Hsp110 cooperates with different cytosolic HSP70 systems in a pathway for de novo folding.

Abstract

Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bind mostly post-translationally to a more restricted subset of newly translated polypeptides, suggesting a downstream function in the folding pathway. Notably, loss of SSB dramatically enhances the cotranslational association of SSA with nascent chains, suggesting SSA can partially fulfill an SSB-like function. On the other hand, the absence of SSE1 enhances polypeptide binding to both SSB and SSA and impairs cell growth. It, thus, appears that Hsp110 is an important regulator of Hsp70-substrate interactions. Based on our data, we propose that Hsp110 cooperates with the SSB and SSA Hsp70 subfamilies, which act sequentially during de novo folding.

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Hsp110 Cooperates with Different Cytosolic Hsp70 Systems
in a Pathway for de Novo Folding*
Receivedforpublication,April1,2005,andinrevisedform,August8,2005 Published,JBCPapersinPress,October11,2005,DOI10.1074/jbc.M503615200
Alice Yen-Wen Yam‡1, Ve ´ronique Albane `se‡1,2, Hen-Tzu Jill Lin‡§, and Judith Frydman‡3
Fromthe‡DepartmentofBiologicalSciencesandBioXProgram,StanfordUniversity,Stanford,California94305-5020and
§UniversityofCaliforniaatSanFranciscoMassSpectrometryFacility,UniversityofCalifornia,San Francisco, California 94143
Molecular chaperones such as Hsp70 use ATP binding and
hydrolysistopreventaggregationandensuretheefficientfoldingof
newly translated and stress-denatured polypeptides. Eukaryotic
cells contain several cytosolic Hsp70 subfamilies. In yeast, these
includetheHsp70sSSBandSSAaswellastheHsp110-likeSse1/2p.
The cellular functions and interplay between these different Hsp70
systems remain ill-defined. Here we show that the different cytoso-
licHsp70systemsfunctionallyinteractwithHsp110toformachap-
erone network that interacts with newly translated polypeptides
duringtheirbiogenesis.BothSSBandSSAHsp70sformstablecom-
plexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that
these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently
associate with newly synthesized polypeptides with different kinet-
ics. SSB Hsp70s bind cotranslationally to a large fraction of nascent
chains, suggesting an early role in the stabilization of nascent
chains. SSA Hsp70s bind mostly post-translationally to a more
restricted subset of newly translated polypeptides, suggesting a
downstream function in the folding pathway. Notably, loss of SSB
dramatically enhances the cotranslational association of SSA with
nascentchains,suggestingSSAcanpartiallyfulfillanSSB-likefunc-
tion. On the other hand, the absence of SSE1 enhances polypeptide
binding to both SSB and SSA and impairs cell growth. It, thus,
appears that Hsp110 is an important regulator of Hsp70-substrate
interactions.Basedonourdata,weproposethatHsp110cooperates
with the SSB and SSA Hsp70 subfamilies, which act sequentially
during de novo folding.
Newly synthesized proteins are faced with the difficult task of trans-
forming their linear amino acid sequence into a three-dimensional
folded structure within the complex and crowded environment of the
cell (1–3). During translation, molecular chaperones, such as Hsp70,4
use ATP binding and hydrolysis to protect the emerging polypeptide
from aggregation and facilitate its correct folding (1, 4, 5). Hsp70s are a
highlyconservedfamilyofproteinsubiquitouslydistributedinallcellu-
lar compartments of prokaryotic and eukaryotic organisms. Most
Hsp70s have a molecular mass of ?70 kDa and consist of two function-
ally coupled domains, a 44-kDa N-terminal domain that mediates ATP
binding and an 18-kDa C-terminal domain that binds the substrate
polypeptide (for reviews, see Refs. 2, 6, and 7). A unique subclass of
Hsp70s called Hsp110s, found only in eukaryotic cells, is distinguished
by an insertion of flexible loops after the polypeptide binding domain
and an extended tail at the C terminus (8, 9). The function of these
additional domains in Hsp110 remains unclear.
Binding and release of an unfolded polypeptide by all Hsp70s rely on
the modulation of the intrinsic peptide affinity of Hsp70 by cycles of
ATP binding and hydrolysis (2, 6, 7). Cycling of Hsp70s between the
different nucleotide states is regulated by an expanding list of cofactors
(1,7,10),includingJ-domainproteinsthatstimulatetheATPaseactivity
of Hsp70 and several structurally distinct nucleotide exchange factors
(6, 11, 12).
An intriguing aspect of the eukaryotic cytosol is that it contains mul-
tiple Hsp70 subfamilies (5). For instance, the cytosol of the yeast Sac-
charomyces cerevisiae contains four functionally redundant Hsp70
homologues called Ssa1–4p, sharing more than 85% identity (herein
SSA) and three ribosome-associated Hsp70s, Ssz1p, and the function-
allyredundantSsb1pandSsb2p,sharingmorethan99%identity(herein
SSB).Inaddition,yeasthastwoHsp110-likehomologuescalledSse1/2p,
sharing 76% identity (5, 9, 13). It is unclear why eukaryotic cells have so
many Hsp70 homologues. Although they appear to have distinct cellu-
larfunctions,asonlySSAisessentialforviabilityandcannotbereplaced
bySSB(5,14),thepreciseroleofthedifferentHsp70sincellularfolding
remains undefined. Genetic and biochemical studies indicate that SSA
isrequiredforthefoldingofsomenewlysynthesizedproteins,including
TRP3 (tryptophan-requiring protein 3) and the heterologous proteins
ornithine transcarbamoylase and the von Hippel-Lindau tumor sup-
pressorprotein(15–17).SSAalsofacilitatesinimportofprecursorpro-
teins into both the endoplasmic reticulum and mitochondria (18, 19)
and is important for rescue of stress-denatured proteins (5). On the
other hand, in vitro experiments showed that the ribosome-bound SSB
canbecross-linkedtonascentchainsincloseproximitytothepolypep-
tide exit channel (20, 21). However, association of SSB with nascent
chains has not yet been observed in vivo. Although not essential, dele-
tionofSSBresultsinaslowgrowthphenotype(14).Ithasrecentlybeen
suggested that SSB function at the ribosome is modulated by another
Hsp70,Ssz1p,inacomplexwiththeJ-domainproteinZuo1p(21–23).It
is further proposed that the Ssz1p?Zuo1p complex is essential for SSB
interaction with nascent chains (21).
The cellular function of Hsp110s is also unclear. Hsp110s have been
showntopreventaggregationofmodelsubstratesinvitro(24)andhave
been implicated in thermotolerance (25–27). In S. cerevisiae, cells lack-
ing Sse1p are slow growing and temperature-sensitive (13), and genetic
studies also implicate Sse1p in Hsp90-dependent processes (24, 28).
Interestingly, biochemical studies suggest that Hsp110 chaperones may
be involved in the regulation of Hsp70 ATP hydrolysis and that Hsp70
may have some stimulatory activity on Hsp110 ATPase as well (29, 30).
* This work was supported by National Institutes of Health Grant GM56433. Mass spec-
trometry analysis at the University of California at San Francisco Mass Spectrometry
Facility was supported by National Institutes of Health Grant RR01614. The costs of
publicationofthisarticleweredefrayedinpartbythepaymentofpagecharges.This
articlemustthereforebeherebymarked“advertisement”inaccordancewith18U.S.C.
Section 1734 solely to indicate this fact.
1These authors contributed equally to this work.
2Recipient of postdoctoral fellowships from the Fondation pour la Recherche Me ´dicale
and the Association pour la Recherche contre le Cancer, France.
3Towhomcorrespondenceshouldbeaddressed.Tel.:650-725-7833;Fax:650-724-4927;
E-mail: jfrydman@stanford.edu.
4The abbreviations used are: Hsp: heat shock protein; SSA/B/E, Stress Seventy A/B/E/;
HA, hemagglutinin.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 50, pp. 41252–41261, December 16, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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However, the ATPase activity of Sse1p appears to be dispensable for its
function (31).
To obtain insights into the function of the different cytosolic Hsp70s
in de novo folding, we searched for cellular binding partners of the
Hsp70s SSB and SSA and examined their interactions with nascent
chains. We find that the major cytosolic Hsp70s, SSA and SSB, both
associate independently with the cytosolic Hsp110 homologue Sse1p.
To evaluate the interplay and relative contribution of each Hsp70 sub-
familytodenovoproteinfolding,weexaminedthefluxofnewlysynthe-
sized proteins through the different Hsp70 chaperones. Our data sug-
gest that the SSB?Sse1p complex plays an early role in cotranslational
nascent chain stabilization, whereas SSA?Sse1p acts mostly post-trans-
lationally to facilitate the folding of a more restricted protein subset.
Analysis of Hsp70 interactions in ?sse1 cells further indicates that
Hsp110 is an important regulator for both SSB- and SSA-substrate
interactions. Our analysis indicates that a network of different cytosolic
Hsp70s cooperates in de novo folding.
EXPERIMENTAL PROCEDURES
Plasmids and Yeast Strains—SSA1, SSB2, and SSE1 were N-terminal
HA-tagged (or 3HA-tagged for SSE1) and expressed in the copper-in-
ducible vector pCu426 (noted as pCu) (32). Importantly, all the tagged
chaperones fully rescued the corresponding null strain (not shown). A
chromosomal copy of SSE1 was also 3HA-tagged at the C terminus via
theintegrationofakanamycincassette(33).Exceptwhereindicated,the
Hsp70s were expressed in the absence of copper. Expression resulting
from leakiness of the promoter closely approached physiological
expression levels, as determined by immunoblot analysis with the cor-
responding antibodies (data not shown). In Fig. 2B, overexpression was
achieved by the addition of 200 ?M CuSO4. All experiments were done
in the BY4743 background (MAT a/? his3?1/his3?1 leu2?0/leu2?0
lys2?0/LYS2 MET15/met15?0 ura3?0/ura3?0) with log-phase cells.
Thedoublemutants?sse1/2and?ssb1/2weregeneratedbymatingand
sporulation of single deletion mutants (34).
Gel Filtration Analysis—Wild type cells with chromosomally tagged
SSE1–3HA were grown in rich media, harvested in log phase, and lysed
at 4 °C in buffer A (50 mM Tris, pH 7.5, 10 mM HEPES, pH 7.5, 100 mM
KCl,5mMMgCl2,10%glycerol,0.1%TritonX-100)bybead-beatingfor
10 min. Clarified extracts (?150 ?g) were then separated by size exclu-
sion chromatography in a Amersham Biosciences Superdex 200 HR
10/30 column equilibrated in buffer A. Fractions were collected at
0.5-ml intervals, and Sse1–3HAp was immunoprecipitated with anti-
bodies against the HA tag. The total elution profile and the immuno-
precipitates were separated by 12% SDS-PAGE and blotted with anti-
bodies against Ssa1/2p, Ssb1/2p, the ribosomal protein Rpl3p, and the
HA tag.
ChaperoneImmunoprecipitationsandMassSpectrometryAnalysis—
All chaperone immunoprecipitations were done via an HA tag located
at the N or C termini of the corresponding chaperone. Yeast lysates
were prepared from log-phase cells as described above. Samples were
supplemented with 20 mg/ml bovine serum albumin and 1 ?l of
anti-HAmonoclonalantibody(BerkeleyAntibodyCo.,Inc.monoclonal
antibody HA.11, 5 mg/ml) and then incubated on ice for 40 min. The
immunoprecipitateswerefurtherclarifiedtoremoveproteinaggregates
before rotating with protein G-Sepharose for 30 min at 4 °C. Sepharose
beads were washed twice with Tris-buffered saline (TBS) plus 0.05%
Tween and then 3 times with TBS plus 1% Tween, as described (35).
Sampleswereseparatedby12%SDS-PAGEgel,andproteinswerevisu-
alized by Coomassie Blue or silver staining. Coomassie-stained bands
were excised and identified by mass spectrometry.
Pulse-Chase Analysis—Cells expressing HA-tagged Hsp70s from the
pCu426 vector were grown to log phase and starved for 30 min in com-
plete synthetic medium without methionine. The cells were then
labeled with 100 ?Ci/ml [35S]methionine for 2.5 min and then chased
with 20 mM cold methionine. At the indicated time points cells were
quickly chilled and harvested in 250 mM cold azide to deplete of ATP
and 0.5 mg/ml cycloheximide to stop protein synthesis. Lysates pre-
pared in buffer A were clarified, and Hsp70-substrate complexes were
isolated by immunoprecipitation using HA-specific antibodies as
described above.
Nascent Chain-Chaperone Interactions—Wild type and Hsp70-de-
leted cells were grown and labeled with [35S]methionine for 1 min as
described above. After lysis in buffer A, ribosome-nascent chain com-
plexeswereisolatedbyultracentrifugationthrougha25%sucrosecush-
ion (25% sucrose, 20 mM HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl2,
0.1% Triton X-100) for 25 min at 200,000 ? g. The ribosomal pellets
were resuspended, and the Hsp70-bound polypeptides in the superna-
tant, sucrose cushion, and ribosomal fractions were immunoprecipi-
tated via the HA tag as described.
Quantitation of Hsp70-associated Newly Made Proteins—Hsp70-as-
sociated polypeptides were quantitated with ImageQuant software on a
Typhoon PhosphorImager (Amersham Biosciences, Version 5.2) by
measuring the radioactivity in each lane in gel exposures that were
within the linear range of the instrument. All quantifications were nor-
malized over the total amount of labeled proteins (i.e. the total radioac-
tivity in the ribosomal fraction for Fig. 4 or total amount of labeled
proteinforFigs.5and6).Forpulse-chasesamples,thelabelcorrespond-
ingtonewlysynthesizedHsp70andHsp110bandswassubtractedfrom
thetotalradioactivityineachlane.Immunoprecipitationswereadjusted
for equivalent protein amounts in each time point. To compare the
effect of chaperone deletions on substrate binding to Hsp70s, the
amount of Hsp70-bound nascent chains within paired experiments in
wild type and mutant cells were expressed as a -fold difference over the
Hsp70-bound nascent chains in wild type cells.
RESULTS
The Hsp70-like Sse1p Associates with the Cytosolic Hsp70s SSB and
SSA—To better understand the function of Hsp70s in de novo folding,
we analyzed the interactions of the ribosome-bound Hsp70 SSB. Upon
immunoprecipitation of Ssb2p, a very prominent associated protein of
higher molecular weight was readily observable (Fig. 1A, lane 1). Mass
spectrometryanalysisindicatedthattheSSB-associatedproteinwasthe
Hsp110 homologue Sse1p. In like fashion, immunoprecipitation of
Sse1p revealed association with two different polypeptides of ?70 kDa
(Fig. 1B, lane 1). Mass spectrometry analysis revealed that one of the
bandscorrespondedtoSsb1/2p,andtheotherbandcontainedthecyto-
solicHsp70sSsa1/2p.TherelativeamountsofSSBandSSAintheSse1p
immunoprecipitation were comparable, suggesting that both Hsp70s
associate to a similar extent with Sse1p (Fig. 1B, lane 1; see also Fig. 1C,
lane2).TheinteractionbetweenSse1pandSsa1/2pwasalsoobservedin
?ssb1/2 cells, indicating that the complexes can form independently of
eachother(Fig.1B,lane2).ImmunoprecipitationofSSAandanalysisof
its associated proteins confirmed that SSA interacts with Sse1p, but it
indicated that only a small fraction of SSA is in a complex with Sse1p
(Fig. 1C, lane 1; see Fig. 2). The association between these Hsp70s was
unaffected by the presence or absence of Mg2?-ATP in the lysis buffer
(not shown) and was independent of the levels of expression of either
Hsp70, as was observed using either plasmid-borne or chromosomally
encoded chaperones (e.g. compare Fig. 1, B (lane 1) and C (lane 2), for
plasmid-borne or chromosomally tagged Sse1p, respectively). Of note,
Hsp110CooperateswithCytosolicHsp70s
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theseimmunoprecipitationexperimentsdidnotdetectadirectinterac-
tion between SSA and SSB, suggesting that Sse1p forms two distinct
complexes with SSA and SSB (Fig. 1, A and C). On the other hand, we
did not observe the other Hsp110 homologue, Sse2p, in any of our
immunoprecipitations. Experiments using tagged Sse2p indicated an
association with SSA Hsp70s but failed to detect association with SSB
(not shown). Because the stress-inducible Sse2p is expressed at much
lower levels than Sse1p (13, 36, 37), it is possible that its function is less
relevant than Sse1p under normal conditions. Alternatively, the two
homologues may have distinct cellular functions whereby only Sse1p
can associate with SSB Hsp70s. Notably, immunoprecipitation of
HA-Ssa1p using antibodies against the epitope tag also isolated
untagged Ssa1/2p, suggesting that this Hsp70 self-associates in vivo, as
suggested by in vitro experiments using purified Hsp70 (38).
The Hsp110 Sse1p Forms Distinct Complexes with the Hsp70s SSA
and SSB—We next examined the distribution of Hsp70-containing
complexesincellextractsusingsizeexclusionchromatography(Fig.2).
Lysates obtained from wild type cells carrying a chromosomally inte-
grated3HAtagattheCterminusofSSE1werefractionatedonaSuper-
dex S200 column, and the elution profiles of the Hsp70s SSA, SSB, and
Sse1pweredeterminedbyimmunoblotanalysis(Fig.2A).Aspreviously
described for mammalian Hsp70 (39), SSA associated with numerous
cellularcomplexesandmigratedverybroadlyintheseparation,ranging
in size from monomeric forms to high molecular weight complexes
eluting in the void of the column (Fig. 2A). In contrast, SSB complexes
werefoundprimarilyintwomajorpeaks.OneSSBcomplexofveryhigh
molecular weight eluted in the void together with the ribosomes (Fig.
2A) and probably corresponds to ribosome-bound SSB (20). The other
broad peak displayed a molecular mass range between 60 and 200 kDa
and probably corresponds to monomeric SSB and soluble complexes
containing SSB. This is consistent with previous observations that SSB
partitionsbetweenribosome-boundandribosome-freeforms(20).The
elutionprofileofSse1pwasmorerestrictedthanthatofSSAorSSB(Fig.
2A).VerylittleSse1pelutedatitsexpectedmonomericmolecularmass;
instead, Sse1p was primarily found in a peak centered around a molec-
ularmassof?180kDa,withaminorshoulderat?210kDa.Becausethe
major Sse1p fraction corresponds in approximate size to a het-
erodimeric complex between Hsp110 and Hsp70 and the immunopre-
cipitation analysis suggests that most Sse1p is associated with either
SSA or SSB (Fig. 1B), it appears that Sse1p is predominantly in complex
withHsp70.Additionally,asmallfractionelutedtogetherwiththeribo-
somal peak.
To further examine the Sse1p-containing complexes, we immuno-
precipitated individual column fractions for the endogenously tagged
Sse1–3HAp followed by immunoblot analysis of SSA and SSB (Fig. 2B).
Sse1p and SSA associated in a major complex of ?180 kDa. SSB also
coimmunoprecipitated with Sse1p in defined complexes. One complex
coeluted with the ribosomal fraction (lane 18), whereas the other com-
plexpeakedat?180kDa,withanelutionprofilealmostidenticaltothat
of Sse1p (Fig. 2A). A minor peak at ?210 kDa, also observed in this
analysis of Sse1p complexes with both Hsp70s, may correspond to
Sse1p?Hsp70 complexes with either additional cofactors or substrates.
The gel filtration analysis of these Hsp70s supports the idea that not all
the SSA or SSB Hsp70s are in a complex with Sse1p, but most Sse1p
forms complexes with either SSA or SSB. Because we did not detect a
ternary SSA?SSE?SSB complex (Fig. 1), it is likely that SSA and SSB
complexes with Sse1p are mutually exclusive.
GeneticInteractionbetweenCytosolicHsp70s—Giventhehighdegree
of homology between Sse1p and Sse2p (76% identity), the differences
between their association with SSA and SSB prompted us to examine
thegeneticinteractionsbetweenthetwoHsp110homologues(Fig.3A).
Deletion of SSE1, although viable, results in a very severe growth phe-
FIGURE 1. Interaction of Sse1p with the Hsp70 proteins Ssa1/2p and Ssb1/2p. A, SSB associates with Sse1p but not with SSA-Hsp70s. Wild type cells expressing HA-Ssb2p were
lysed and immunoprecipitated with anti-HA antibodies as described under “Experimental Procedures.” As a control a parallel analysis was carried out on cells transformed with the
backbonevectoralone(pCu).Ssb2pbindingpartnerswereidentifiedbymassspectrometry.MW,molecularweight.B,Sse1pinteractsdirectlywithbothHsp70sSsa1/2p,andSsb1/2p
and can associate with Ssa1/2p independently of Ssb1/2p. Wild type (WT) or ?ssb1/2 cells expressing 3HA-SSE1 from the pCu plasmid were analyzed for Sse1p-interacting proteins
asinA.C,HA-Ssa1passociateswithSse1pbutnotwithSSB.WildtypecellscarryingpCuSSA1orthepCuvectoralone(lanes1and3)wereanalyzedforSsa1p-interactingproteinsas
in A. In parallel, Sse1p-interacting proteins were also immunoprecipitated from cells carrying a chromosomally tagged copy of SSE1–3HA (lane 2). All immunoprecipitations were
separated by SDS-PAGE and either Coomassie-stained (A and B) or silver-stained (C). Positions of tagged and untagged Hsp70 chaperones are indicated as are the positions of the
heavy chain (HC), light chain (LC), and bovine serum albumin (asterisk) that is added to the immunoprecipitation.
Hsp110CooperateswithCytosolicHsp70s
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notype at all temperatures examined (Fig. 3A). In contrast, deletion of
SSE2 has no detectable growth defect, even at 37 °C. The severity of
phenotype of the doubly deleted ?sse1?sse2 was comparable with that
of the ?sse1 strain. Although these results are in agreement with previ-
ousobservations(13),asimilaranalysisinadifferentstrainbackground
observed a less severe phenotype for the ?sse1 cells and a synthetic
lethal effect for ?sse1?sse2 (40). In any case, the genetic analysis dem-
onstrates that SSE function plays a fundamental role in normal cell
growth. Furthermore, in our genetic background, Sse1p is the major
source of Hsp110 activity, in agreement with estimates that Sse1p is
?10-fold more abundant that Sse2p (37).
The severity of the ?sse1 phenotype together with the observation
that most Sse1p is associated with either SSB or SSA raised the possi-
bilitythatSse1pplaysanimportantroleinthefunctionoftheseHsp70s.
Indeed, analysis of the consequences of Ssa1p or Sse1p overexpression
in ?ssb1/2 cells indicated that the interplay between these three cyto-
solic Hsp70 systems is important for cellular function (Fig. 3B). Loss of
SSBfunctionresultsinaslowgrowthphenotype.Althoughlowlevelsof
expressionofSsa1porSse1pdidnotaffectviabilityofeitherwildtypeor
?ssb1/2cells(Fig.3B,?copper),overexpressionofeitherSsa1porSse1p
in the ?ssb1/2 background has a severe toxic effect on cell growth (Fig.
3B,?copper).Thus,thereisanoptimumbalancebetweenthefunctions
of these cytosolic Hsp70s that has to be maintained for cell viability.
The SSB and SSA Hsp70s Associate with Distinct Subsets of Nascent
Chains—Previous studies in mammalian systems found that Hsp70
assists de novo folding by interacting co- and post-translationally with
newly made proteins (4, 35). To better understand the functional inter-
play between SSB, SSA, and Sse1p, we next examined their interaction
with newly translated polypeptides. The SSB known ribosome associa-
tiontogetherwithSse1pmigrationinribosome-containingfractionsled
us to initially assess Hsp70 substrate interactions that may occur either
during or soon after polypeptide synthesis. Accordingly, we initially
tested chaperone binding to both ribosome-associated nascent chains
as well as completed full-length polypeptides already released from the
ribosome (Fig. 4). Newly translated polypeptides were specifically
labeledbyaveryshortpulsewith[35S]methioninetopreferentiallylabel
polypeptides that are still bound to the ribosome (Fig. 4B, lane 3),
although some proteins are completed and released during this pulse
(Fig. 4B, lane 1). Cells were then depleted of ATP to stabilize Hsp70-
substrate interactions and lysed in the presence of cycloheximide to
stabilize ribosome-nascent chain complexes. To distinguish between
co- and post-translational interactions, we separated the ribosome-
bound nascent chains (R) from the released polypeptides in the soluble
fraction (S) by ultrasedimentation through dense sucrose cushions (C)
(Fig. 4, B–F, see Totals in lanes 1–6). After separation, ribosomal (R),
sucrose (C), and supernatant (S) fractions were either directly analyzed
by SDS-PAGE (Fig. 4, B–F, lanes 1–6) or subjected to immunoprecipi-
tation to assess nascent chain interactions with the indicated chaper-
ones (Fig. 4, B–F, lanes 7–15). Newly synthesized polypeptides were
then detected by autoradiography.
WefirstexaminednascentchainbindingtoSSBinwildtypecells(Fig.
4B). We found that SSB associates mostly to ribosome-bound nascent
chainsfoundintheribosomalfractions(Fig.4B,lane9),consistentwith
the results that SSB binds ribosomes in close proximity of the exit site
(20). Notably, although many completed
already released into the soluble fraction (Fig. 4B, lane 1), only newly
made SSB, Sse1p, and an additional polypeptide were observed in the
SSB immunoprecipitation of the supernatant of the centrifugation (Fig.
4B,lane7),suggestingthatSSBassociatespreferentiallywithribosome-
bound nascent chains but less effectively with released full-length
polypeptides.Becauseinvitroexperimentssuggestedthatanothercyto-
solicHsp70,Ssz1p,isrequiredfornascentchainbindingtoSSB(21),we
nextexaminedSSBnascentchainbindingina?ssz1strain.Notably,loss
of Ssz1p did not diminish the proportion of SSB-bound polypeptides
(Fig. 4B, compare lanes 9 and 12; Fig. 4G), indicating that this Hsp70 is
35S-labeled proteins were
FIGURE2.Sse1pformsdistinctcomplexeswithSsa1pandSsb1/2p.Lysatesfromwild
type cells carrying chromosomally tagged Sse1–3HAp were fractionated (fr) by size
exclusionchromatographyonaSuperdex200column(AmershamBiosciences).A,total
elution profile of each Hsp70 chaperone was determined by immunoblot (Imm.) analy-
sis. B, Sse1p complexes with SSA and SSB. Sse1–3HAp immunoprecipitations (IP) with
anti-HAantibodieswereanalyzedbySDS-PAGEfollowedbyWesternblottingwithanti-
bodiesagainstSsa1/2p,Ssb1/2p,theribosomalproteinRpl3p,andtheHAtag.Elutionof
molecular weight standards are indicated above the elution volumes.
FIGURE 3. Genetic interactions between cytosolic Hsp70s. A, relative growth rates of
wild type (WT), ?sse1, ?sse2, and ?sse1?sse2 cells assessed by serial dilution on yeast
extract/peptone/dextrose plates at 30 and 37 °C. B, toxicity of Ssa1p or Sse1p overex-
pression in ?ssb1/2 cells. Overexpression was achieved by induction of copper-regu-
latedSsa1porSse1pinthepresenceof200?MCuSO4ineitherwildtypeor?ssb1/2cells.
The empty backbone vector (pCu) served as a control.
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FIGURE 4. Nascent chain interaction with SSA, SSB, and Sse1p. A, experimental design. Wild type (WT) or mutant cells were pulse-labeled for 1 min with [35S]methionine to
preferentially label nascent chains. Ribosome-bound (R) and soluble (S) nascent chains were separated by ultracentrifugation through a sucrose cushion (C). Chaperone-substrate
interactions were determined after immunoprecipitation (IP) of the various fractions with anti-HA antibodies, as described under “Experimental Procedures.” B and C, binding of
Ssb2ptonascentchainswasassessedinwildtypeversus?ssz1cells(B)andwildtypeversus?sse1cells(C).DandE,bindingofSsa1ptonascentchainswasassessedinwildtypeversus
?ssb1/2cells(D)andwildtypeversus?sse1cells(E).F,bindingofSse1ptonascentchainswasassessedinwildtypeand?ssb1/2cells.Pairedexperimentsforeachpanelweredone
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not required for association of SSB with nascent chains in vivo. How-
ever,thisdoesnotexcludethatSsz1pmayregulateotheraspectsofSSB
function, such as polypeptide release or ATPase activity (22).
Because a substantial proportion of SSB was found in a complex with
Sse1p, we also considered that Sse1p may regulate polypeptide binding
to SSB. Interestingly, we observed a marked increase in the proportion
of SSB-bound nascent chains in ?sse1 cells over that observed in wild
type cells (Fig. 4, C (compare lanes 9 and 12) and G). This suggests
that Sse1p is not required for SSB binding to newly synthesized pro-
teins but instead may assist in SSB-mediated processing of unfolded
polypeptides.
We next examined the contribution of SSA to nascent chain binding
in wild type and chaperone-deleted strains. To facilitate comparison
between experiments, all Hsp70s contained the same epitope tag;
accordingly, all chaperone immunoprecipitations were carried out
using the same antibody on cell lysates containing comparable propor-
tions of35S-labeled proteins. In wild type cells immunoprecipitation of
SSA in the ribosomal fractions revealed an interaction with nascent
chains (Fig. 4D, lane 9), albeit at a much lower level than observed for
SSB. In further contrast to SSB, the immunoprecipitation of SSA in the
soluble fraction (S) revealed an association of this Hsp70 with many
full-length polypeptides released from the ribosome (Fig. 4D, lane 7).
This analysis, thus, suggests that SSA does not bind as efficiently as SSB
to ribosome-bound chains but does associate post-translationally with
newly made proteins. Strikingly, in ?ssb1/2 cells the association of SSA
with nascent chains increased about 10-fold (Fig. 4, C (compare lanes 9
and 12) and G). Therefore, it does appear that SSA can also interact
cotranslationally with a broad spectrum of nascent chains when SSB is
absent. Accordingly, the ribosome-bound SSB may effectively prevent
SSAfromearlycotranslationalbinding.TheseresultssuggestthatSSBis
specifically recruited to the ribosome, whereas SSA is not and instead
interacts post-translationally with newly made proteins.
We next tested whether SSA polypeptide association was affected in
?sse1 cells. Unlike what was observed for SSB, the level of SSA nascent
chainbindingwasnotsubstantiallyalteredin?sse1cellsrelativetowild
type cells (Fig. 4, E (compare lanes 9 and 12) and G). Thus, although
Sse1pisnotrequiredforassociationofnewlymadeproteinstoSSA,this
experiment does not reveal a significant role of Sse1p in the early inter-
action of SSA with ribosome-bound nascent chains.
Finally, we examined the interactions of Sse1p with newly synthe-
sized proteins. Analysis in wild type cells revealed an association of
Sse1p with both ribosome-bound nascent chains (Fig. 4F, lane 9) and
released full-length proteins (Fig. 4F, lane 7), consistent with the idea
that Sse1p functionally interacts with both SSB and SSA. However, the
extent of interaction between newly made polypeptides and Sse1p was
reducedrelativetothatofSSBandSSA.Thiscouldbecausedbyahigher
substrate dissociation rate from this chaperone or could indicate that
the association of Sse1p with substrate is indirect, possibly mediated by
SSA or SSB. A parallel analysis of Sse1p interactions in ?ssb1/2 cells
indicated that Sse1p is not exclusively recruited to translating polypep-
tides by SSB. Thus, Sse1p is still associated with nascent chains and
full-length proteins in ?ssb1/2 cells (Fig. 4F). This association is proba-
bly mediated through a complex with SSA, since SSA takes over most
nascent chain binding in the absence of SSB (Fig. 4D).
TheHsp70ChaperonesSSB,SSA,andSse1pInteractTransientlywith
Newly Translated Polypeptides during Their Biogenesis—Molecular
chaperones have been shown to facilitate de novo folding by transiently
associating with newly synthesized proteins until they reach the native
state (2, 35, 41, 42). To examine the relative contributions of SSB, SSA,
and Sse1p to de novo folding, we analyzed the flux of newly translated
polypeptides through these Hsp70 proteins (Fig. 5). Newly translated
proteinswere35S-labeledwithashortpulse,andchaperoneinteractions
duringfoldingwerefollowedbyachaseinunlabeledmedia.Thekinetics
of transit through different Hsp70 systems was examined by immuno-
precipitation of HA-tagged chaperones.
Immunoprecipitation of SSB-associated polypeptides revealed SSB
binding to a broad spectrum of newly made polypeptides at the earliest
timepoint(Fig.5B,lane1).However,theserapidlydissociatedfromSSB
afteronly1minofchase(Fig.5,B(comparelanes1and2)andE;seealso
Fig. 6A). The diffuse nature of the coimmunoprecipitated polypeptides
in the earliest time point together with the rapid dissociation kinetics is
consistent with our observation that SSB interacts with a large propor-
tion of ribosome-bound polypeptides (Fig. 4). Only few full-length pro-
teins remained associated to SSB during the later chase times and dis-
sociatedwithslowerkinetics(Fig.5B,lanes2–7).Togethertheseresults
indicate that SSB associates with nascent chains and that few proteins
stay bound to SSB after completion of synthesis.
SSAalsoassociatedtransientlywithnewlymadepolypeptides(Fig.5,
C and E). However, the spectrum of SSA-bound polypeptides and the
kinetics of interaction differed from those observed for SSB (Fig. 5C,
lanes 1–7; see also Fig. 6B). Polypeptides dissociated more slowly from
SSA than from SSB, with many proteins still bound after 6 min of chase
(Fig. 5, C (lane 5) and E). In addition, most SSA-bound proteins were
larger than 36 kDa. Given that a single folding domain is ?25–30 kDa,
this suggests most SSA-bound polypeptides are slow folding multido-
mainproteins.Interestingly,similarresultswereobtaineduponanalysis
of protein flux through mammalian Hsp70 (35). A similar pulse-chase
analysismeasuringthefluxofnewlysynthesizedproteinsthroughSse1p
indicated that Sse1p also interacts transiently with newly synthesized
proteins (Fig. 5D, lanes 1–6). Sse1p bound a diffuse smear of proteins
ranging from ?20–200 kDa early in the time course (Fig. 5D, lanes 1
and 2). At later chase times Sse1p interacted with full-length proteins
with dissociation kinetics similar to those observed for SSA (Fig. 5, D
and E).
Sse1p Modulates the Association of SSB and SSA with Newly Synthe-
sized Polypeptides—Our finding that loss of Sse1p altered the interac-
tion between SSB and ribosome-bound nascent chains (Fig. 4) led us to
examine the effect of SSE1 on the flux of nascent chains through SSB
and SSA. Initially, we examined the transit of newly translated polypep-
tides through SSB in both wild type and ?sse1 cells (Fig. 6A). Although
SSB still interacted transiently with newly made proteins in ?sse1 cells,
loss of Sse1p caused more than a 4-fold increase in the fraction of SSB-
bound polypeptides over that bound in wild type cells (Fig. 6A). This
analysis indicates that Sse1p is not required for polypeptide binding to
SSB nor is it essential for polypeptide release. However, Sse1p modu-
latestheSSB-substrateinteractioneitherbyregulatingitsaffinityorthe
rate of release during folding of newly synthesized proteins.
in parallel, and 5% of each fraction was analyzed (Totals) to account for the small variability in the short labeling pulse. To control for nonspecific immunoprecipitation, a parallel
experimentwascarriedoutonwildtypecellscarryingthepCuvectoralone.G,effectofvariousHsp70deletionsontheinteractionsofSSBandSSAwithnascentchains.35S-Labeled
chains bound to either SSB or SSA in the chaperone-deleted and wild type strains were quantified and normalized to the total amount of labeled proteins in the ribosomal fraction
asdescribedunder“ExperimentalProcedures.”Chaperone-bound35S-labeledchainsinthemutantcellsarerepresentedas-folddifferenceoverchainsboundtothesechaperones
in wild type cells. Analysis from B to E presented here is representative of at least three independent experiments. The position of immunoprecipitated newly made35S-labeled
Hsp70s is indicated by arrows. Note that SSA and SSB are not resolved by the gel in F.
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We next examined the effect of SSE1 on SSA-substrate interac-
tions. We reasoned that the very short pulse used in Fig. 4, which
primarily labeled partially synthesized ribosome-bound polypep-
tides, may not have revealed the effects of SSE1 on SSA, which
appears to binds primarily to full-length polypeptides. Instead, the
pulse-chase analysis used to assess the flux of newly made polypep-
tides through Hsp70 uses a longer pulse (2.5 min) that already labels
full-length polypeptides and allows us to observe later effects,
including changes in the rates of polypeptide release. Strikingly,
when we compared polypeptide transit through SSA in wild type and
?sse1cellsweobservedthatSSAassociatedtransientlywithasimilar
spectrum of proteins in wild type and ?sse1 cells, but the level of
association was 3–4-fold higher in cells lacking Sse1p (Fig. 6B).
Thus, the absence of Sse1p increased the proportion of newly made
polypeptides associated to SSA, as observed for SSB. These experi-
ments indicate that Sse1p enhances substrate binding to both SSB
and SSA by comparable levels without noticeably affecting the spec-
ificity of the Hsp70s for their client proteins. These findings are
consistent with our observations that Sse1p is not required to recruit
these Hsp70s to bind polypeptide substrates (Fig. 4, C and E) and
indicate that Sse1p modulates the activity and substrate interactions
of both Hsp70s SSA and SSB by a similar mechanism of action.
FIGURE 5. Newly synthesized proteins transit with different kinetics through the cytosolic Hsp70s SSB, SSA, and Sse1p. A, experimental design. Cells expressing HA-tagged
chaperones were pulse-labeled with [35S]methionine, chased with cold methionine for the indicated time points, and analyzed as described under “Experimental Procedures.”
Chaperone-substrateinteractionsattheindicatedchasetimesweredeterminedafterimmunoprecipitationwithanti-HAantibodies.Controlimmunoprecipitationswerecarriedout
with a nonimmune antibody. B–D, the flux of newly translated proteins through SSB (B), SSA (C), and Sse1p (D) was assessed in wild type cells. NI, nonimmune antibody. The total
fraction (Tot, lane 8) represents 5% of the total labeled sample at 0 min of chase and highlights the reproducibility of35S labeling between experiments. E, quantification of the flux
of newly translated proteins through the different Hsp70s. Results are representative of at least three independent experiments.
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DISCUSSION
In this study we characterize the interplay of the major cytosolic
Hsp70 systems during de novo folding. We find that most of the cyto-
solic pool of the yeast Hsp110 homologue Sse1p is found in complexes
with either of the two major cytosolic Hsp70s, SSB and SSA (Figs. 1 and
2). Consistent with these findings, non-denaturing PAGE analysis by
Shaneretal.(Ref.49,thecompanionstudy)alsoindicatesthatcytosolic
Sse1pisfoundexclusivelyincomplexeswitheitherSSAorSSB.It,thus,
appears that the cellular role of Sse1p is functionally linked to these
Hsp70s. The severe growth defect of ?sse1 cells indicates that the
Hsp110 Sse1p plays an important role regulating the function of both
Hsp70s (Fig. 3A). The idea of a functional interplay between cytosolic
Hsp110s and the Hsp70s SSB and SSA is further supported by our find-
ings that Sse1p or Ssa1p overexpression is toxic in ?ssb1/2 cells (Fig.
3B). All three Hsp70s, SSB, SSA, and Sse1p, transiently associate with
newly made proteins (Fig. 5), which suggests a role in de novo folding.
SSB appears to interact primarily with ribosome-bound chains,
although SSA instead associates mostly post-translationally with newly
made proteins (Fig. 4). Although Sse1p is not essential for polypeptide
binding by either SSB or SSA (Figs. 4 and 6), we find that polypeptide
association to the Hsp70s SSA and SSB is considerably enhanced in
?sse1 cells. Accordingly, we propose that Sse1p functions to modulate
the activities of SSA and SSB during folding of newly made proteins.
Distinct Roles of Cytosolic Hsp70s in de Novo Folding—We find that
the three cytosolic Hsp70s, SSA, SSB, and Sse1p, transiently associate
withnewlysynthesizedpolypeptides(Fig.5).Notably,thedistinctspec-
trum and kinetics of polypeptide flux through these Hsp70s suggest
distinct functions in de novo folding. The predominant association of
SSB with ribosome-bound nascent chains (Fig. 4B) together with the
rapid kinetics of polypeptide dissociation from SSB (Fig. 5B) suggest
that the primary function of this Hsp70 occurs early during synthesis
and is probably to protect emerging ribosome-bound nascent chains
(Fig. 6). However, SSB function can be partially replaced by SSA (Fig.
4D) or by other chaperone systems (43). This may explain why full-
length proteins such as ornithine transcarbamoylase or von Hippel-
Lindau tumor suppressor protein are observed to need SSA for folding
(16, 17), whereas, no proteins have been shown to require SSB. On the
other hand, SSA associates with few ribosome-bound nascent chains
andbindspredominantlypolypeptidesreleasedfromtheribosome(Fig.
4D). Notably, SSA can switch to a cotranslational mode of interaction
when SSB is absent, suggesting this chaperone may block access of SSA
to the ribosome exit site (Fig. 4D). In pulse-chase analysis the spectrum
of SSA-bound polypeptides consists mostly of full-length proteins of
greater than 40 kDa; these polypeptides dissociate from SSA with sig-
nificantly slower kinetics than for SSB (Fig. 5E). Sse1p also associates
with both ribosome-bound nascent chains and with full-length
FIGURE6.Sse1pmodulatesthefluxofnewlysynthesizedproteinsthroughSSAandSSB.TodeterminetheeffectofSSE1ontheassociationofnewlysynthesizedpolypeptides
withSSBandSSA,wildtype(WT)and?sse1cellsexpressingHA-SSB2(A)orHA-SSA1(B)werepulse-labeledwith[35S]methionine,andchaperone-substrateinteractionswereanalyzed
and quantified as in Fig. 5. NI, control immunoprecipitations (IP) with a nonimmune antibody. Chaperone-bound polypeptides were quantified as described under “Experimental
Procedures” and expressed as a -fold difference over SSB- or SSA-bound polypeptides at 0 min in wild type cells.
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polypeptides (Figs. 4F and 5E). As discussed below, it is unclear at this
point whether the interaction is direct or only mediated by its associa-
tion with either SSB or SSA.
Our data argue for a sequential pathway of interactions of different
Hsp70s with newly made polypeptides. It is likely that the early interac-
tionofSSBwithmostnascentchainsismediatedbyitsdirectaffinityfor
the ribosome (20). This interaction may suffice to fold smaller proteins,
whereas larger multidomain proteins may require SSA to prevent fold-
ingintermediatesfromengagingininter-molecular“domainswapping”
that may lead to aggregation. Although early interaction with SSB is
clearly optimal for folding, as evidenced by the slow growth phenotype
of ?ssb1/2 cells, the sequential pathway is not obligate because SSA can
interact with nascent chains when SSB is deleted.
The Hsp110 Sse1 Regulates Hsp70-Substrate Interactions—We find
that Sse1p binds to two different cytosolic Hsp70s and regulates their
interactionswithsubstrates.OurfindingthatbothSSBandSSAhavean
enhanced association with newly made polypeptides in the absence of
Sse1p raises several possible mechanisms for Hsp110-mediated regula-
tion of Hsp70s. One option is that the Sse1p/Hsp70 heterodimer has
reduced affinity or a slower binding kinetics for polypeptide binding
than Hsp70 homodimer complexes. However, this scenario is unlikely
given the large excess of SSB and SSA over Sse1p in the cytosol (4- and
9-fold, respectively (37)). Because even in wild type cells most SSB
and/or SSA are not in a heterodimer with Sse1p and would be available
to bind polypeptides, a lower substrate affinity or slower on-rate Sse1p-
containing complex would be unable to compete effectively for
polypeptide binding. More plausibly, Sse1p may modulate the Hsp70-
substrate interaction once it is established. For instance, it may cooper-
ate with SSA and SSB to promote folding such that folded polypeptides
would not return to Hsp70s for binding. Alternatively, Sse1p may act
catalyticallytoregulatethereleaseofsubstratefromSSBandSSA,based
on the reported effects of Hsp110s on Hsp70 ATPase activity. These
possibilities are not mutually exclusive, and it is conceivable that more
than one describes the function of Hsp110s in the cell.
Because ATP binding and hydrolysis are essential to regulate Hsp70
substratebindingandrelease,ourfindingsontheroleofSse1presonate
withstudiessuggestingthatHsp110scanregulateHsp70ATPaseactiv-
ity.Forinstance,intheendoplasmicreticulum,adistantHsp110homo-
logue, Lhs1p, stimulates the ATPase activity of the endoplasmic reticu-
lum Hsp70 Kar2p by acting as a nucleotide exchange factor, whereas
Kar2p coordinately regulates Lhs1p activity by stimulating its ATPase
(30). Interestingly, mammalian Hsp110 has been reported to inhibit the
ATPase activity of nucleotide-loaded Hsp70 (29), which could be con-
sistentwithanHsp110nucleotideexchangeactivity.Inaddition,Shaner
et al. (49) report that Sse1p can stimulate the ATPase of SSA in con-
junctionwithitsJ-proteinYdj1.BecausenucleotideexchangebyHsp70
promotes polypeptide release, the suggestion that Sse1p may act as a
nucleotide exchange factor is consistent with our functional analysis.
Indeed, the enhanced association of newly made proteins with SSA and
SSB in ?sse1 cells (Fig. 6) is consistent with the idea that Sse1p could
function as a nucleotide exchange factor that causes substrate release.
A Pathway of Hsp70 Interactions during de Novo Folding—Previous
studies showing that different chaperone systems act sequentially to
promote folding in the cell focused on the cooperation of Hsp70 with
other classes of chaperones such as chaperonins, Hsp90 or Hsp104 (4,
17, 44, 45). Remarkably, our data suggest sequential cooperation
between different types of Hsp70s whereby SSB interacts first with nas-
cent chains as they emerge from the ribosome, whereas SSA interacts
later with a more restricted subset of larger proteins (Fig. 7). What may
be the function of such a pathway? Coupling of SSB binding to transla-
tion may protect newly synthesized proteins from the bulk cytosol (35,
43), although post-translational transfer of larger proteins to SSA may
facilitatethefinalstagesoffolding.Sse1pappearstomodulatebothSSA
and SSB activity. Our results indicate that neither SSB nor Sse1p is
requiredforSSAassociationwithnewlysynthesizedpolypeptides(Figs.
4Dand6B),althoughitisinprinciplepossiblethatSse1pstimulatesthe
handover of polypeptides from SSB to SSA. Alternatively, Sse1p may
promote polypeptide release and facilitate the transfer of SSA-bound
polypeptides to more downstream chaperone systems.
Notably,ourfindingsofsequentialcooperationbetweenSSBandSSA
bear striking parallels to the Escherichia coli chaperones trigger factor
and the Hsp70 DnaK (41, 42). In E. coli, the trigger factor binds directly
totheribosomewhereitrecognizesshorternascentchains,whereasthe
Hsp70 DnaK binds mostly post-translationally to larger proteins. Fur-
thermore, the substrates of DnaK are also predominantly large proteins
that probably have more than one folding domain (41, 42), similar to
whatisobservedforSSA-Hsp70(Fig.5)andformammalianHsp70(35).
Therefore, two structurally different chaperones, trigger factor and
DnaK, stabilize and fold newly synthesized proteins in E. coli. In con-
trast, in the eukaryotic cytosol these functions appear to involve the
cooperation of different Hsp70s. This feature of eukaryotes may afford
thepossibilityofregulatedbindingandrelease,asHsp70sareinfluenced
by various cofactors, such as J-domain and nucleotide exchange factors
(2, 7, 10, 46). Thus, substrate binding to SSA is regulated by the J-do-
main proteins Ydj1p and Sis1p, whereas substrate binding to SSB is
thoughtoberegulatedbytheJ-domainproteinZuo1p.Ourobservation
thatsubstrateinteractionswithbothSSBandSSAisinturnregulatedby
anotherHsp70-likeprotein,theHsp110Sse1p,mayaddanotherlayerof
complexity and regulation to de novo folding in the eukaryotic cytosol.
Interestingly, there are no identified homologues of SSB in mammalian
cells. Instead, Hsp70 binds to polypeptides both co- and post-transla-
tionally (4), while probably also interacting with Hsp110 (39). Addi-
FIGURE 7. A model for sequential chaperone interactions during de novo folding.
SSB and SSA form complexes with Sse1p, where Sse1p may have a role in modulating
Hsp70activity.Thesechaperonesystemstogethercancreateanetworkforthefoldingof
newly synthesized proteins. Ribosome-associated SSB Hsp70 has first contact with the
emergingnascentchain.Restrictedsubsetsofslowfoldingpolypeptidesmaytheninter-
act co- or post-translationally with downstream chaperones such as the Hsp70-SSA or
theTRiC/CCT(chaperonintaillesscomplexpolypeptide1(TCP1)ringcomplex/chapero-
nin containing TCP1).
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tional regulatory features are probably also conserved in mammalian
cells in light of the recent characterization of the mammalian Zuo1p
homologue (47, 48). Perhaps the functions of SSB and SSA are encom-
passedbythemammalianHsp/Hsc70homologues,raisingthepossibil-
ity of a handover between ribosome-bound Hsp70 and soluble Hsp70.
Acknowledgments—We thank Drs. Douglas Cyr, Elizabeth Craig, Ron Davis,
JonathanWarner,andKevinMoranoforgenerouscontributionsofantibodies,
strains, and/or plasmids. We also thank members of the Frydman laboratory
and Raul Andino for discussions and comments on the manuscript.
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