.DNAextractionmethodfor Adams, I.R.,andJ.V.Kilmartin.1999.Localizationofcorespindlepolebody References Accepted: 20August2003 Submitted: 10July2003 with either1mMCaCl binding buffer(50mMTris-Cl,pH7.4,0.15MNaCl,and0.05%Tween20 and fusion10(1100–1242).Pull-downswereinatotalvolumeof40 6 (205–309),fusion7(332–470),8(464–612),9(613–805), (643–677), fusion4b(676–710),and4c(739–811);forhSfi1: fusion 2c(279–386),3a(451–474),3b(469–507),4a (551–803), fusion5(796–947),2a(181–250),2b(246–281), for Sfi1p:fusion1(1–187),2(181–386),3(379–552),4 U13852), andweredesignedtoexpresstheaminoacidsshowninbrackets, shown inFig.7werepGEX-3X(GenBank/EMBL/DDBJaccessionno. of FrangioniandNeel(1993)wasfollowed.FusionsSfi1phSfi1 2 (Fig.8c),theCdc31pconcentrationwasbetween10and100 Araki, M.,C.Masutani,M.Takemura,A.Uchida,K.Sugasawa,J.Kondoh,Y. Amos, W.B.,L.M.Routledge,andF.F.Yew.1975.Calcium-bindingproteinsina lays. when scanningthegelsbecausethesefailedtoreactwithCdc31pinover- ware (AmershamBiosciences).BandsinthefreeGSTpositionwereignored eter SI;MolecularDynamics)andprocessedwithImageQuant™1.2soft- ple buffer.SDSgelswerescannedwithadensitometer(PersonalDensitom- and equivalentamountsofsupernatantbeadswereboiledinSDSsam- were rotatedfor3–4hatRT,washedfourorfivetimeswith0.5mlbuffer, orotic acidresistance. lacking orotidine-5 139:23–36. by modulatingthefunctionoftwoTGN localizationsignals. promotes TGN-endosomalcyclingofKex2p andothermembraneproteins 175. as aselectiveagentinyeastmoleculargenetics. ating non-neuronalcells. nens. 1998.Glutamylationofcentrioleandcytoplasmictubulininprolifer- pole body. body components:Kar1pisrequiredforCdc31plocalizationtothespindle in greenalgaeandlandplants. evolutionary analysisofthecalcium-modulatedcontractileprotein,centrin, Yeast. cient andversatilePCR-basedgenetargetingin Wach, P.Philippsen,andJ.R.Pringle.1998.Heterologousmodulesforeffi- cella. screening yeastclonesbyPCR. Cell Biol. (SPB) componentsduringSPBduplicationin genome nucleotideexcisionrepair. is partofthexerodermapigmentosumgroupCcomplexthatinitiatesglobal Ohkuma, andF.Hanaoka.2001.Centrosomeproteincentrin2/caltractin1 Vorticellid contractileorganelle. Gen. Physiol.Ser.

Cetn2, BernardEddé(CNRS,Montpellier,France)forGT335, Nature. 14:943–951. 145:809–823. J. CellBiol. 229:127–128. Yeast spindlepolebodyduplication| 30:411–436. 2 or10mMEGTA)with3 -phosphate decarboxylaseactivityinyeast:5-fluoro- Mol. Gen.Genet. 125:843–852. Cell Motil.Cytoskeleton. Plant Mol.Biol. SOI1 Biotechniques. J. CellSci. J. Biol.Chem. encodesanovel,conservedproteinthat 197:345–346. M. Forthesaturationoffusion 19:203–213. 28:668–674. 23:1243–1254. Methods Enzymol. 276:18665–18672. 39:223–232. Schizosaccharomyces pombe. Saccharomyces cerevisiae. l packedbeadsanda Kilmartin J. CellBiol. 154:164– M. Beads Saccha- Vorti- 1219 Soc.

J. l The Journal of Cell Biology 1220 Gavet, O.,C.Alvarez,P.Gaspar,andM.Bornens.2003.Centrin4p,anovelmam- Gattiker, A.,E.Gasteiger,andA.Bairoch.2002.ScanProsite:areferenceimple- Garcia-Higuera, I.,C.Gaitatzes,T.F.Smith,andE.J.Neer.1998.FoldingaWD Hagan, I.,andM.Yanagida.1995.Theproductofthespindleformationgene Grandi, P.,V.Doye,andE.C.Hurt.1993.PurificationofNSP1revealscomplex Gorodkin, J.,L.J.Heyer,S.S.Brunak,andG.D.Stormo.1997.Displayingthein- Geier, B.M.,H.Wiech,andE.Schiebel.1996.Bindingofcentrinsyeastcal- Gallardo, M.,andA.Aguilera.2001.Anewhyperrecombinationmutationidenti- Frangioni, J.V.,andB.G.Neel.1993.Solubilizationpurificationofenzymati- Houdusse, A.,M.Silver,andC.Cohen.1996.AmodelofCa Houdusse, A.,andC.Cohen.1996.Structureoftheregulatorydomainscallop Errabolu, R.,M.A.Sanders,andJ.L.Salisbury.1994.CloningofacDNAencod- Hoeflich, K.P.,andM.Ikura.2002.Calmodulininaction:diversitytargetrec- Hart, P.E.,G.M.Poynter,C.M.Whitehead,J.D.Orth,J.N.Glantz,R.C.Busby, Labbe-Bois, R.1990.Theferrochelatase from Knop, M.,andE.Schiebel.1997.Spc98p andSpc97poftheyeast Klotz, C.,N.GarreaudeLoubresse,F.Ruiz,andJ.Beisson.1997.Geneticevi- Kilmartin, J.V.,S.L.Dyos,D.Kershaw,andJ.T.Finch.1993.Aspacerprotein in Jaspersen, S.L.,T.H.Giddings,Jr.,andM.Winey.2002.Mps3pisanovelcompo- Elliott, S.,M.Knop,G.Schlenstedt,andE.Schiebel.1999.Spc29pisacompo- Donaldson, A.D.,andJ.V.Kilmartin.1996.Spc42p:aphosphorylatedcomponent Cheney, R.E.,andM.S.Mooseker.1992.Unconventionalmyosins. Byers, B.,andL.Goetsch.1974.Duplicationofspindleplaquesintegration Cliften, P.,P.Sudarsanam,A.Desikan,L.Fulton,B.J.Majors,R.Water- malian centrinspecificallyexpressedinciliatedcells. mentation ofaPROSITEscanningtool. protein betasubunitandSec13. repeat propeller.RoleofhighlyconservedasparticacidresiduesintheG sad1 NIC96. formation with‘GLFG’nucleoporinsandanovelnuclearporeprotein put. Appl.Biosci. formation contentsofstructuralRNAalignments:thestructurelogos. 28374. pole bodycomponentsKar1pandSpc110p. modulin tosyntheticpeptidescorrespondingbindingsitesinthespindle 1818–1834. totic recombination. fies anovelyeastgene, 210:179–187. cally activeglutathioneS-transferase(pGEX)fusionproteins. myosin at2Åresolution:implicationsforregulation. poles. ing humancentrin,anEF-handproteinofcentrosomesandmitoticspindle ognition andactivationmechanisms. rine centrinCetn2gene. S.L. Barrett,andJ.L.Salisbury.2001.CharacterizationoftheX-linkedmu- viability. Spc110p. plex mediatebindingtothespindlepole bodyviatheirinteractionwith 172–186. namics oftheinfraciliarylatticein dence foraroleofcentrin-associatedproteinsintheorganizationanddy- the mologue Cdc31p. nent oftheyeastspindlepolebodythatinteractswithcentrinho- tory switch. binding tounconventionalmyosinsrevealshowcalmodulinactsasaregula- cation. nent oftheSpc110psubcomplexandisessentialforspindlepolebodydupli- SPB duplication. of the the yeastcellcycle. 16, Munksgaard,Copenhagen.119–131. M. Kielland-Brand,andA.Stenderup,editors. Alfred BenzonSymposium romyces cerevisiae 7278–7283. disruption, andexpression ofitsstructuralgene regulated. Saccharomyces ston, B.A.Cohen,andM.Johnston.2003.Findingfunctionalfeaturesin Cell Biol. The JournalofCellBiology Saccharomyces cerevisiae associateswiththefissionyeastspindlepolebodyandisessentialfor J. CellSci. S. cerevisiae Proc. Natl.Acad.Sci.USA. EMBO J. 4:27–35. J. CellBiol. EMBO J. J. CellBiol. Structure. genomesbyphylogeneticfootprinting. 13:583–586. 107:9–16. . J. CellBiol. spindlepolebody(SPB)withanessentialfunctionduring 12:3061–3071. In J. CellBiol. 16:6985–6995. Cold SpringHarb.Symp.Quant.Biol. MolecularGeneticsinYeast.D.vonWettstein,J.Friis, 129:1033–1047. Genetics. 123:1175–1184. 4:1475–1490. THP1 Gene. spindlepolebodywhosetranscriptiscellcycle- 132:887–901. , connectingtranscriptionelongationwithmi- 157:79–89. 159:945–956.

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J. Biol.Chem. Volume 162,Number7,2003 96:6205–6210. Paramecium. Cell. Appl. Bioinformatics. Saccharomyces cerevisiae 108:739–742. 273:9041–9049. HEM15.

J. Biol.Chem. Cell Motil.Cytoskeleton. Science. Structure. Mol. Biol.Cell.

38:123–131. J. Biol.Chem. 2 -free calmodulin 301:71–76. Anal. Biochem. 1:107–108. -tubulin com- 271:28366– 4:21–32. Curr. Opin. . Sequence, Com- 265: 14: 38: Schiebel, E.,andM.Bornens.1995.Insearchofafunctionforcentrins. Salisbury, J.,K.Suino,R.Busby,andM.Springett.2002.Centrin-2isrequiredfor Salisbury, J.L.,A.Baron,B.Surek,andM.Melkonian.1984.Striatedflagellar Rout, M.P.,andJ.V.Kilmartin.1990.Componentsoftheyeastspindlespin- Schumacher, M.A.,A.F.Rivard,H.P.Bachinger,andJ.P.Adelman.2001.Struc- Pulvermuller, A.,A.Giessl,M.Heck,R.Wottrich,Schmitt,O.P.Ernst,H.W. O’Toole, E.T.,M.Winey,andJ.R.McIntosh.1999.High-voltageelectrontomog- Nishikawa, S.,Y.Terazawa,T.Nakayama,A.Hirata,Makio,andEndo. Moriyama, Y.,H.Okamoto,andAsai.1999.Rubber-likeelasticityvolume Neer, E.J.,C.J.Schmidt,R.Nambudripad,andT.F.Smith.1994.Theancient Nasmyth, K.,G.Adolf,D.Lydall,andA.Seddon.1990.Theidentificationofa Muhlrad, D.,R.Hunter,andParker.1992.Arapidmethodforlocalizedmu- Nilsson, B.,T.Moks,B.Jansson,L.Abrahmsen,A.Elmblad,E.Holmgren,C. Tatham, A.S.,andP.R. Shewry.2000.Elastomericproteins: biologicalroles,struc- Li, H.,A.F.Oberhauser,S.B.Fowler,J.Clarke,andJ.M.Fernandez.2000.Atomic Levy, Y.Y.,E.Y.Lai,S.P.Remillard,M.B.Heintzelman,andC.Fulton.1996. Ma, P.,J.Winderickx,D.Nauwelaers,F.Dumortier,A.DeDoncker,J.M.Thev- Middendorp, S.,T.Kuntziger,Y.Abraham,S.Holmes,N.Bordes,M.Paintrand, Sikorski, R.S.,andP.Hieter.1989.Asystemofshuttlevectorsyeasthost Sullivan, D.S.,S.Biggins,andM.D.Rose. 1998.Theyeastcentrin,Cdc31p,and Lee, V.D.,andB.Huang.1993.Molecularcloningcentrosomallocalizationof Maciejewski, J.J.,E.J.Vacchiano,S.M.McCutcheon,andH.E.Buhse.1999. Middendorp, S.,A.Paoletti,E.Schiebel,andM.Bornens.1997.Identificationofa Spang, A.,I.Courtney,K.Grein,M. Matzner,andE.Schiebel.1995.The Spang, A.,I.Courtney,U.Fackler,M.Matzner,andE.Schiebel.1993.Thecal- duplicationinmammaliancells. tile organelle. roots: isolationandpartialcharacterizationofacalcium-modulatedcontrac- dle polebody. 2194–2203. ture ofthegatingdomainaCa Cell Biol. bly ofcentrin-G-proteincomplexinphotoreceptorcells. Choe, K.P.Hofmann,andU.Wolfrum.2002.Calcium-dependentassem- myces cerevisiae. raphy ofspindlepolebodiesandearlymitoticspindlesintheyeast for nucleardivisionandfusion. 2003. Nep98pisacomponentoftheyeastspindlepolebodyandessential changes intheisolatedspasmonemeofgiant regulatory-protein familyofWD-repeatproteins. of SWI5nuclearentry. second cellcyclecontrolonthe tagenesis ofyeastgenes. main basedonStaphylococcalproteinA. Henrichson, T.A.Jones,andM.Uhlen.1987.AsyntheticIgG-bindingdo- tial Ras-cAMPpathwaydeficiencyintheyeast Natl. Acad.Sci.USA. force microscopyrevealsthemechanicaldesignofamodularprotein. 298–323. and MTOCsin Centrin isaconservedproteinthatformsdiverseassociationswithcentrioles elein, andP.VanDijck.1999.Deletionof duction. A. Paoletti,andM.Bornens.2000.Aroleforcentrin3incentrosomerepro- induced contraction. siae. strains designedforefficientmanipulationofDNAin Biol. the interactingproteinkinase,Kic1p,are requiredforcellintegrity. causes G human caltractin. Cloning andexpressionofacDNAencoding Ca siae CDC31 new mammaliancentringene,morecloselyrelatedto 173. min: anEF-handcalcium-bindingprotein. yeast spindlepolebody. Cdc31p-binding proteinKar1pisacomponent ofthehalfbridge 416. cium-binding proteincelldivisioncycle31of component ofthehalfbridgespindlepolebody. 2

143:751–765. Genetics. /calmodulin. 2 5:197–201. J. CellBiol. arrest. gene. 122:19–27. J. CellBiol. J. CellBiol.

Mol. Biol.Cell. Naegleria Yeast. Proc. Natl.Acad.Sci.USA. Proc. Natl.Acad.Sci.USA. Nature. 148:405–416. 97:6527–6531. Biophys. J. 15:1097–1109. Cell. Yeast. J. CellBiol. 99:962–970. andotherorganisms. 410:1120–1124. 111:1913–1927. 62:631–647. 8:79–82. 10:2017–2031. 76:993–1000. J. Biol.Chem. HO 2 128:863–877. promoterinyeast:cellcycleregulation -activated K Protein Eng. Curr. Biol. SFI1 J. Eukaryot.Microbiol. 90:11039–11043. 94:9141–9146. 278:9938–9943. Zoothamnium Saccharomyces cerevisiae Cell Motil.Cytoskeleton. , anovelsuppressorofpar- Vorticella convallaria Nature. channelcomplexedwith Saccharomyces cerevisiae 12:1287–1292. 1:107–113. J. CellBiol. Saccharomyces cerevi- Saccharomyces cerevi- Mol. Cell.Biol. 371:297–300. sp.underCa 123:405– Saccharo- 46:165– Trends J. Cell spas- Proc. isa 33: 22: 2 - , The Journal of Cell Biology Vallen, E.A.,W.Ho,M.Winey,andM.D.Rose.1994.Geneticinteractionsbe- Terrak, M.,G.Wu,W.F.Stafford,R.C.Lu,andR.Dominguez.2003.Twodis- Wach, A.,A.Brachat,C.Alberti-Segui,Rebischung,andP.Philippsen.1997. Wigge, P.A.,O.N.Jensen,S.Holmes,Souès,M.Mann,andJ.V.Kilmartin. Weis-Fogh, T.,andW.B.Amos.1972.Evidencefornewmechanismsofcellmotil- tubule organizingcenterin tween the boundIQmotifs-functionalimplications. tinct myosinlightchainstructuresareinducedbyspecificvariationswithin tures andmechanisms. Saccharomyces cerevisiae. Heterologous 1998. Analysisofthe ity. Nature. CDC31 236:301–304. HIS3 and markerandGFPreportermodulesforPCR-targetingin KAR1 Saccharomyces Trends Biochem.Sci.

Yeast. , twogenesrequiredforduplicationofthemicro- Saccharomyces cerevisiae. 13:1065–1075. spindlepolebymatrix-assistedlaserdes- 25:567–571. EMBO J.

Genetics. 22:362–371. 137:407–422. Xie, X.,D.H.Harrison,I.Schlichting,R.M.Sweet,V.N.Kalabokis,A.G.Szent- Wolff, A.,B.deNechaud,D.Chillet,H.Mazarguil,E.Desbruyeres,S.Audebert, Winey, M.,L.Goetsch,P.Baum,andB.Byers.1991. Wigge, P.A.,andJ.V.Kilmartin.2001.TheNdc80pcomplexfrom myosin at2.8Åresolution. Gyorgyi, andC.Cohen.1994.Structureoftheregulatorydomainscallop GT335. pha andbeta-tubulininmousetissuesusingaspecificmonoclonalantibody, B. Edde,F.Gros,andP.Denoulet.1992.Distributionofglutamylatedal- orption/ionization (MALDI)massspectrometry. Biol. yeast genesdefiningdistinctstepsofspindlepolebodyduplication. segregation. cerevisiae 114:745–754. containsconservedcentromerecomponentsandhasafunctionin Eur. J.CellBiol. Yeast spindlepolebodyduplication| 59:425–432. J. CellBiol. Nature. 368:306–312. 152:349–360. J. CellBiol. MPS1 and Kilmartin 141:967–977. Saccharomyces MPS2 : novel J. Cell 1221