Propagation of Saccharomyces cerevisiae [PSI] Prion Is Impaired by Factors That Regulate Hsp70 Substrate Binding

MOLECULAR AND CELLULAR BIOLOGY, May 2004, p. 3928⫺3937 Vol. 24, No. 9 0270-7306/04/$08.00⫹0 DOI: 10.1128/MCB.24.9.3928–3937.2004 P opaga ion o Saccha omyces ce e isiae [PSI ⫹ ] P ion Is Impai ed by Fac o s Tha Regula e Hsp70 Subs a e Binding Ga y Jones,† You ao Song, Seyung Chung, and Daniel C. Masison* Labo a o y o Biochemis y and Gene ics, Na ional Ins i u e o Diabe es and Diges i e and Kidney Diseases, Na ional Ins i u es o Heal h, Be hesda, Ma yland 20892-0851 Recei ed 2 Oc obe 2003/Re u ned o modi ica ion 1 No embe 2003/Accep ed 9 Feb ua y 2004 The Saccha omyces ce e isiae [PSI ⴙ ] p ion is belie ed o be a sel -p opaga ing cy oplasmic amyloid. Ea lie cha ac e iza ion o HSP70 (SSA1) mu a ions sugges ed ha [PSI ⴙ ] p opaga ion is impai ed by al e a ions ha enhance Ssa1p’s subs a e binding. This impai men is o e come by second-si e mu a ions in Ssa1p’s con- se ed C- e minal mo i (GPTVEEVD), which media es in e ac ions wi h e a icopep ide epea (TPR) cochape ones. S i1p, a TPR cochape one homolog o mammalian Hop1 (Hsp70/90 o ganizing p o ein), ac i- a es Ssa1p ATPase, which p omo es subs a e binding by Ssa1p. He e we ind ha in SSA1-21 cells deple ion o S i1p imp o ed [PSI ⴙ ] p opaga ion, while excess S i1p weakened i . In con as , deple ion o Fes1p, a nucleo ide exchange ac o o Ssa1p ha acili a es subs a e elease, weakened [PSI ⴙ ] p opaga ion, while o e p oducing Fes1p imp o ed i . The e o e, al e a ions o Hsp70 cochape ones ha p omo e o p olong Hsp70 subs a e binding impai [PSI ⴙ ] p opaga ion. We also ind ha he GPTVEEVD mo i is impo an o physical in e ac ion wi h Hsp40 (Ydj1p), ano he Hsp70 cochape one ha p omo es subs a e binding bu is dispensable o iabili y. We u he ind ha deple ing Cp 7p, an Hsp90 TPR cochape one and CyP-40 cyclophilin homolog, imp o ed [PSI ⴙ ] p opaga ion in SSA1 mu an s. Al hough Cp 7p and S i1p a e Hsp90 cochape ones, we p o ide e idence ha Hsp90 is no in ol ed in [PSI ⴙ ] p opaga ion, sugges ing ha S i1p and Cp 7p unc ionally in e ac wi h Hsp70 independen ly o Hsp90. Hsp70 is a uni e sally conse ed essen ial p o ein ha ac s in many cellula p ocesses whe e p o eins a e incomple ely olded, such as ansla ion and memb ane anspo (7, 25). Addi ionally, Hsp70 exp ession is inc eased by s ess, whe e- upon i p o ec s cells by binding o pa ially un olded p o eins and p e en ing hyd ophobic in e ac ions ha lead o agg ega- ion. P ope unc ioning o Hsp70 in i s di e se oles depends upon a inely uned cycle o binding and elease o he sub- s a e ha is egula ed by ATP hyd olysis and nucleo ide ex- change. Hsp70 has an amino- e minal ATPase domain ha egula es he unc ion o an adjacen subs a e-binding domain (SBD). When ATP is bound, he SBD is “open” and apidly binds and eleases he subs a e. Hyd olysis o ATP o ADP con e s he SBD o a “closed” con o ma ion, es ablishing a igh associa- ion wi h he subs a e. Nucleo ide exchange es o es he ATP- bound s a e, e u ning he SBD o he open con o ma ion and allowing elease o he subs a e. Hsp70’s ATPase ac i i y is s imula ed by subs a e binding (41), indica ing wo-way communica ion be ween he domains, and is in luenced by in e ac ions wi h cochape one p o eins. Hsp40s, a conse ed amily o Hsp70 cochape ones ha also bind pa ially olded p o eins, s imula e Hsp70’s ATPase ac- i i y. This s imula ion is ma kedly ele a ed in he p esence o he subs a e, coupling ATP hyd olysis wi h subs a e cap u e (23, 65). Re u n o he ATP-bound s a e is egula ed by nu- cleo ide exchange ac o s. Func ion o euka yo ic Hsp70 is also egula ed by cochap- e ones ha con ain e a icopep ide epea (TPR) mo i s (52). Mammalian Hop1 (Hsp70/90-o ganizing p o ein) has wo TPR egions ha dis inc ly and simul aneously bind Hsp70 and Hsp90, o ming a physical link be ween hem (49). Hsp90 is ano he essen ial and s ess-induced p o ein chape one. Al- hough i s unc ion du ing s ess is unclea , Hsp90 has a well- de ined ole in assis ing olding o s e oid ecep o s and sig- naling kinases (48). Hsp70 ac s in his p ocess and has been shown o “accep ” subs a es om Hsp40 and “p esen ” hem o Hsp90 h ough an in e ac ion acili a ed by Hop1 (26). O he p o eins in ol ed in his pa hway include CyP-40 cyclo- philins, which a e pep idyl-p olyl isome ases ha ha e high a ini y o immunosupp essan s. Cyclophilins compe e wi h Hop1 o binding o Hsp90 and displace Hop1 and Hsp70 om he complex. The Hsp90 TPR cochape one machine y is conse ed in yeas and includes he Hop1 homolog S i1p, which was shown ecen ly o s imula e Ssa1p ATPase, and he CyP-40 cyclophi- lin homolog Cp 7p (8, 16, 45, 60). In addi ion o in e ac ing wi h Hsp70 and Hsp90, hese TPR p o eins also in e ac wi h Hsp104, a chape one ha disagg ega es p o ein agg ega es in a eac ion aided by Hsp70 and Hsp40 (1, 20). Hsp70 and Hsp90 ha e simila conse ed amino acid esidues a hei ex eme C e mini ha media e in e ac ions wi h TPR p o eins and Hsp40 (18, 49). The C e minus o Hsp104 is simila bu less conse ed. Yeas p ions a e p o eins ha mis old and o m sel - epli- ca ing agg ega es, which a e belie ed o p opaga e as amyloid (35, 63, 64). U e2p and Sup35p, he p o ein de e minan s o * Co esponding au ho . Mailing add ess: Labo a o y o Biochem- is y and Gene ics, Na ional Ins i u e o Diabe es and Diges i e and Kidney Diseases, Na ional Ins i u es o Heal h, Building 8, Room 407, Be hesda, MD 20892-0851. Phone: (301) 594-1316. Fax: (301) 496- 9431. E-mail: [email p o ec ed]. † P esen add ess: Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland. 3928 he Saccha omyces ce e isiae p ions [URE3] and [PSI ⫹ ], e- spec i ely, apidly and spon aneously o m amyloid when pu- i ied (19, 32, 57). Addi ionally, ib ous s uc u es o U e2p ha e been de ec ed in [URE3] bu no [u e-o] cells (55), and he p o ease diges ion pa e n o U e2p om [URE3] cells is iden- ical o ha o amyloid o med om pu i ied U e2p (40, 57). In a manne analogous o mammalian amyloidoses, he ag- g ega es ac as “seed” o ec ui and con e he soluble o m o he p o ein in o he same mis olded o m as i joins he polyme . Yeas p ions eplica e in he cy oplasm and a e in- ec ious in ha hey a e ansmi ed be ween cells du ing cell di ision and cell usion. Sup35p (eRF3) encodes a ansla ion elease ac o (56, 66). When [PSI ⫹ ] is p esen , much Sup35 p o ein is agg ega ed and una ailable o unc ion in ansla ion e mina ion, which causes a nonsense supp ession pheno ype. [PSI ⫹ ] hus p o ides a simple ye powe ul sys em o s udying amyloid p opaga ion in i o. In line wi h yeas p ions being p o ein olding p oblems, al e ed abundance o unc ion o a a ie y o p o ein chape - ones can a ec hei p opaga ion (9, 28, 33, 34, 42, 54). Mos e ec s a e obse ed upon o e exp ession o he chape ones, and mechanisms unde lying he e ec s emain specula i e. Among he chape ones Hsp104 is special in ha bo h i s lack o i s o e p oduc ion can cause loss o [PSI ⫹ ] (9). So an in e - media e le el o Hsp104’s disagg ega ing ac i i y appea s o be c i ical o e icien [PSI ⫹ ] p opaga ion. Hsp104 plays an im- po an ole in main aining p ion seed numbe , p esumably by b eaking p ion agg ega es in o mo e nume ous sel -p opaga - ing pa icles (9, 43, 47). The elimina ion o [PSI ⫹ ] by o e p o- duced Hsp104 is mode a ed somewha by simul aneously o e - p oducing Hsp70 Ssa1p (44). Ou p e ious cha ac e iza ion o an Ssa1p mu an (Ssa1-21p) showed ha i educed gene a ion o [PSI ⫹ ] seeds (28). Despi e impai ing [PSI ⫹ ] conside ably, Ssa1-21p has li le e ec on cell g ow h unde op imal o s ess- ul condi ions, sugges ing ha i in e ac s di e en ly wi h amy- loid han wi h o he subs a es. We p e iously gene a ed second-si e mu a ions in Ssa1-21p ha es o ed no mal [PSI ⫹ ] p opaga ion o unde s and be e how Hsp70 a ec s yeas p ion p opaga ion (27). Among o h- e s, se e al mu a ions we e loca ed in he conse ed C- e mi- nal mo i (GPTVEEVD) known o be impo an o in e ac- ions wi h TPR cochape ones. He e, we used a candida e gene app oach o iden i y ele an in e ac ing p o eins and ound ha deple ion o speci ic TPR cochape ones imp o ed [PSI ⫹ ] p opaga ion in SSA1-21 cells. Al hough he Hsp90 cochape - ones S i1p and Cp 7p had signi ican e ec s on [PSI ⫹ ], we ind e idence ha Hsp90 is no in ol ed in [PSI ⫹ ] p opaga ion, unco e ing a unc ional in e ac ion be ween hese cochape - ones and Hsp70 ha appea s independen o Hsp90. Ou cha - ac e iza ion o Hsp70 in e ac ions wi h cochape ones e eals he al e ed Hsp70 unc ion ha an agonizes amyloid p opaga- ion and p o ides an explana ion o e ec s seen in some ea - lie o e exp ession s udies. MATERIALS AND METHODS S ains, media, g ow h condi ions, and plasmids. Yeas s ains a e lis ed in Table 1. Excep o s ains G658 h ough G663, which a e ans o man s o G400-1C (MATaka 1-1 SUQ5 his3 leu2 lys2 p1 u a3 ssa1::KanMX ssa2::HIS3 ssa3::TRP1 ssa4::u a3-1 /pRDW10 [27]), all a e isogenic o 779-6A (MAT␣ ka 1-1 SUQ5 ade2-1 his3⌬202 leu2⌬1 p1⌬63 u a3-52 [29]). Because Ssap unc- ion is essen ial, plasmids wi h SSA alleles can be main ained in G400-1C cells on nonselec i e media. Simila ly, plasmids wi h HSC82 alleles can be main ained in hsc82⌬hsp82⌬cells wi hou selec ion. SSA1 gene eplacemen s we e made as desc ibed p e iously (29) by ans o ma ion using alleles on BglII-SphI ag- men s om plasmids pJ126 and pJ127 (see below). These agmen s con ain he SSA coding egion and 500 bp o 5⬘and 3⬘ lanking DNA. The coding egion o SSA2 was p ecisely eplaced wi h HIS3 by ans o ma ion wi h DNA ob ained by PCR-ampli ying HIS3 using p ime s wi h 5⬘homology o SSA2 un ansla ed DNA (3). Remaining genes we e dis up ed by ans o ma ion using KanMX casse es (59). YPAD (excess adenine), 1/2YPD (limi ing adenine), and syn he ic media we e as desc ibed p e iously (50, 51). Unless indica ed o he wise, cells we e g own a 30°C. Gene ic me hods we e as desc ibed p e iously (22, 28). The p esence o absence o [PSI ⫹ ] was con i med by bo h guanidine cu ing (below) and cy oduc ion, which is ansmission o cy oplasm be ween s ains h ough abo i e ma ing (10, 28). The pRS se ies plasmids ha e been desc ibed (53). Plasmids pRDW10 (URA3) and pJ120 (LEU2) a e single-copy ec o s wi h SSA1 (27, 28). Plasmid pJ121 is pJ120 wi h L483W ( he SSA1-21 mu a ion) in SSA1 (27). Plasmids pJ126 and pJ127 a e pJ120 and pJ121, espec i ely, wi h a hisG::URA3::hisG casse e (2) inse ed a an Aa II si e enginee ed 200 bp 3⬘ o he SSA1 e mina ion codon. SSA1⌬8and SSA1-21⌬8a e SSA1 and SSA1-21, espec i ely, which lack he eigh C- e minal codons (GPTVEEVD) and we e made by si e-di ec ed mu- agenesis o pJ120 and pJ121. The URA3-based plasmid pHSSA1, a gi om E. C aig (Uni e si y o Wisconsin, Madison; named pRSETB-SSA1 by he ), has an amino- e minal His 6 - agged SSA1 unde con ol o he TEF2 p omo e . Plasmids pHSSA1-21,pHSSA1⌬8, and pHSSA1-21⌬8a e pHSSA1 wi h he indica ed allele in place o SSA1. Mul icopy plasmids p423STI1 and p423FES1 a e pRS423 wi h STI1 and FES1, espec i ely. The single-copy plasmid pL116 is pRS314 wi h he in ac HSC82(1-709) gene and 500 bp o 5⬘and 3⬘ lanking DNA, PCR ampli ied om s ain 779-6A, on a BamHI agmen . Plasmid pL118 con ains he unca ed HSC82(1-704) allele lacking he i e C- e minal codons (MEEVD). Mu agenesis. The SSA1-21 allele o SSA1 has a yp ophan in place o leucine a codon 483 (L483W). A sc een o second-si e supp esso s o L483W was done as desc ibed p e iously (27). B ie ly, plasmid pJ121 was andomly mu agenized wi h hyd oxylamine and hen used o ans o m s ain G400-1C o leucine p o- o ophy. T ans o man colonies we e eplica ed on o pla es lacking adenine (⫺ade) and con aining 5- luo o-o o ic acid o simul aneously selec o cells ha p opaga ed [PSI ⫹ ] and had los pRDW10, which equi es ha he mu an p o- eins bo h es o e [PSI ⫹ ] p opaga ion and p o ide essen ial Ssa1p unc ion. Si e-di ec ed mu agenesis on pJ126 and pJ127 using he QuikChange ki (S a - agene) and app op ia e p ime s was done o c ea e alleles o genomic gene eplacemen s o SSA1. Nonsense supp ession ( ead- h ough) assays. A dual-luci e ase assay sys em was used (24). The sys em consis s o bicis onic mRNAs encoding a ansla- ional usion o Renilla and i e ly luci e ase genes wi h UGG o UAA a he six h codon o he i e ly gene (J. W. Ha ge and J. D. Dinman, unpublished da a). Because he enzymes a e exp essed om he same mRNA and he p oduc is a usion p o ein, abundance o mRNA and p o ein a e con olled o in e nally. Cells wi h plasmids pYDL505 (UGG) and pYDL506 (UAA) we e g own in plasmid selec i e medium o an op ical densi y a 600 nm (OD 600 ) o 0.5 o 1. Cells in 1 ml o cul u e we e cen i uged, washed, and hen b oken by agi a ion wi h glass beads in 0.3 ml o lysis bu e . B oken cells we e cen i uged o 5 min a 8,000 pm in a mic ocen i uge (So all; MC12) and luci e ase ac i i y o 5 ml o supe na an was assayed using he P omega dual-luci e ase assay sys em in a Zylux FB15 luminome e . Pu i ica ion o His- agged Ssa1p and Wes e n blo ing. His 6 - agged Ssa1p and Ssa1-21p we e indis inguishable om he un agged p o eins wi h espec o unc ions in g ow h and [PSI ⫹ ] p opaga ion (da a no shown). S ain 1012 [psi ⫺ ] ans o man s wi h he pHSSA1 se ies plasmids we e ans e ed om solid medium lacking u acil o YPAD liquid. Cul u es (OD 600 ⫽1.0) we e washed, suspended in lysis bu e (0.5⫻phospha e-bu e ed saline [pH 7.4], 50 mM KCl, 5 mM MgCl2, EDTA- ee p o ease inhibi o cock ail able s [Roche]), and b oken by agi a ion wi h glass beads. P o eins we e c oss-linked o 60 min a 4°C wi h 1 mM di hiobis(succinimidylp opiona e) (Sigma). The eac ion was s opped wi h 5 mM lysine o 30 min a 4°C. Lysa es (3 mg o p o ein) we e dilu ed wi h equal olumes o binding bu e (1⫻phospha e bu e [pH 7.4], 10 mM imida- zole, HisT ap ki [Ame sham]) and incuba ed wi h nickel-cha ged esin o 1 h a 4°C. A e i e washes wi h binding bu e and hen i e washes wi h washing bu e (1⫻phospha e bu e [pH 7.4], 40 mM imidazole, HisT ap ki [Ame - sham]), esin-bound p o eins we e elu ed wi h 120 ␮l o elu ion bu e (1⫻ phospha e bu e [pH 7.4], 500 mM imidazole, HisT ap ki [Ame sham]). Sam- ples we e boiled in 30 ␮lo 5⫻sodium dodecyl sul a e-polyac ylamide gel elec opho esis sample bu e o 5 min, and 20-␮l aliquo s we e subjec ed o VOL. 24, 2004 Hsp70 COCHAPERONES IMPAIR AMYLOID PROPAGATION 3929 Wes e n analysis. Mouse an i-Hsp70 and abbi an i-Hsp104 an ibodies (SPA- 822 and SPA-1040) we e om S essgen, abbi an i-Ydj1p polyclonal an ibodies we e a gi om J. B odsky (Uni e si y o Pi sbu gh), and abbi an i-S i1p and an i-Cp 7p polyclonal an ibodies we e gene a ed o his s udy. Guanidine cu ing o [PSI ⴙ ]. [PSI ⫹ ] cells g own on ⫺ade pla es a 25°C we e used o inocula e liquid YPAD cul u es, which we e g own o e nigh a 30°C. These cul u es we e dilu ed in YPAD con aining 3 mM guanidine-hyd ochlo ide and main ained a an OD 600 o ⱕ0.8 by dilu ion in o esh guanidine-con aining medium. Samples we e emo ed a each cell numbe doubling, measu ed as doubling o OD 600 , and sp ead on o h ee YPD pla es a dilu ions p oducing 300 o 500 colonies pe pla e. En i ely ed colonies we e sco ed as [psi ⫺ ]. When guanidine cu ing was used as a con i ma ion o he p esence o [PSI ⫹ ], cells we e g own o colonies on YPAD pla es con aining 3 mM guanidine and hen assayed o he [PSI ⫹ ] pheno ype on YPD. RESULTS Impai men o [PSI ⴙ ] p ion p opaga ion by Hsp70 mu a- ion. [PSI ⫹ ] is a sel - eplica ing agg ega ed o m o Sup35p hough o be amyloid. Agg ega ion o Sup35p in [PSI ⫹ ] cells causes nonsense supp ession because much Sup35 p o ein is una ailable o unc ion in ansla ion e mina ion. Mu an s wi h he ade2-1 nonsense allele canno g ow wi hou adenine and a e ed when g own on limi ing amoun s o adenine (e.g., on 1/2YPD) because o he accumula ion o a pigmen ed sub- s a e o Ade2p. Pa ial supp ession o ade2-1 by [PSI ⫹ ], which also equi es he weakly UAA-supp essing RNA SUQ5/ SUP16, allows g ow h wi hou adenine and elimina es he pig- men a ion (11). The ela i e s eng h o [PSI ⫹ ] can be es i- ma ed om he deg ee o pigmen a ion and a e o g ow h wi hou adenine, which e lec he deg ee o nonsense supp es- sion caused by [PSI ⫹ ] (13, 28). Ano he indica o o obus ness o [PSI ⫹ ] p opaga ion is i s mi o ic s abili y, which ypically co ela es wi h nonsense supp ession. Spon aneous mi o ic loss o [PSI ⫹ ] is seen as appea ance o ed, [psi ⫺ ] colonies. Figu e 1 shows he a ious [PSI ⫹ ] pheno ypes o ou s ains, se e al o which we e abno mal. To ule ou he possibili y ha mu a ions we e gene a ing a ian s o [PSI ⫹ ] ha p opaga ed a ypically, all s ains wi h abno mal [PSI ⫹ ] pheno ypes we e used as cy oplasm dono s in cy oduc ion c osses wi h wild- ype [psi ⫺ ] cells (see Ma e ials and Me hods). In all c osses, he wild- ype ecipien s had a no mal [PSI ⫹ ] pheno ype, indica ing ha he mu a ions weakened p opaga ion o a no mal o m o [PSI ⫹ ]. This es also con i ms absence o [PSI ⫹ ]in[psi ⫺ ] cells. Whe e app op ia e, we con i m ha colony colo e lec ed le els o nonsense supp ession by quan i ying s op codon ead- h ough (Fig. 2). Ou wild- ype [PSI ⫹ ] cells a e whi e on YPD medium and g ow well wi hou adenine a bo h 25°C and he mo e s ingen 30°C (Fig. 1, column 1, uppe panels). [PSI ⫹ ] is e y s able in hese cells, and we do no obse e spon aneous appea ance o TABLE 1. Yeas s ains and g ow h a es S ain Rele an geno ype a Gene a ion ime (min) b SSA1 SSA2 CPR7 STI1 [psi ⫺ ][PSI ⫹ ] 1001 ⫹⫹⫹⫹99 100 1002 ⫹ssa2⌬⫹ ⫹ 100 109 1003 ⫹⫹cp 7⌬⫹ 144 145 1004 ⫹⫹⫹s i1⌬93 110 1005 ⫹ssa2⌬cp 7⌬⫹ 117 122 1006 ⫹ssa2⌬⫹ s i1⌬108 111 1007 ⫹⫹cp 7⌬s i1⌬175 151 1008 ⫹ssa2⌬cp 7⌬s i1⌬192 167 1009 SSA1 636S ⫹⫹⫹108 106 1010 SSA1 636S ssa2⌬⫹ ⫹ 108 111 1011 ssa1⌬⫹⫹⫹93 96 1012 ssa1⌬ssa2⌬⫹ ⫹ 141 173 1013 SSA1-21 ⫹⫹⫹ 84 84 1014 SSA1-21 ssa2⌬⫹ ⫹ 102 NA 1015 SSA1-21 ⫹cp 7⌬⫹ 127 125 1016 SSA1-21 ⫹⫹s i1⌬90 108 1017 SSA1-21 ssa2⌬cp 7⌬⫹ 133 NA 1018 SSA1-21 ssa2⌬⫹ s i1⌬111 NA 1019 SSA1-21 ⫹cp 7⌬s i1⌬170 153 1020 SSA1-21 ssa2⌬cp 7⌬s i1⌬168 208 1021 SSA1-21 636S ⫹⫹⫹ 94 103 1022 SSA1-21 636S ssa2⌬⫹ ⫹ 95 108 919 es1⌬SSA1 145 126 921 es1⌬SSA1-21 123 NA G612 hsc82⌬hsp82⌬/pHSC82(1-709) ND ND G616 hsc82⌬hsp82⌬/pHSC82(1-704) ND ND G658 ssa1,2,3,4/pSSA1 90 ND G659 ssa1,2,3,4/pSSA1-21 92 NA G660 ssa1,2,3,4/pSSA1 P636S 105 ND G661 ssa1,2,3,4/pSSA1-21 P636S 98 ND G662 ssa1,2,3,4/pSSA1⌬8136 165 G663 ssa1,2,3,4/pSSA1-21⌬8265 NA a All excep G658 h ough G663 a e isogenic o wild- ype s ain 779-6A (see Ma e ials and Me hods). Va ious allele combina ions a e indica ed unde ele an geno ype. G658 h ough G663 a e G400-1C (see Ma e ials and Me hods [27]) wi h he indica ed plasmids in place o pRDW10. b G ow h a es we e measu ed in liquid YPAD a 30°C. ND, no de e mined; NA, no applicable, since [PSI ⫹ ] is unable o p opaga e in hese s ains. 3930 JONES ET AL. MOL.CELL.BIOL. ed [psi ⫺ ] colonies du ing ou ine handling. SSA1-21, an allele o he HSP70 gene SSA1 wi h he L483W mu a ion, causes equen mi o ic loss o [PSI ⫹ ] and educes nonsense supp es- sion (Fig. 1, column 1, lowe panels; Fig. 2). E ec s o Ssa1-21p on [PSI ⫹ ] a e dominan o e hose o Ssa2p, which is ano he cons i u i ely exp essed cy osolic Hsp70 ha is 97% iden ical o Ssa1p. When Ssa2p exp ession is abolished, Ssa1p abun- dance is ele a ed in a compensa o y manne . While ssa2⌬cells ha e a no mal [PSI ⫹ ] pheno ype, [PSI ⫹ ] canno p opaga e in SSA1-21 ssa2⌬cells (28) (Fig. 1, column 2). C- e minal second-si e supp esso s o Ssa1-21p. To unde - s and be e how Ssa1-21p unc ion was al e ed, we p e iously isola ed second-si e mu a ions in Ssa1-21p ha es o ed [PSI ⫹ ] p opaga ion in SSA1-21 ssa2⌬cells (27). Among o he s, h ee (P636S, E639K, and E640K) we e in he ex eme C e minus o Ssa1-21p. The conse ed C- e minal oc apep ide o Hsp70 (GPTVEEVD) media es in e ac ions wi h TPR-con aining co- chape ones and is necessa y o op imal Hsp70 unc ion (18, 49). Assuming ha he h ee mu a ions simila ly supp essed he impai ed [PSI ⫹ ] pheno ype by al e ing Ssa1-21p in e ac- ion wi h TPR cochape ones, we chose P636S o u he s udy. Isogenic s ains ha con ained ch omosomal SSA1 and SSA1-21 alleles wi h o wi hou he P636S subs i u ion (SSA1- 21 P636S has bo h L483W and P636S) we e cons uc ed. An ssa1⌬s ain and an isogenic ssa2⌬se ies we e also es ed. As o iginally isola ed, SSA1-21 P636S ssa2⌬cells had a no mal [PSI ⫹ ] pheno ype (Fig. 1, compa e column 4, lowe panels, o column 1, uppe panels). The e o e, he P636S subs i u ion comple ely supp essed he [PSI ⫹ ]-inhibi o y e ec s o he Ssa1p L483W subs i u ion in ssa2⌬cells. The p esence o Ssa2p in SSA1 P636S o SSA1-21 P636S cells educed abili y o [PSI ⫹ ] o cause nonsense supp ession (Fig. 1, compa e columns 3 and 4; Fig. 2). The [PSI ⫹ ] pheno ype was also sligh ly weakened in ssa1⌬cells bu was no mal ssa2⌬cells (Fig. 1, columns 11 and 2, uppe panels; Fig. 2). These esul s show ha Ssa2p weakened [PSI ⫹ ] in cells wi h comp omised Ssa1p unc ion and e eal a unc ional dis inc ion be ween Ssa1p and Ssa2p wi h ega d o [PSI ⫹ ] p opaga ion. Deple ion o TPR cochape ones es o es [PSI ⴙ ] p opaga- ion in SSA1-21 cells. Since he ex eme C e minus o Hsp70 is impo an o physical in e ac ion wi h TPR mo i s o co- chape ones, we expec ed ha he C- e minal supp essing mu- a ions o Ssa1-21p we e dis up ing such in e ac ions. Taking a candida e gene app oach o iden i ying TPR cochape ones in ol ed in he e ec s on [PSI ⫹ ], we dele ed sepa a ely he nonessen ial cyclophilin homologs CPR6 and CPR7 and HOP1 homologs SGT2 and STI1. Dele ing CPR6 o SGT2 had no e ec on g ow h o [PSI ⫹ ] pheno ype o wild- ype o SSA1-21 s ains. Dele ing CPR7 o STI1 in wild- ype cells also had no ob ious e ec . In con as , dele ing ei he o hem in SSA1-21 cells educed pigmen accumula ion and imp o ed mi o ic s a- bili y o [PSI ⫹ ] (Fig. 1, lowe panels, compa e columns 5 and 7 o column 1). Dele ing STI1 imp o ed [PSI ⫹ ] p opaga ion be e han dele ing CPR7. On medium lacking adenine a he mo e s ingen 30°C, SSA1-21 and SSA1-21 cp 7⌬cells we e unable o o m colonies bu SSA1-21 s i1⌬cells g ew well. As FIG. 1. E ec s o SSA1 and cochape one mu a ions on [PSI ⫹ ] pheno ype. Cells we e g own as colonies on YPD o 2 days a 30°C ollowed by 3 days a 25°C o as pa ches o cells eplica pla ed on o ⫺ade pla es, which we e incuba ed o 5 days a 25°C(⫺ade 25°) o o 3 days a he mo e s ingen 30°C(⫺ade 30°). Rep esen a i e a eas o g ow h on pla es a e shown. Excep o hose in column 11, s ains in uppe panels ha e wild- ype SSA1 and s ains in lowe panels ha e SSA1-21, as indica ed. S ains in column 11 uppe panels a e ssa1⌬, and hose in lowe panels a e ssa1⌬ssa2⌬. Rele an alleles o s ains in each column a e indica ed; w is wild ype. Colo s on YPD ange om whi e o ed, e lec ing om mos o leas supp ession o ade2-1 (see he ex ). The ex en o ade2-1 supp ession is also e lec ed as densi y o g ow h on ⫺ade pla es. Red cell colo is enhanced on ⫺ade pla es compa ed wi h YPD pla es, especially o nong owing s ains. VOL. 24, 2004 Hsp70 COCHAPERONES IMPAIR AMYLOID PROPAGATION 3931 obse ed p e iously (16), dele ing CPR7 educed he g ow h a e (Table 1). These esul s show ha S i1p and Cp 7p we e equi ed o Ssa1-21p o ha e i s ull e ec s on impai ing [PSI ⫹ ]. Unlike he P636S mu a ion, dele ion o STI1 o CPR7 did no es o e [PSI ⫹ ] p opaga ion in SSA1-21 ssa2⌬cells. When bo h STI1 and CPR7 we e dele ed in SSA1-21 ssa2⌬cells, howe e , [PSI ⫹ ] p opaga ed s ably (Fig. 1, lowe panels, col- umn 10). These [PSI ⫹ ] cells did no g ow wi hou adenine, howe e , indica ing ha es o a ion o [PSI ⫹ ] p opaga ion was incomple e. Compa ed wi h he comple e es o a ion o [PSI ⫹ ] in SSA1-21 ssa2⌬cells by he P636S subs i u ion in Ssa1-21p, his esul sugges s ha o he TPR cochape ones may a ec [PSI ⫹ ] p opaga ion. One candida e is he essen ial Cns1p, which is unc ionally edundan wi h Cp 7p (14, 39). O e exp ession o STI1 weakens [PSI ⴙ ] and enhances SSA1-21 e ec s. Since deple ing Cp 7p and S i1p imp o ed [PSI ⫹ ] p opaga ion, we an icipa ed ha inc easing hei abun- dance would ha e he opposi e e ec . In ac , o he s ha e shown ha o e p oduc ion o S i1p can weaken p opaga ion o weak o hyb id o ms o [PSI ⫹ ] (33). We es ed o e p oduc ion by ans o ming [PSI ⫹ ] cells wi h high-copy-numbe plasmids ca ying CPR7 and STI1. We also es ed CNS1 because excess Cns1p es o es no mal g ow h o cp 7⌬cells (39). In wild- ype cells, [PSI ⫹ ] was no a ec ed by excess Cp 7p o Cns1p, bu o e p oduced S i1p weakened [PSI ⫹ ] p opaga ion, inc easing pigmen a ion and educing mi o ic s abili y o [PSI ⫹ ] (Fig. 3A). Simila ly, only excess S i1p enhanced he impai men o [PSI ⫹ ] p opaga ion in SSA1-21 cells, inc easing bo h pigmen accumula ion and equency o mi o ic loss o [PSI ⫹ ] (Fig. 3A). In SSA1-21 cp 7⌬cells, o e p oduced Cns1p es o ed bo h g ow h a e and impai men o [PSI ⫹ ] by Ssa1-21p (da a no shown). Thus, in addi ion o being edundan wi h Cp 7p o FIG. 2. Quan i ied le els o [PSI ⫹ ]-media ed nonsense supp es- sion. S ains ca y plasmids exp essing a ansla ional usion o Renilla (ups eam) and i e ly (downs eam) luci e ases, wi h o wi hou a e mina ion codon (UAA) ea ly in he i e ly coding egion. The a io o ac i i ies o i e ly oRenilla enzymes o he UAA cons uc e sus he con ol cons uc [( i e ly/Renilla) UAA /( i e ly/Renilla) con ol ] p o- ides a measu emen o UAA ead- h ough. Read- h ough alues o mu an s ains a e exp essed as pe cen ages ela i e o alues o he wild- ype s ain. Assays we e done in iplica e a leas h ee imes. SSA1-21 de i a i es o ssa2⌬and es1⌬s ains a e no ep esen ed, since hey a e unable o p opaga e [PSI ⫹ ]. FIG. 3. Opposing e ec s o S i1p and Fes1p on [PSI ⫹ ]. (A) In- c eased exp ession o S i1p and Fes1p. Pa ches o colonies o [PSI ⫹ ] SSA1 and SSA1-21 ans o man s o a high-copy-numbe plasmid, wi h o wi hou he indica ed genes, we e g own on selec ion pla es wi h limi ing adenine o 2 days a 30°C. While [PSI ⫹ ] can p opaga e in SSA1-21 cells o e p oducing S i1p, he enhanced weakening o [PSI ⫹ ] makes i di icul o dis inguish i s p esence a he le el o indi idual colonies. (B) Pa ches o SSA1 and SSA1-21 cells, ha ing o lacking FES1 as indica ed, we e eplica pla ed on o pla es lacking adenine and incuba ed a he indica ed empe a u e (gi en in deg ees Celsius) o 2 days. 3932 JONES ET AL. MOL.CELL.BIOL. g ow h, Cns1p also o e laps unc ionally wi h Cp 7p wi h e- ga d o [PSI ⫹ ] p opaga ion h ough e ec s on Hsp70. Dele ion o FES1 weakens [PSI ⴙ ] and enhances SSA1-21 e ec s. While i is no known how he Hsp90 cochape one Cp 7p a ec s Hsp70 unc ion, S i1p was ecen ly shown o be an ac i a o o Ssa1p ATPase (60) and hus p omo es Ssa1p subs a e binding. In con as , he nucleo ide exchange ac o Fes1p accele a es elease o ADP om Ssa1p (30), he eby acili a ing Ssa1p’s e u n o he open s a e and subs a e e- lease. The e o e, dele ing Fes1p should p olong Ssa1p’s closed s a e, which we p edic ed would ad e sely a ec [PSI ⫹ ], while excess Fes1p was expec ed o ha e he opposi e e ec s. Indeed, we ound ha [PSI ⫹ ] was unable o p opaga e in SSA1-21 cells lacking FES1 and p opaga ed be e in SSA1-21 cells ha o e - p oduced Fes1p (Fig. 3A and B). Mo eo e , dele ing FES1 p edic ably weakened [PSI ⫹ ] in wild- ype cells, educing bo h nonsense supp ession and mi o ic s abili y o [PSI ⫹ ] (Figs. 2 and 3, da a no shown). TPR cochape one dele ion o P636S es o es [PSI ⴙ ] seed numbe in SSA1-21 cells. In o de o [PSI ⫹ ] o be main ained in a popula ion, he numbe o ansmissible p ion pa icles, o seeds, mus double, on a e age, in he ime i akes cells o di ide. SSA1-21 signi ican ly educes he a e age numbe o [PSI ⫹ ] seeds pe cell (28), which explains why i causes e- quen loss o [PSI ⫹ ]. We used guanidine cu ing o [PSI ⫹ ] o de e mine i P636S and he cochape one dele ions es o ed [PSI ⫹ ] seed gene a ion in SSA1-21 cells. A millimola concen- a ions, guanidine in g ow h media a es s eplica ion o [PSI ⫹ ] seeds, which hen become dilu ed as cells di ide, e en- ually gi ing ise o cells ha ing los [PSI ⫹ ] (17). A e adding guanidine o g owing cul u es, he a e o appea ance o [psi ⫺ ] cells p o ides an es ima e o he a e age numbe o seeds pe cell. SSA1-21 P636S cells had a nea -wild- ype cu ing p o ile, show- ing ha he P636S subs i u ion signi ican ly es o ed seed num- be (Fig. 4). Dele ing STI1 o CPR7 had no e ec on [PSI ⫹ ] cu ing in wild- ype cells. In SSA1-21 cells, dele ing STI1 e- s o ed he cu ing p o ile, simila o ha o SSA1-21 P636S cells, while dele ion o CPR7 es o ed seed numbe less well (Fig. 4). When bo h CPR7 and STI1 we e dele ed in SSA1-21 cells, [PSI ⫹ ] seed numbe was es o ed comple ely. These esul s show ha Cp 7p and S i1p a ec Hsp70 unc ion wi h espec o [PSI ⫹ ] seed gene a ion and a e consis en wi h ou o he indings ha al e ing S i1p abundance had g ea e e ec s on [PSI ⫹ ]. Hsp70 C- e minal mu a ions a iably a ec in e ac ion wi h cochape ones. To assess he e ec s o he C- e minal mu a- ions on cochape one in e ac ions, we compa ed abundance o p o eins ha copu i ied wi h Ssa1p. Amino- e minal His 6 - agged e sions o Ssa1p and Ssa1-21p we e used o speci ically isola e Ssa1p iso o ms o Hsp70. Binding o S i1p o Ssa1p and Ssa1-21p was educed bu no elimina ed by bo h he P636S subs i u ion and C- e minal dele ion (Fig. 5). While hese mu- a ions modes ly educed he amoun o Cp 7p associa ed wi h Ssa1p, hey did no ha e a signi ican e ec on he binding o Cp 7 o Ssa1-21p. The e o e, al hough he TPR in e ac ion mo i is impo an o binding o S i1p and Cp 7p o Ssa1p, hese cochape ones can bind Ssa1p a a di e en si e. Ne e - heless, since bo h poin mu a ions and comple e dele ion o he C- e minal TPR in e ac ion mo i supp ess Ssa1-21p im- pai men o [PSI ⫹ ], his seconda y in e ac ion mus ha e in- comple e o no unc ionali y wi h ega d o e ec s on [PSI ⫹ ]. Also, since mo e Cp 7p was associa ed wi h Ssa1-21p, he L483W subs i u ion may enhance he seconda y in e ac ion wi h Cp 7. Dele ing he C- e minal esidues signi ican ly educed Hsp40 (Ydj1p) in e ac ion, which ag ees wi h ea lie wo k showing ha Hsp40 in e ac ion wi h Hsp70 is educed when Hsp70 lacks i s ou C- e minal esidues (EEVD) (12). Hsp40s also in e ac wi h he ATPase domain o Hsp70 (31), and FIG. 4. Guanidine cu ing o [PSI ⫹ ]. The pe cen age o [PSI ⫹ ] cells emaining in log-phase cul u es was moni o ed as a unc ion o cell di isions a e addi ion o guanidine-hyd ochlo ide o a inal concen- a ion o 3 mM. FIG. 5. Copu i ica ion o Hsp70-associa ed p o eins. His 6 - agged e sions o Ssa1p and Ssa1-21p we e pu i ied on nickel a ini y esin and subjec ed o Wes e n analysis. Pu i ied His 6 - agged p o eins a e indica ed a he op, and lane C is om a simila pu i ica ion using lysa es o cells exp essing non agged Ssa1p. The Hsp70 and S i1p panels a e om sepa a e blo s loaded wi h iden ical aliquo s o he same samples. The Cp 7p and Ydj1p panels a e om he Hsp70 and S i1p blo s, espec i ely, which we e s ipped and ep obed. VOL. 24, 2004 Hsp70 COCHAPERONES IMPAIR AMYLOID PROPAGATION 3933 esidual binding o Hsp40 o he dele ion mu an s is likely media ed by his in e ac ion. The P636S mu a ion had li le a ec on binding o Hsp40 o Ssa1p o Ssa1-21p, sugges ing ha supp ession o L483W e ec s by P636S is no due o educed abili y o Hsp40 o in e ac wi h Ssa1p. Dele ing Hsp90’s TPR in e ac ion mo i has no e ec on [PSI ⴙ ]. Because Cp 7p and S i1p a e Hsp90 cochape ones, we an icipa ed ha Hsp90 migh be in ol ed in he SSA1-21 e - ec s on [PSI ⫹ ]. I so, hen dis up ing he in e ac ion be ween Hsp90 and TPR cochape ones would imp o e [PSI ⫹ ] p opa- ga ion in SSA1-21 cells. We es ed his p edic ion by cons uc - ing s ains ha lacked bo h ch omosomal HSP90 genes (HSC82 and HSP82) and exp essed in ac (Hsc82(1-709) o unca ed (Hsc82(1-704) o ms o Hsc82p. Exp ession o ei he Hsc82p o Hsp82, collec i ely e e ed o as Hsp90, is essen ial o iabili y. The Hsc82(1-704) p o ein lacks he C- e minal MEEVD amino acids, which media e TPR in e ac ion bu a e dispensable o iabili y (37). The [PSI ⫹ ] pheno ypes o bo h wild- ype and SSA1-21 cells we e unchanged when he unca ed o m o Hsc82p was ex- p essed in place o in ac Hsc82p (da a no shown). These esul s demons a e ha he Hsp90 TPR in e ac ion mo i is dispensable o [PSI ⫹ ] p opaga ion and sugges ha Hsp70 can unc ionally in e ac wi h TPR cochape ones independen ly o Hsp90. Al e ed Hsp70 and Hsp104 abundance does no co ela e wi h e ec s on [PSI ⴙ ]. Since al e ed abundance o Hsp70 and Hsp104 can ha e signi ican e ec s on [PSI ⫹ ] p opaga ion (9, 34, 44), we add essed he possibili y ha he SSA and cochap- e one e ec s on [PSI ⫹ ] we e due o al e ed exp ession o Hsp70 and Hsp104 by compa ing hei abundance in ou s ains (Fig. 6). Al hough he e was a complex pa e n o Hsp70 and Hsp104 exp ession, he e was no co ela ion be- ween he le els o ei he o hese p o eins and [PSI ⫹ ] pheno- ype. Also, he e was no co ela ion be ween Hsp70 o Hsp104 abundance and abili y o p opaga e [PSI ⫹ ]. These esul s sug- ges ha e ec s o SSA and cochape one mu a ions on [PSI ⫹ ] p opaga ion we e due o al e ed ac i i y o indi idual p o eins o complexes a he han al e ed abundance o Hsp70 and Hsp104. Dele ion o CPR7 co ela ed wi h ele a ed Hsp104 abun- dance, which is consis en wi h ea lie da a (15). Hsp104 was also ele a ed o mos ssa2⌬s ains bu no o ssa1⌬s ains, which e eals ano he unc ional dis inc ion be ween Ssa1p and Ssa2p. The Ssa1p C- e minal TPR in e ac ion mo i is dispensable o iabili y. The SSA sub amily o cy osolic Hsp70 genes, which consis s o ou membe s (Ssa3p and Ssa4p a e ex- p essed only unde nonop imal g ow h condi ions), a e he only S. ce e isiae Hsp70s wi h he GPTVEEVD mo i . Exp es- sion o a leas one SSA gene is essen ial o g ow h (62). To de e mine he impo ance o he GPTVEEVD mo i o es- sen ial Ssap unc ion, plasmids ca ying SSA1 and SSA1-21 alleles wi hou i (SSA1⌬8and SSA1-21⌬8, espec i ely) we e exp essed in a s ain lacking all ch omosomal SSA genes. Cells exp essing in ac SSA1 o SSA1-21 as he only SSA gene g ew as well as he wild ype (Table 1). Those exp essing only SSA1⌬8g ew mo e slowly (Table 1). Thus, he TPR in e ac- ion mo i was impo an bu dispensable o essen ial Ssap unc ion. [PSI ⫹ ] p opaga ed s ably in his s ain, bu i s p es- ence slowed g ow h u he . The SSA1-21⌬8allele also sup- po ed g ow h, bu only e y weakly and i cells we e [psi ⫺ ] (Table 1). Because SSA1-21⌬8did no suppo g ow h o [PSI ⫹ ] cells, we could no es i he dele ion o e came SSA1-21 e ec s on [PSI ⫹ ] in he absence o o he Ssap. How- e e , as wi h he P636S subs i u ion, he C- e minal dele ion supp essed he e ec o Ssa1-21p on [PSI ⫹ ] in cells exp essing Ssa2p (da a no shown). DISCUSSION We iden i y new p o eins whose al e ed abundance a ec s [PSI ⫹ ] p opaga ion and implies ha amyloid p opaga ion in i o is impai ed by al e a ions ha p omo e con e sion o, o s abilize, he subs a e-bound s a e o Hsp70. Figu e 7 depic s he Hsp70 eac ion cycle and whe e hese p o eins ac on i . Ou ea lie wo k showed ha second-si e mu a ions in Ssa1- 21p ha o e come he [PSI ⫹ ]-impai ing e ec o he L483W subs i u ion weaken subs a e binding, which indica es ha L483W enhances subs a e binding o Ssa1p (27). Consis en FIG. 6. Rela i e abundance o Hsp70 and Hsp104. Abundance o Hsp70 and Hsp104 was examined by Wes e n analysis. Blo s p obed wi h an i-Hsp70 an ibodies we e s ipped and ep obed wi h an i-Hsp104 an ibodies. Rep esen a i e po ions o memb anes, s ained by amido black as a loading and ans e con ol, a e shown (Load). Rele an alleles o s ains a e indica ed a he op; w is wild ype. The ssa1⌬ssa2⌬sample was dilu ed 1:3, as indica ed, because Hsp104 abundance in his s ain is e y high (compa e amoun s in he Load panel). The signal in he Hsp70 blo o his s ain ep esen s Ssa3p and Ssa4p, which a e exp essed when Ssa1p and Ssa2p bo h a e absen . 3934 JONES ET AL. MOL.CELL.BIOL. wi h his in e p e a ion, o e p oducing S i1p, which p omo es subs a e binding by ac i a ing Ssa1p ATPase, also impai ed [PSI ⫹ ] p opaga ion. Con e sely, dele ion o S i1p es o ed [PSI ⫹ ] p opaga ion in SSA1-21 cells. As p edic ed and in con- as o he case wi h S i1p, o e p oduc ion o Fes1p, which acili a es subs a e elease (30), coun e ac ed he impai men o [PSI ⫹ ] by Ssa1-21p, while deple ion o Fes1p, which should p olong he subs a e-bound s a e o Ssa1p, impai ed [PSI ⫹ ]in bo h wild- ype and SSA1-21 cells. Thus, in addi ion o mu a- ions in Hsp70 ha al e i s unc ion di ec ly, an imbalance o Hsp70 cochape ones can p oduce a simila [PSI ⫹ ]-impai ing e ec by dis up ing egula ion o he Hsp70 eac ion cycle. Ou esul s ag ee wi h p e ious da a showing ha excess S i1p des abilizes some a ian s o [PSI ⫹ ] (33) and sugges ha his des abiliza ion is media ed h ough e ec s on Ssa1p ac i - i y a he han al e ed exp ession o o he Hsps as p e iously sugges ed. Hsp40s also s imula e Hsp70 ATPase, and in pa - icula Ydj1p and Sis1p s imula e Ssa1p ATPase (38). Consis- en wi h ou conclusion, o e p oduc ion o he Hsp40 ho- mologs Ydj1p, Sis1p, and Apj1p impai s p opaga ion o yeas p ions (33, 34, 42), and a supp essing mu a ion in Ssa1-21p desc ibed ea lie is in a esidue expec ed o dis up Hsp40 in e ac ion (27). Addi ionally, weakening o [PSI ⫹ ] by Ydj1p o e p oduc ion is inc eased when Ssa1p is simul aneously el- e a ed. Ou da a sugges ha he p e iously desc ibed e ec s o S i1p and Hsp40 o e p oduc ion on yeas p ions can be a ibu ed o s imula ed Ssa1p ATPase. Da a p esen ed he e suppo ou ea lie in e p e a ion ha o he mu a ions in Ssa1p ha inhibi [PSI ⫹ ] p opaga ion also inc ease ATPase ac i i y o Ssa1p. Aside om L483W, all o eigh p e iously desc ibed Ssa1p mu a ions ha impai [PSI ⫹ ] a e in he ATPase domain and canno di ec ly a ec in e ac- ion wi h he subs a e (27). The L483W subs i u ion is wi hin he subs a e-binding domain bu dis an om he subs a e- binding pocke , so i s e ec s should also be indi ec , pe haps al e ing in e domain communica ion. Toge he , e ec s o he Hsp70 and cochape one mu a ions on [PSI ⫹ ] sugges ha he inc ease in ATPase is e lec ed in he educed gene a ion o amyloid seeds om p eexis ing ma e ial. Since cells exp essing Ssa1-21p as he only essen ial Ssap a e unable o main ain [PSI ⫹ ] bu g ow as well as wild- ype cells, an implica ion o ou esul s is ha i migh be possible o he apeu ically modula e ATPase ac i i y o Hsp70 in o he sys ems, di ec ly o h ough cochape ones, in a way ha would impai cy osolic amyloid p opaga ion wi h minimal side e ec s. How does p omo ing o p olonging subs a e binding by Hsp70 impai [PSI ⫹ ] p opaga ion? In addi ion o he con in- ued g ow h o polyme s, p opaga ion o yeas p ions (in ec- ious amyloid) equi es he gene a ion o new sel - eplica ing seeds om p eexis ing ma e ial and e icien dis ibu ion o seeds be ween mo he and daugh e cells. Gene a ion o yeas p ion seeds equi es Hsp104 p o ein disagg ega ing unc ion, which may b eak he polyme s in o smalle , mo e nume ous pieces ha con inue o p opaga e he s uc u e and a e mo e eadily ansmi ed o daugh e cells (4, 43, 47). Since SSA1-21 mu an s ha e se e al old ewe seeds pe cell, al e ed Hsp70 unc ion can in e e e wi h amyloid seeding. Because Ssa1-21p does no a ec Hsp104’s abili y o p o ide he mo ole ance (28) o o eac i a e hea -dena u ed luci e ase in i o (G. Jung, unpublished da a), i s e ec on [PSI ⫹ ] migh be di ec . The simples way o explain ou da a is ha Ssa1-21p educes gen- e a ion o [PSI ⫹ ] seeds by binding oo a idly o Sup35p agg e- ga es, which s e ically es ic s access o Hsp104 (27). I his explana ion we e ue, hen his al e ed unc ion mus a ec disassembly o b eakage o he p esumed highly o de ed amy- loid ibe s o yeas p ions di e en ly han disagg ega ion o amo phous, he mally dena u ed subs a es. A way o explain his di e ence is ha amyloid is ecognized di e en ly as a subs a e by Hsp70 and Hsp104. Since Hsp40 is hough o be able o p esen a subs a e o Hsp70, and i s subs a e ecogni ion o e laps ha o Hsp70, Hsp40 also may con ibu e o he appa en subs a e-speci ic e ec s on amy- loid. In line wi h his idea, p ope ies o a speci ic Hsp40 (Sis1p) ha e been sugges ed o enhance in e ac ion o he yeas p ion [PIN ⫹ ]/[RNQ ⫹ ] de e minan Rnq1p wi h Hsp70 (36). Ano he way ha enhanced subs a e binding by Hsp70 migh impai [PSI ⫹ ] is ha Hsp70’s abili y o assis Hsp104 in esolubilizing p o ein om agg ega es is al e ed. Al hough he mechanism o p o ein disagg ega ion by Hsp104 is unknown, Hsp70 and Hsp40 coope a e in his p ocess, and oge he hese h ee chape ones can esolubilize la ge p o ein agg ega es in i o (20). The al e ed unc ion o he mu an Hsp70 may a ec an aspec o a ansien physical in e ac ion be ween Hsp70 and Hsp104 o o hei abili y o ac sequen ially o simul a- neously on a subs a e. In he homologous bichape one sys em o Esche ichia coli, he Hsp104 homolog ClpB binds he sub- s a e i s , inc easing exposu e o su aces o subsequen Hsp70 in e ac ion (21, 61). I he eac ion is simila in yeas , a subs a e p oduced om Hsp104 in e ac ion wi h amyloid may in e ac wi h Hsp70 in a way ha allows i o e ain he abili y o p opaga e he sel - eplica ing s uc u e. By binding oo a - idly wi h his subs a e, Ssa1-21p may in e e e wi h he abili y o he sel - eplica ing con o ma ion o be main ained. Such a scena io may explain why Ssa1-21p has a de imen al e ec on amyloid wi hou g ea ly a ec ing o he cellula p ocesses ha equi e Hsp70 unc ion. Ou da a sugges ha Cp 7p, whose e ec on Hsp70’s enzy- ma ic unc ion is unknown, may also ac o enhance subs a e FIG. 7. Regula ion o Hsp70 (Ssa1p) eac ion cycle by cochape - ones. Hsp70 unc ion is inely uned by ATP hyd olysis and nucleo ide exchange, which egula e subs a e binding. I is known ha Hsp40 (Ydj1p, Sis1p) and S i1p s imula e ATP hyd olysis, p omo ing sub- s a e binding, and ha Fes1p accele a es ADP elease and nucleo ide exchange, p omo ing subs a e elease. Ou da a sugges ha enhanc- ing con e sion o Hsp70 o he ADP-bound s a e o s abilizing his s a e impai s [PSI ⫹ ] p opaga ion. Ou da a also sugges ha Cp 7p and Cns1p migh p omo e o s abilize his s ep in he cycle. VOL. 24, 2004 Hsp70 COCHAPERONES IMPAIR AMYLOID PROPAGATION 3935 binding by Ssa1p. Since mo e Cp 7p was associa ed wi h Ssa1- 21p han wi h Ssa1p, and mo e Ssa1-21p is expec ed o be in he closed ADP-bound s a e, Cp 7p may p e e en ially bind he ADP-bound o m o Hsp70. I so, hen Cp 7p may be ac ing o s abilize he closed con o ma ion a he han o in- duce con e sion o his s a e. The addi i e e ec o deple ing bo h Cp 7p and S i1p is consis en wi h his in e p e a ion. Dele ing CPR6 o SGT2, o he yeas cyclophilin and Hop1 homologs, espec i ely, had no e ec on [PSI ⫹ ] p opaga ion in wild- ype o SSA1-21 cells, showing ha he e ec o TPR cochape ones on [PSI ⫹ ] was no a gene al one. Thus, wi h espec o [PSI ⫹ ] he e a e clea unc ional di e ences in TPR cochape one in e ac ions wi h Ssa1p, which a e likely oo ed in di e ences in speci ici y o a ini y. Such di e ences also may unde lie unc ional dis inc ions be ween Ssa1p and Ssa2p e- ga ding [PSI ⫹ ] p opaga ion ( his s udy), he abili y o excess Ssa1p bu no Ssa2p o cu e cells o he yeas [URE3] p ion (50), o he speci ic equi emen o Ssa2p o anspo o ce ain p o eins in o p e acuola (Vid) esicles (6). Ou inding ha dele ing he TPR in e ac ion mo i (MEEVD) o Hsp90 had no e ec on [PSI ⫹ ] sugges s ha Hsp90 is no in ol ed in S i1p and Cp 7p e ec s on [PSI ⫹ ] p opaga ion. Despi e wide in e es in yeas p ions and Hsp90 and he a ailabili y o many Hsp90 mu an s, he only o he expe imen s desc ibing e ec s o Hsp90 on yeas p ions we e also nega i e, showing a lack o e ec o o e p oduced Hsp90 on [PSI ⫹ ] (44). Whe he Hsp90 is equi ed o [PSI ⫹ ] p opa- ga ion canno be es ed di ec ly because i is essen ial o iabili y. Howe e , we also ound ha [PSI ⫹ ] p opaga ion was una ec ed in bo h wild- ype and SSA1-21 cells by dele ion o only HSC82, which educes he o e all abundance o Hsp90 o oughly 10% o wild- ype le els (5), o when cells we e ea ed wi h a wide ange o concen a ions o he Hsp90 inhibi o s geldanamycin and adicicol (G. Jones, unpublished da a). To- ge he hese obse a ions sugges ha Hsp90 unc ion is no impo an o [PSI ⫹ ] p opaga ion. Ou da a he e o e p o ide e idence sugges ing ha S i1p and Cp 7p can unc ionally in- e ac wi h Ssa1p independen ly o Hsp90. Cp 7 and CNS1 ha e been ound oge he in complexes wi hou Hsp90 (58), and ou esul s may e eal e ec s o pe u ba ion o such a complex. Ou indings illus a e he u ili y o yeas p ions as a sys em o s udying bo h amyloid p opaga ion and p o ein chape one unc ion. In addi ion o ad ancing he unde s anding o how chape ones a ec amyloid p opaga ion in i o, con inued s udy wi h his unique sys em will p o ide new insigh s in o he unc ions o he chape one machine y in gene al. ACKNOWLEDGMENTS We hank Andy Golden, Will P inz, and Ke in O’Connell o help ul commen s on he manusc ip . REFERENCES 1. Abbas-Te ki, T., O. Donze, P. A. B iand, and D. Pica d. 2001. Hsp104 in e ac s wi h Hsp90 cochape ones in espi ing yeas . Mol. Cell. Biol. 21: 7569–7575. 2. Alani, E., L. Cao, and N. Kleckne . 1987. A me hod o gene dis up ion ha allows epea ed use o URA3 selec ion in he cons uc ion o mul iply dis- up ed yeas s ains. Gene ics 116:541–545. 3. Baudin, A., O. Ozie -Kaloge opoulos, A. Denouel, F. Lac ou e, and C. Cul- lin. 1993. A simple and e icien me hod o di ec gene dele ion in Saccha- omyces ce e isiae. Nucleic Acids Res. 21:3329–3330. 4. Bo chsenius, A. S., R. D. Weg zyn, G. P. Newnam, S. G. Inge-Vech omo , and Y. O. Che no . 2001. Yeas p ion p o ein de i a i e de ec i e in agg e- ga e shea ing and p oduc ion o new ⬘seeds.’EMBO J. 20:6683–6691. 5. Bo ko ich, K. A., F. W. Fa elly, D. B. Finkels ein, J. Taulien, and S. Lindquis . 1989. Hsp82 is an essen ial p o ein ha is equi ed in highe concen a ions o g ow h o cells a highe empe a u es. Mol. Cell. Biol. 9:3919–3930. 6. B own, C. R., J. A. McCann, and H. L. Chiang. 2000. The hea shock p o ein Ssa2p is equi ed o impo o uc ose-1,6-bisphospha ase in o Vid esicles. J. Cell Biol. 150:65–76. 7. Bukau, B., and A. L. Ho wich. 1998. The Hsp70 and Hsp60 chape one machines. Cell 92:351–366. 8. Chang, H. C., and S. Lindquis . 1994. Conse a ion o Hsp90 mac omolec- ula complexes in Saccha omyces ce e isiae. J. Biol. Chem. 269:24983–24998. 9. Che no , Y. O., S. L. Lindquis , B. Ono, S. G. Inge-Vech omo , and S. W. Liebman. 1995. Role o he chape one p o ein Hsp104 in p opaga ion o he yeas p ion-like ac o [psi⫹]. Science 268:880–884. 10. Conde, J., and G. R. Fink. 1976. A mu an o Saccha omyces ce e isiae de ec i e o nuclea usion. P oc. Na l. Acad. Sci. USA 73:3651–3655. 11. Cox, B. S. 1965. “⌿”a cy oplasmic supp esso o supe -supp esso in yeas . He edi y 20:505–521. 12. Demand, J., J. Lude s, and J. Hoh eld. 1998. The ca boxy- e minal domain o Hsc70 p o ides binding si es o a dis inc se o chape one co ac o s. Mol. Cell. Biol. 18:2023–2028. 13. De ka ch, I. L., Y. O. Che no , V. V. Kushni o , S. G. Inge-Vech omo , and S. W. Liebman. 1996. Genesis and a iabili y o [PSI] p ion ac o s in Sac- cha omyces ce e isiae. Gene ics 144:1375–1386. 14. Dolinski, K. J., M. E. Ca denas, and J. Hei man. 1998. CNS1 encodes an essen ial p60/S i1 homolog in Saccha omyces ce e isiae ha supp esses cy- clophilin 40 mu a ions and in e ac s wi h Hsp90. Mol. Cell. Biol. 18:7344– 7352. 15. Duina, A. A., H. M. Kal on, and R. F. Gabe . 1998. Requi emen o Hsp90 and a CyP-40- ype cyclophilin in nega i e egula ion o he hea shock e- sponse. J. Biol. Chem. 273:18974–18978. 16. Duina, A. A., J. A. Ma sh, and R. F. Gabe . 1996. Iden i ica ion o wo CyP-40-like cyclophilins in Saccha omyces ce e isiae, one o which is equi ed o no mal g ow h. Yeas 12:943–952. 17. Eagles one, S. S., L. W. Ruddock, B. S. Cox, and M. F. Tui e. 2000. Guani- dine hyd ochlo ide blocks a c i ical s ep in he p opaga ion o he p ion-like de e minan [PSI ⫹ ]o Saccha omyces ce e isiae. P oc. Na l. Acad. Sci. USA 97:240–244. 18. F eeman, B. C., M. P. Mye s, R. Schumache , and R. I. Mo imo o. 1995. Iden i ica ion o a egula o y mo i in Hsp70 ha a ec s ATPase ac i i y, subs a e binding and in e ac ion wi h HDJ-1. EMBO J. 14:2281–2292. 19. Glo e , J. R., A. S. Kowal, E. C. Schi me , M. M. Pa ino, J. J. Liu, and S. Lindquis . 1997. Sel -seeded ibe s o med by Sup35, he p o ein de e mi- nan o [PSI ⫹ ], a he i able p ion-like ac o o S. ce e isiae. Cell 89:811–819. 20. Glo e , J. R., and S. Lindquis . 1998. Hsp104, Hsp70, and Hsp40: a no el chape one sys em ha escues p e iously agg ega ed p o eins. Cell 94:73–82. 21. Goloubino , P., A. Mogk, A. P. Z i, T. Tomoyasu, and B. Bukau. 1999. Sequen ial mechanism o solubiliza ion and e olding o s able p o ein ag- g ega es by a bichape one ne wo k. P oc. Na l. Acad. Sci. USA 96:13732– 13737. 22. Gu h ie, C., and G. R. Fink (ed.). 1991. Guide o yeas gene ics and molec- ula biology, ol. 194. Academic P ess, San Diego, Cali . 23. Han, W., and P. Ch is en. 2003. Mechanism o he a ge ing ac ion o DnaJ in he DnaK molecula chape one sys em. J. Biol. Chem. 278:19038–19043. 24. Ha ge , J. W., and J. D. Dinman. 2003. An in i o dual-luci e ase assay sys em o s udying ansla ional ecoding in he yeas Saccha omyces ce e i- siae. RNA 9:1019–1024. 25. Ha l, F. U., and M. Haye -Ha l. 2002. Molecula chape ones in he cy osol: om nascen chain o olded p o ein. Science 295:1852–1858. 26. He nandez, M. P., A. Chadli, and D. O. To . 2002. Hsp40 binding is he i s s ep in he Hsp90 chape oning pa hway o he p oges e one ecep o . J. Biol. Chem. 277:11873–11881. 27. Jones, G. W., and D. C. Masison. 2003. Saccha omyces ce e isiae Hsp70 mu a ions a ec [PSI ⫹ ] p ion p opaga ion and cell g ow h di e en ly and implica e Hsp40 and e a icopep ide epea cochape ones in impai men o [PSI ⫹ ]. Gene ics 163:495–506. 28. Jung, G., G. Jones, R. D. Weg zyn, and D. C. Masison. 2000. A ole o cy osolic Hsp70 in yeas [PSI ⫹ ] p ion p opaga ion and [PSI ⫹ ] as a cellula s ess. Gene ics 156:559–570. 29. Jung, G., and D. C. Masison. 2001. Guanidine hyd ochlo ide inhibi s Hsp104 ac i i y in i o: a possible explana ion o i s e ec in cu ing yeas p ions. Cu . Mic obiol. 43:7–10. 30. Kabani, M., J. M. Becke ich, and J. L. B odsky. 2002. Nucleo ide exchange ac o o he yeas Hsp70 molecula chape one Ssa1p. Mol. Cell. Biol. 22:4677–4689. 31. Kelley, W. L. 1999. Molecula chape ones: How J domains u n on Hsp70s. Cu . Biol. 9:R305–R308. 32. King, C. Y., P. Ti mann, H. G oss, R. Gebe , M. Aebi, and K. Wu h ich. 3936 JONES ET AL. MOL.CELL.BIOL.