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 .
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