The Hsp90 Cochaperones Cpr6, Cpr7, and Cns1 Interact with theIntact Ribosome

Victoria R. Tenge, Abbey D. Zuehlke,* Neelima Shrestha, Jill L. Johnson

Department of Biological Sciences, University of Idaho, Moscow, Idaho, USA

The abundant molecular chaperone Hsp90 is essential for the folding and stabilization of hundreds of distinct client proteins.Hsp90 is assisted by multiple cochaperones that modulate Hsp90’s ATPase activity and/or promote client interaction, but the invivo functions of many of these cochaperones are largely unknown. We found that Cpr6, Cpr7, and Cns1 interact with the intactribosome and that Saccharomyces cerevisiae lacking CPR7 or containing mutations in CNS1 exhibited sensitivity to the transla-tion inhibitor hygromycin. Cpr6 contains a peptidyl-prolyl isomerase (PPIase) domain and a tetratricopeptide repeat (TPR) do-main flanked by charged regions. Truncation or alteration of basic residues near the carboxy terminus of Cpr6 disrupted ribo-some interaction. Cns1 contains an amino-terminal TPR domain and a poorly characterized carboxy-terminal domain. Theisolated carboxy-terminal domain was able to interact with the ribosome. Although loss of CPR6 does not cause noticeablegrowth defects, overexpression of CPR6 results in enhanced growth defects in cells expressing the temperature-sensitive cns1-G90D mutation (the G-to-D change at position 90 encoded by cns1). Cpr6 mutants that exhibit reduced ribosome interactionfailed to cause growth defects, indicating that ribosome interaction is required for in vivo functions of Cpr6. Together, theseresults represent a novel link between the Hsp90 molecular-chaperone machine and protein synthesis.

Molecular chaperones assist in the folding of other proteinsduring synthesis, as well as upon denaturation or misfold-

ing. Prior results suggest that one type of molecular chaperoneassists in folding of newly translated proteins, while a different setof molecular chaperones help proteins fold after denaturation (1).The abundant essential chaperone Hsp90 does not associate withnascent chains and does not appear to have a general role in pro-tein folding. Instead, Hsp90 is required for the folding and activa-tion of a specific subset of cellular proteins (2–4). Although it isunlikely that it plays a general role in ribosomal function, Hsp90has been shown to be involved in polysome stability and to havephysical or genetic interactions with select proteins with ribo-somal or preribosomal functions (5–8).

In Saccharomyces cerevisiae, Hsp90 is assisted by over 10 co-chaperones that target clients to Hsp90; directly or indirectlymodulate Hsp90 ATPase activity; or have other, less defined func-tions (9). Many of the cochaperones contain tetratricopeptide re-peat (TPR) domains and compete for binding to the conservedcarboxy-terminal MEEVD sequence of Hsp90. Three of the TPR-containing cochaperones are Cpr6, Cpr7, and Cns1. Loss of CPR6does not cause noticeable growth defects; loss of CPR7 results inslow, temperature-sensitive growth; and CNS1 is essential for vi-ability (10–12). Cpr6 and Cpr7 share 38% amino acid identity yetexhibit different functions in vivo and in vitro (13–16). In partic-ular, overexpression of CPR7 rescues the temperature-sensitivedefect of cns1-G90D (the G-to-D change at position 90 encoded bycns1) cells (17), while overexpression of CPR6 exacerbates thegrowth defects (18). Further, overexpression of CNS1, but notCPR6, was able to rescue growth defects of cpr7 cells (11–13).Yeast chaperones have been characterized as chaperones linked toprotein synthesis (CLIPS) and heat shock proteins (HSPs). Tran-scription of HSPs is induced by multiple environmental stressors,while transcription of CLIPS is repressed under the same condi-tions. The pattern of transcriptional activation of CPR6 was verysimilar to that of HSP82, one of two Hsp90 genes in yeast, as wellas genes encoding the Sti1 and Sba1 cochaperones (1). However,

Cpr6 and/or Cpr7 was previously shown to associate with trans-lating ribosomes, and loss of CPR7 conferred hypersensitivity tothe translation inhibitor hygromycin (1), suggesting a potentiallink between Hsp90 cochaperones and the ribosome.

We previously demonstrated that isolation of Cpr6 results incopurification of Hsp90, Hsp70, and Ura2, an Hsp90 client pro-tein required for pyrimidine biosynthesis (14). In this study, wefound that isolation of Cpr6 also results in copurification of com-ponents of both the large and small ribosomal subunits. Further,select mutations within the TPR domain that disrupt Cpr6 inter-action with Hsp90 stabilized ribosome interaction. Cns1 and Cpr7also interacted with the ribosome, although detection of Cpr7interaction required the presence of the stabilizing mutation in theTPR domain. Although the functional significance of the interac-tion remains unclear, our results indicate a novel link betweenHsp90 cochaperones and the intact ribosome.

MATERIALS AND METHODSMedia, chemicals, and antibodies. Yeast cells were grown in either YPD(yeast extract-peptone-dextrose) or defined synthetic complete mediumsupplemented with 2% dextrose. Growth was examined by spotting 10-fold serial dilutions of yeast cultures onto the appropriate media, followed

Received 16 July 2014 Accepted 5 November 2014

Accepted manuscript posted online 7 November 2014

Citation Tenge VR, Zuehlke AD, Shrestha N, Johnson JL. 2015. The Hsp90cochaperones Cpr6, Cpr7, and Cns1 interact with the intact ribosome. EukaryotCell 14:55–63. doi:10.1128/EC.00170-14.

Address correspondence to Jill L. Johnson, jilljohn@uidaho.edu.

* Present address: Abbey D. Zuehlke, Urologic Oncology Branch, Center for CancerResearch, National Cancer Institute, Bethesda, Maryland, USA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.00170-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/EC.00170-14

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by incubation for 2 days at the indicated temperature. 5-Fluoroorotic acid(5-FOA) was obtained from Toronto Research Chemicals. Anti-Zuo1 an-tiserum was a gift from Elizabeth Craig (University of Wisconsin—Mad-ison) (19). Hygromycin B was obtained from Sigma. Anti-TAP antibodywas obtained from Pierce.

Yeast strains. Cpr6-ribosome interaction was observed in three dif-ferent strain backgrounds: S288C, W303, and BY4741. S288C strains ex-pressing RPS0A-TAP or RPL8A-TAP were obtained from Open Biosys-tems (20). Wild-type (WT), ssz1, zuo1, egd1, egd2, and btt1 strains in theBY4741 background were also from Open Biosystems. Strains JJ762(WT), JJ1138 (cpr6), JJ110 (cpr6 hsc82 hsp82/YEp24HSP82), JJ1093(ura2), and JJ21 (cns1::TRP1/YCp50-CNS1), JJ1115 (cpr7), and JJ816(hsc82 hsp82/YEp24HSP82), which are isogenic to W303, have been de-scribed previously (13, 14, 18, 21).

Plasmids. pRS416GPDHis-Cpr6, pRS414GPDHis-Cpr6, pRS415GPDHis-Sti1, pRS416GPDHis-Cpr7 (WT and Cpr7 193–393), pRS416GPDHis-Cns1, YEP24-CNS1, and pRS317-CNS1 (WT and mutant) werepreviously described (13, 14, 18, 22). Plasmids expressing untagged WT ormutant untagged Hsp90 (the Hsp82 isoform) (pRS314 HSP82 WT or�MEEVD) were transformed into the indicated cpr6 hsc82 hsp82 strainharboring the YEp24-HSP82 plasmid. Transformants were grown in thepresence of 5-FOA to lose the YEp24-HSP82 plasmid. To assess hygromy-cin sensitivity, His-tagged WT or mutant Hsc82 (pRS313GPDHis-HSC82) was transformed into JJ816, and the YEp24-HSP82 plasmid wascured by plating in the presence of 5-FOA. SBA1 was cloned intopRS416GPDHis using engineered BamHI and EcoRI sites. pRS416GPDHis-Zuo1 was a gift from Elizabeth Craig (University of Wisconsin).The plasmid expressing His-Hsp82 WT was a gift from Len Neckers (Na-tional Cancer Institute). Amino acid mutations were constructed usingsite-directed mutagenesis. All mutations were confirmed using DNA se-quencing. The mutagenic oligonucleotide sequences are available uponrequest.

Isolation of His-Cpr6 or other His-tagged complexes. Briefly, cellswere grown overnight and harvested at an optical density at 600 nm(OD600) of 1.2 to 2.0. The cell pellets were resuspended in lysis buffer (20mM Tris, pH 7.5, 100 mM KCl [or other concentrations of KCl as noted],5 mM MgCl2, 5 mM imidazole containing a protease inhibitor tablet[Roche Applied Science]) and were disrupted in the presence of glassbeads with 8 30-s pulses. The cell lysates were incubated with nickel resin(1 h with rocking; 4°C), followed by washes with lysis buffer plus 35 mMimidazole and 0.1% Tween 20. Proteins were eluted from the nickel resinby boiling in SDS-PAGE sample buffer, and protein complexes were sep-arated by gel electrophoresis (generally 12.5% acrylamide), followed byCoomassie blue staining. Alternatively, proteins were transferred to nitro-cellulose and chemiluminescence immunoblots were performed accord-ing to the manufacture’s suggestions (Pierce, Rockford, IL).

Mass spectrometry fingerprint analysis. Ribosomal proteins wereidentified at the Environmental Biotechnology Institute at the Universityof Idaho. The analysis of peptides was done using reverse-phase liquidchromatography on a Waters nanoAcquity Ultra Performance LiquidChromatograph (UPLC). This was followed by tandem mass spectrome-try (MS-MS) using a Waters Micromass Q-Tof Premier quadrupole-timeof flight mass spectrometer using a nanospray electrospray ionizationinlet. The smear of proteins in the 15- to 40-kDa range was excised fromthe Coomassie-stained gel and dried using acetonitrile. Sequence gradetrypsin (Sigma-Aldrich) was used to digest the bands at 37°C for 20 h.Peptides were extracted three times using 100 mM ammonium bicarbon-ate for one extraction and 50% acetonitrile containing 5% formic acid forthe other two extractions. The extracts were evaporated to complete dry-ness and concentrated in 20 �l 5% acetonitrile and 0.1% formic acid.Peptides were identified using the MASCOT program with a peptide andMS-MS tolerance of 0.2 Da. The complete list of proteins with scores over25 is in Table S1 in the supplemental material.

RESULTSThe Hsp90 cochaperone Cpr6 binds the intact ribosome. Wepreviously showed that purification of the Hsp90 cochaperoneCpr6 from yeast results in the copurification of Hsp90, Hsp70 (Ssafamily), and the Hsp90 client Ura2 (14). Basic residues in the TPRdomain of Cpr6 interact with carboxy-terminal EEVD residues ofHsp70 and/or Hsp90 in what has been termed a carboxylate clamp(23). The crystal structure of bovine Cyp40, the homolog of Cpr6,is available, and prior analysis identified amino acids in the pre-dicted EEVD-binding groove of Cyp40 that were important forHsp90 and Hsp70 binding (24–26). Mutation of a Cpr6 residuepredicted to contact the EEVD sequences (K309) disrupted Hsp70(Ssa family) and Hsp90 (Hsc82 and Hsp82) interaction withoutdisrupting Cpr6-Ura2 interaction (14).

Because the TPR domain plus flanking regions of Cpr6 wasrequired for interaction with Ura2, Hsp90, and Hsp70 (14), weexamined the impact of alanine substitution for additional resi-dues within the TPR domain. In agreement with a prior study(25), the K223A, F230A, L281A, and K309A alterations disruptedHsp90 and Hsp70 interaction, and K231A resulted in reducedinteraction (Fig. 1A). In contrast, alteration of residue E300,which is outside the predicted EEVD-binding groove, had little orno effect. None of the mutations disrupted the Cpr6-Ura2 inter-action. Surprisingly, isolation of His–Cpr6-F230A also resulted incopurification of multiple proteins in the 15- to 40-kDa range.Reduced levels of the same proteins were observed in cells express-ing K309A. Mass spectrometry analysis identified those proteinsas components of both the large and small ribosomal subunits(Fig. 1B; see Table S1 in the supplemental material). Zuo1 is achaperone that binds the large subunit near the exit tunnel (19,27). An immunoblot confirmed a low level of Zuo1 interactionwith WT Cpr6 and enhanced interaction with Cpr6-F230A and-K309A.

We next isolated His-Cpr6 from a commercially availablestrain expressing TAP-tagged RPS0A, a component of the smallsubunit, from the normal chromosomal location (20). As shownin Fig. 2A, low levels of RPS0A were present in WT His-Cpr6complexes, whereas greatly enhanced levels copurified with Cpr6-F230A. Similar results were obtained in a strain expressingRPL8A-TAP, a component of the large subunit (data not shown;

FIG 1 Cpr6 alteration affects Hsp90, Hsp70, and ribosome interaction. (A)WT or mutant His-Cpr6 complexes were isolated from �cpr6 strain JJ1138 inbuffer containing 100 mM KCl. Proteins eluted from the nickel resin wereanalyzed using Coomassie blue staining (top) or immunoblot analysis (bot-tom). The bands marked with a bracket are unidentified proteins that bindnonspecifically to nickel resin used as internal loading controls. Lane �, cellscontained empty vector. (B) Mass spectrometry analysis identified multiplecomponents of the large and small ribosomal subunits.

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see Fig. 7B). Reducing the salt concentration in the lysis bufferresulted in similar levels of ribosome interaction with Cpr6 WTand -F230A (Fig. 2B). Together, these results indicate that Cpr6WT and -F230A interact with the assembled 80S ribosome in bothW303 and S288C backgrounds.

The results shown in Fig. 2A and B suggest that WT Cpr6 isloosely bound to intact ribosomes, dissociating above 10 mM KCl.We examined the effect of increasing salt on Cpr6-F230A–ribo-some interaction. As shown in Fig. 2C, Cpr6-F230A dissociatedfrom the ribosome at concentrations over 100 mM KCl. Similarresults were obtained in strain JJ1138 (not shown). The reasonthat the F230A mutation stabilizes ribosome interaction againstincreasing salt concentrations remains unknown. For compari-son, interaction of the Hsp70 Ssb1 with the ribosome is very sta-ble, persisting in the presence of 1 M KCl (19). Surprisingly, theCpr6-Ura2 interaction was not disrupted by increasing salt con-centrations.

We conducted additional experiments to determine whetherCpr6-ribosome interaction was regulated by Hsp90, other ri-bosome-associated chaperones, or Ura2. The terminal MEEVDresidues of Hsp90 are critical for stable interaction with Sti1,Ppt1, Cpr6, and Cpr7 (9). Hsp82�MEEVD and WT Hsp82exhibit similar steady-state levels (14, 28), and cells expressingHsp82�MEEVD as the only Hsp90 in the cell do not exhibit anyobvious growth defects. Since ribosome interaction was stabilizedupon alteration of residues in the TPR-binding groove, we examinedthe impact of deletion of the MEEVD sequence. Although Cpr6 failedto stably interact with Hsp82�MEEVD, ribosome interaction wasnot affected (Fig. 3A).

Yeast contains two ribosome-tethered chaperone complexes,RAC and NAC, which interact with nascent chains (29). We testedthe possibility that Cpr6 loosely binds to RAC or NAC compo-nents in order to facilitate interaction with newly synthetized pro-teins. As shown in Fig. 3B, deletion of individual components ofRAC (SSZ1 and ZUO1) or NAC (EDG1, EDG2, and BTT1) hadlittle or no effect on Cpr6-F230A interaction with the ribosome.Similar effects were observed with WT Cpr6 (not shown). SinceCpr6 appears to directly interact with Ura2, yet another possibilitywas that Cpr6 bound nascent Ura2, resulting in coisolation of theribosome. Although the interaction of WT Cpr6 with the ribo-some was slightly reduced, ribosome association of Cpr6-F230Awas unaltered in cells that lack URA2 (Fig. 3C). Together, theseresults indicate that Cpr6-ribosome interaction is not dependenton Hsp90 and Ura2 interaction and that it is unlikely that RAC orNAC mediates ribosome interaction.

Basic amino acids in the carboxy terminus of Cpr6 are re-quired for ribosome interaction. We took advantage of availabletruncated forms of His-Cpr6 (Fig. 4A) (14) to determine whichpart of Cpr6 interacts with the ribosome. As shown in Fig. 4B,Cpr6 171–371, but not Cpr6 1–212, bound Hsp90, Hsp70, andUra2, as well as WT Cpr6. Cpr6 1–358 bound Hsp90 at a reducedlevel. None of the truncated constructs bound the ribosome aswell as intact Cpr6. We introduced the F230A alteration to facili-tate narrowing down the interacting region. As shown in Fig. 4C,Cpr6 171–371:F230A was able to interact with the ribosome, butCpr6 1–358:F230A was not. This suggests that the ribosome-bind-

FIG 2 Effect of altered KCl concentration on Cpr6-ribosome interaction. (A)His-Cpr6 complexes were isolated from a strain expressing RPS0A-TAP fromthe endogenous chromosomal location. RPS0A-TAP bound to resin was rec-ognized by an anti-TAP antibody. (B) As in panel A, except that 10 mM KClwas used in the lysis and wash buffers instead of 100 mM KCl. (C) His–Cpr6-F230A complexes were isolated from the RPS0A-TAP strain lysed in the pres-ence of the indicated concentrations of KCl. Lanes �, cells contained emptyvector.

FIG 3 Cpr6-ribosome interaction was unaffected by mutation of Hsp90 or deletion of genes encoding ribosome-associated chaperones or Ura2. (A) His-Cpr6complexes were isolated from a cpr6 hsc82 hsp82 strain expressing WT Hsp82 or Hsp82�MEEVD. (B) His-Cpr6 WT or -F230A complexes were isolated fromstrain JJ1138 (�cpr6), BY4741, or BY4741 containing deletions in SSZ1, ZUO1, EDG1, EDG2, or BTT1. (C) His-Cpr6 complexes were isolated from strain JJ762(WT) or JJ1093 (ura2::HIS3). Lanes �, cells contained empty vector.

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ing site is located near the carboxy terminus of Cpr6. Of note,Cpr6 1–358 bound Hsp90 better under low-salt conditions (Fig.4B) than under higher-salt conditions (Fig. 4C).

An earlier study showed that Cpr6 and/or Cpr7 specificallycomigrated with polysomes, but the antibody used in those exper-iments did not distinguish between Cpr6 and Cpr7 (1). As previ-ously shown, isolation of His-Cpr7 does not result in isolation ofHsp90 or Ura2 (14). Cpr7 also failed to interact with the ribosomeunless it contained the F251A alteration that is homologous toCpr6-F230A (Fig. 4D). This also indicates that Cpr6 and Cpr7 aresimilarly affected by alteration of a homologous residue in theTPR domain.

Cpr6 1–358, which contains a deletion of the terminal 13

amino acid residues (aa) of Cpr6, exhibited reduced ribosomeinteraction. Eight of the last 20 amino acids of Cpr6 are lysine orarginine residues. As shown in Fig. 5A, only some of them areconserved in Cpr7. We tested the impact of mutation of basicresidues on ribosome interaction in the context of WT Cpr6 (Fig.5B) or Cpr6-F230A (Fig. 5C). Either changing the three lysines inthe KAKK sequence to alanines (AAAA) or reversing the chargesof the two adjacent lysine residues (KAEE) reduced ribosome in-teraction without affecting Ura2, Hsp90, or Hsp70 interaction.The DMFS mutant, which reverses the charge of the conservedlysine closest to the carboxy terminus, had little effect in the con-text of WT Cpr6. However, all three mutations disrupted ribo-some interaction under the more stringent conditions (Fig. 5C).

FIG 4 The carboxy terminus of Cpr6 is required for ribosome interaction. (A) Schematic of WT and truncated Cpr6 constructs. (B) His-Cpr6 complexes wereisolated from the RPS0A-TAP strain in the presence of 10 mM KCl. (C) Full-length or truncated His-Cpr6 complexes were isolated from the cpr6 strain in thepresence of 100 mM KCl. (D) The indicated His-tagged Cpr6 or Cpr7 constructs were isolated from the RPS0A-TAP strain in the presence of 10 mM KCl. Lanes�, cells contained empty vector.

FIG 5 Identification of basic residues required for ribosome interaction. (A) Sequences of the carboxy termini of Cpr7 and Cpr6 with targeted residues of Cpr6underlined. (B) His-Cpr6 containing the indicated mutations in basic residues isolated from the RPS0A-TAP strain in the presence of 10 mM KCl. (C)His–Cpr6-F230A cells containing additional mutations in basic residues were isolated from the the RPS0A-TAP strain in the presence of 100 mM KCl. Lanes �,cells contained empty vector.

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We recently showed that overexpression of CPR6 results innegative growth in cells expressing a temperature-sensitive muta-tion in CNS1, cns1-G90D (18). We examined whether there is acorrelation between ribosome interaction and ability to cause neg-ative growth. As shown in Fig. 6A, cpr6-F230A and cpr6 1-358failed to cause negative growth in cns1-G90D cells. Similar effectswere observed with the cpr6-KAEE and -DMFS mutants. This in-dicates that disruption of either Hsp90 or ribosome interaction isable to relieve the negative effects of CPR6 overexpression. We alsoexamined whether the peptidyl-prolyl isomerase (PPIase) domainwas required for Cpr6 to cause negative growth. However, Cpr6171–371 and 212–371, both of which lack the PPIase domain,caused a strong negative effect (Fig. 6B).

Cns1 also interacts with the ribosome. Since the ability ofCpr6 to cause a growth defect in cns1-G90D cells is linked to ribo-some interaction, we determined whether Cns1 also interacts withthe ribosome. As shown in Fig. 7A, isolation of His-Cns1 results incopurification of Hsp90. His-Cns1 also interacts with the ribo-some, as judged by gel staining and copurification of RPS0A. Wenext examined the abilities of altered forms of Cns1 to interactwith the ribosome. Cns1-G90D, which contains an alteration in aconserved residue of the TPR domain (30), exhibited reducedHsp90 interaction but maintained ribosome interaction. In con-trast, Cns1 1–212, which contains the TPR domain plus the flank-ing region, interacted with Hsp90 but not the ribosome. Cns1213–385, which lacks the TPR domain, interacted with the ribo-some. We further examined whether Hsp90, Sti1, or Sba1 inter-acted with the ribosome. For comparison, we included the knownribosome-associated chaperone Zuo1. As expected, His-Sti1bound Hsp90 and Hsp70. His-Sba1 interaction with Hsp90 wasnot observed due to the lack of ATP (9). Similar levels of Cpr6,Sti1, Sba1, Hsp82, and Zuo1 bound nickel resin (Fig. 7B). How-ever, only Cpr6 and Zuo1 showed robust ribosome interaction.

Cells containing deletions of ribosome-associated chaperonesare sensitive to hygromycin, a translation inhibitor (31). A priorstudy showed that, unlike cells lacking HSP82, STI1, or CPR6, cellslacking CPR7 were sensitive to hygromycin (1). As shown in Fig.8A, cpr7 cells exhibited slow growth at all three temperatures andwere unable to grow in the presence of hygromycin. Transforma-tion with WT CPR7, cpr7 193-393 (analogous to Cpr6 171–371)(13), or cpr7-F251A, rescued all the growth defects. This is consis-tent with a prior report that the PPIase domain of Cpr7 is dispens-able for most known functions (32). The cns1 1-121 andcns1-G90D mutations were previously known to cause tempera-ture-sensitive growth (17). Both mutations also caused stronggrowth defects in the presence of hygromycin, indicating that theability of Cns1 to interact with both Hsp90 and the ribosome isrequired for growth in the presence of the drug. Cells lackingCPR6 were not sensitive to hygromycin under these conditions(not shown).

To assess whether Hsp90 is involved in the ribosomal functionsof cochaperones, we examined previously characterized hsc82

FIG 6 Effect of CPR6 mutation on growth of cns1-G90D cells. (A and B) StrainJJ21 (cns1::TRP1) expressing cns1-G90D under its own promoter was trans-formed with plasmids expressing WT or mutant His-Cpr6 or empty vector(�). After overnight growth in selective media, 10-fold serial dilutions wereplated and grown for 2 days at the indicated temperatures.

FIG 7 Cns1 also interacts with the ribosome. (A) WT or mutant His-Cns1complexes were isolated from the RPS0A-TAP strain in the presence of 10 mMKCl. (B) The indicated His-tagged protein complexes were isolated from theRPL8A-TAP strain in the presence of 10 mM KCl. Lanes �, cells containedempty vector.

FIG 8 Growth defects of cochaperone and Hsp90 mutant cells. (A) cpr7(JJ1115) cells expressing WT or mutant His-tagged Cpr7 or JJ21 (cns1::TRP1)cells expressing WT or mutant CNS1 under its own promoter were grown inrich media for 2 days at the indicated temperatures. YPD plates containing 60mM hygromycin B were grown for 2 days at 30°C. (B) As in panel A, except aplasmid expressing WT or mutant His-tagged HSC82 was expressed in hsc82hsp82 cells (JJ816).

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mutations (6, 21). When expressed as the only Hsp90 in the cell,hsc82-G309S, hsc82-A583T, and hsc82-I588AM589A cause similargrowth defects at 37°C. Of the three mutants, only hsc82-I588AM589A also showed a strong growth defect in the presenceof hygromycin. Of note, hsc82-I588AM589A, cpr7, and cns1 mu-tant cells also exhibit slow growth at 25°C (Fig. 8A). Yeast lackingindividual components of RAC also exhibits cold sensitivity (33).It is tempting to speculate that the cold sensitivity of cpr7, cns1,and hsc82 mutant strains is related to defects in ribosomal func-tions, but more work is needed to establish that connection.Regardless, the hygromycin defect of hsc82-I588AM589A cellssuggests that Hsp90 and cochaperones cooperate to promote ri-bosome function.

DISCUSSION

Hsp90 mediates the folding and activation of proteins as a com-plex molecular machine, requiring ATP binding and hydrolysis,posttranslational modifications, and multiple cochaperones (9,34). Cochaperones bind distinct domains and/or specific confor-mations of Hsp90. In addition to interacting with Hsp90, somecochaperones interact directly with clients. Cdc37 and Sgt1 haveimportant roles targeting protein kinases or leucine-rich-repeat-containing proteins, respectively, to Hsp90 (35, 36). Cpr6 or ho-mologs have been shown to be important for RNA-induced si-lencing, lipid trafficking, and viral function (37–39). Here, weshow that Cpr6, Cns1, and Cpr7, but not other tested cochaper-ones, interact with intact ribosomes, providing new evidence forspecialized cochaperone functions.

Cpr6 contains a PPIase domain and a TPR domain flanked bycharged regions. The TPR domain plus flanking regions (aa 171 to371) contains separate binding sites for Hsp90, Ura2, and the ri-bosome. Hsp90 binds residues in the established EEVD-bindingcleft (23, 25). Basic residues in the last 15 amino acids of Cpr6 wererequired for ribosome interaction. A chimeric protein that con-tains the first 271 amino acids of Cpr6 and the last �100 aminoacids of Cpr7 (His-Cpr6-6A/7B) failed to interact with Ura2 or torescue growth defects caused by loss of CPR6 but was able torescue growth defects caused by loss of CPR7 (14). This suggeststhat the last 100 aa of Cpr6 dictate Ura2 interaction and Cpr6-specific functions. Preliminary results also suggest that, similar toCpr7, Cpr6-6A/7B failed to interact with the ribosome unless itcontained the F230A alteration (not shown). Further analysis isrequired to determine whether basic amino acid residues in thecarboxy terminus of Cpr7 also mediate ribosome interaction. TheTPR domains and/or flanking regions of three additional Hsp90cochaperones, Sgt1, Tah1, and AIP, are also critical for interac-tions with proteins other than Hsp90 (40–42). Surprisingly, thePPIase domains of both Cpr6 and Cpr7 appear to be dispensablefor known functions. However, it is possible that the PPIase do-main stabilizes the interaction of WT Cpr6 with the ribosome,since Cpr6 171–373 exhibited a slight reduction in ribosome in-teraction compared to WT Cpr6. The PPIase domain of Cpr6 wasnot required to cause negative growth in the cns1-G90D strain.Mutation of a residue (R64) predicted to be required for catalyticactivity (32) did not disrupt Cpr6-ribosome interaction, and over-expression of cpr6-R64A, like WT CPR6, caused growth defects ina cns1-G90D strain (not shown). The ribosome-associated chap-erone trigger factor of Escherichia coli also contains a PPIase do-main. Although not essential for folding of cytosolic proteins, thePPIase domain of the trigger factor assists in folding some sub-

strates (43, 44). More recently, part of the PPIase domain thatoverlaps the site of enzymatic activity was shown to interact withan unfolded substrate (45).

Mutation or overexpression of CNS1 is known to affect Hsp90function in yeast (30, 46, 47). Human and Drosophila homologs(TTC4 and Dpit47, respectively), interact with Hsp90, as well asDNA polymerase alpha, the replication protein Cdc6, the histoneacetyltransferase MYST/MOF, and the transcriptional initiationfactor TFIIIB (48–52). No specific clients of yeast Cns1 have beenpreviously identified. Sequences outside the TPR domain are re-quired for growth at elevated temperature, ribosome interaction,and growth in the presence of hygromycin. There is limited ho-mology between the carboxy terminus of Cpr6 and sequences ad-jacent to the TPR domain of Cns1. However, most of those resi-dues are missing in Cns1 212–385, which was sufficient forribosome interaction. Thus, Cpr6 and Cns1 appear to use differ-ent sequences to interact with the ribosome. An altered version ofTTC4 found in cancer cells contained a mutation located outsidethe TPR domain (51), and the temperature-sensitive mutationcns1-3C contains three amino acid alterations in the carboxy ter-minus, D260G, E324G, and L330S (17). Additional work isneeded to narrow down the site of ribosomal interaction in Cns1and to determine whether any of those mutations disrupt ribo-some interaction.

Interaction of Cpr6, Cns1, and Cpr7 with the intact ribosomesuggests Hsp90 involvement. Although we did not detect Hsp90interaction with intact ribosomes, cells expressing the hsc82-I588AM589A mutations exhibited growth defects in the presenceof hygromycin, suggesting that mutation may affect ribosomalfunctions. Hsc82 residues I588 and M589 are homologous toHsp82 I592 and M593, which are located in a putative client-binding site (53). Interestingly, similar to hsc82-I588AM589A,some mutations in hsp82 residues in the client-binding site alsoresulted in cold-sensitive growth. Further work is needed to de-termine whether those mutations result in sensitivity to hygromy-cin and/or ribosomal defects. Alternatively, ribosomal functionsof Cns1 and Cpr6 may be related to the ability of both of theproteins to bind Hsp70 of the Ssa family (14, 26, 54). The Hsp70nucleotide exchange factor Snl1 was recently found to interactwith intact ribosomes, with a presumed binding site on the largesubunit (55). The function of that interaction is unclear, but en-doplasmic reticulum (ER) membrane localization of Snl1 suggestsa potential role in protein transport. Alternatively, both Cpr7 andCns1 interact with Hsp104 during respiratory growth (56), and wehave detected Hsp104 in complex with His-Cpr6 (not shown), soit is possible that Hsp104 mediates ribosome interaction.

The gene expression tool SPELL (Serial Pattern of ExpressionLevels Locator) (57) indicates that CNS1 and CPR7 share tran-scriptional regulation patterns similar to those of genes encodingproteins required for ribosome function (see Table S2 in the sup-plemental material). Both the cns1-G90D and cns1 1-212 mutantsshowed hypersensitivity to hygromycin, indicating that loss ofHsp90 interaction or ribosome interaction affects Cns1 functionin that assay. Deletion of CPR7 also confers sensitivity to hygro-mycin. Cpr6 is functionally linked to the ribosome, since Cpr6mutants that fail to interact with the ribosome are unable to causegrowth defects in cns1-G90D cells. Independent evidence comesfrom a prior study that found that Cpr6 and/or Cpr7 bound topolysomes (1). Our model is that Cns1 and Cpr7 have ribosomalfunctions in unstressed cells. Overexpression of the stress-induc-

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ible CPR6 may disrupt Cpr7 or Cns1 functions by competing foreither Hsp90 or ribosomes. Enhanced ribosomal interaction ofCpr6-F230A and Cpr7-F251A suggests that Cpr6 and Cpr7 inter-action may also be regulated by posttranslational modification ofnearby residues. Preliminary results suggest that a mild heat shock(10 min at 42°C) had no effect on Cpr6-ribosome interaction, butwe have not tested more severe conditions. Two recent papersindicate that ribosome pausing occurs during heat shock (58, 59).Although Hsp90 does not appear to play a significant role, re-duced levels of Hsp70-ribosome interaction play a role in ribo-somal pausing. It is possible that Cpr6 and/or Cns1 modulatesHsp70 function in that process by targeting Hsp70 to misfoldedproteins during times of cellular stress.

Further analysis is needed to determine whether Cns1, Cpr6,and Cpr7 are involved in previously described ribosomal func-tions linked to Hsp90 or other cochaperones (5–8, 60). Possiblecochaperone functions are interactions with nascent chains thatare also Hsp90 clients, regulating polysome stability, assisting inthe folding of a ribosomal protein, or differentially modulatingribosomal function in response to stress. Our results do not sup-port a direct interaction with components of RAC and NAC, butfurther experiments are required to determine whether the co-chaperones associate with ribosome-bound nascent chains. It isintriguing that the interaction of WT Cpr6 with the ribosomeappeared to be stabilized by Ura2, but further studies are neededto confirm those results. Two-hybrid analysis and other genome-wide analyses uncovered an interaction of Cns1 with elongationfactor 2, which catalyzes ribosomal translocation during proteinsynthesis (30, 61). We have not examined whether Cpr6, Cpr7,and/or Cns1 directly interacts with elongation factor 2. We also donot yet know whether ribosome interaction is linked to the chap-eroning ability of Cpr6 and Cpr7 (15).

TPR-containing cochaperones competitively interact with thecarboxy-terminal EEVD sequences of Hsp90. Each of the cochap-erones has varied abilities to modulate the ATPase activity ofHsp90 (or Hsp70) and may have additional functionalities, suchas PPIase domains (Cpr6 and Cpr7) or phosphatase domains(Ppt1). Despite having sequence similarity, Cpr6 and Cpr7 havedifferent in vivo functions. A dimer of Hsp90 is able to bind twodifferent TPR cochaperones at the same time, resulting in a pro-gression of different Hsp90-cochaperone complexes during clientfolding (9, 62). However, only some ternary complexes appearpossible. For instance, Cpr6 is able to form ternary complexes withHsp90 and Aha1, but Cpr7 is not (16). Cns1 and Cpr7 are found inthe same complexes in the cell and have overlapping cellular func-tions. There is also evidence that Cns1 and Cpr7 interact in theabsence of Hsp90 (11, 12, 17).

The ability of CPR6 to cause growth defects in cns1 mutantstrains requires ribosome interaction. This suggests that cochap-erones may compete with one another, either by binding Hsp90 orby binding to the same ribosomal client. We do not have evidencethat Cpr6 and Cns1 directly compete for interaction with the sameribosomal protein, and experiments to determine whether over-expression of Cns1 disrupted Cpr6 interaction, or vice versa, didnot provide consistent results. Alternatively, Cns1 interactionmay cause a conformational change or steric hindrance that pre-vents Cpr6 interaction, or vice versa. However, differential expres-sion patterns of these cochaperones raises the intriguing possibil-ity that cochaperone-client complexes vary depending on growthconditions. This finding is similar to a prior report that some

Hsp90 cochaperones interact with Hsp104 during respiratorygrowth (56). Either of these cases likely results in altered activity ofsubsets of client proteins. In summary, these studies indicate thatthere is much to be learned about the specificity and in vivo func-tions of TPR-containing Hsp90 cochaperones.

ACKNOWLEDGMENTS

This work was funded primarily by a grant from the National Science Foun-dation (MCB-0744522). Additional support was provided by grant P30GM103324 from the NIH National Institute of General Medical Sciences.

Steven Heid and Kyle Odom assisted with construction of CNS1 mu-tants. We thank Elizabeth Craig and members of her laboratory for helpfuladvice, in addition to the anti-Zuo1 antibody, the 416GPDHis-Zuo1 plas-mid, and the ura2::HIS3 strain. Kevin Morano also provided reagents andhelpful discussion. We thank Len Neckers for the plasmid expressing His-Hsp82. We also thank Rick Gaber for providing reagents and access toMarija Tesic’s Ph.D. thesis.

REFERENCES1. Albanese V, Yam AY, Baughman J, Parnot C, Frydman J. 2006. Systems

analyses reveal two chaperone networks with distinct functions in eukary-otic cells. Cell 124:75– 88. http://dx.doi.org/10.1016/j.cell.2005.11.039.

2. Frydman J, Nimmesgern E, Ohtsuka K, Hartl FU. 1994. Folding ofnascent polypeptide chains in a high molecular mass assembly withmolecular chaperones. Nature 370:111–117. http://dx.doi.org/10.1038/370111a0.

3. Nathan DF, Vos MH, Lindquist S. 1997. In vivo functions of the Sac-charomyces cerevisiae Hsp90 chaperone. Proc Natl Acad Sci U S A 94:12949 –12956. http://dx.doi.org/10.1073/pnas.94.24.12949.

4. Zhao R, Davey M, Hsu YC, Kaplanek P, Tong A, Parsons AB, KroganN, Cagney G, Mai D, Greenblatt J, Boone C, Emili A, Houry WA. 2005.Navigating the chaperone network: an integrative map of physical andgenetic interactions mediated by the hsp90 chaperone. Cell 120:715–727.http://dx.doi.org/10.1016/j.cell.2004.12.024.

5. Franzosa EA, Albanese V, Frydman J, Xia Y, McClellan AJ. 2011.Heterozygous yeast deletion collection screens reveal essential targetsof Hsp90. PLoS One 6:e28211. http://dx.doi.org/10.1371/journal.pone.0028211.

6. Tenge VR, Knowles J, Johnson JL. 2014. The ribosomal biogenesisprotein Utp21 interacts with Hsp90 and has differing requirements forHsp90-associated proteins. PLoS One 9:e92569. http://dx.doi.org/10.1371/journal.pone.0092569.

7. Kim TS, Jang CY, Kim HD, Lee JY, Ahn BY, Kim J. 2006. Interaction ofHsp90 with ribosomal proteins protects from ubiquitination and protea-some-dependent degradation. Mol Biol Cell 17:824 – 833. http://dx.doi.org/10.1091/mbc.E05-08-0713.

8. Schlatter H, Langer T, Rosmus S, Onneken ML, Fasold H. 2002. A novelfunction for the 90 kDa heat-shock protein (Hsp90): facilitating nuclearexport of 60 S ribosomal subunits. Biochem J 362:675– 684. http://dx.doi.org/10.1042/0264-6021:3620675.

9. Li J, Soroka J, Buchner J. 2012. The Hsp90 chaperone machinery: con-formational dynamics and regulation by co-chaperones. Biochim BiophysActa 1823:624 – 635. http://dx.doi.org/10.1016/j.bbamcr.2011.09.003.

10. Duina AA, Marsh JA, Gaber RF. 1996. Identification of two CyP-40-likecyclophilins in Saccharomyces cerevisiae, one of which is required fornormal growth. Yeast 12:943–952. http://dx.doi.org/10.1002/(SICI)1097-0061(199608)12:10�943::AID-YEA997�3.0.CO;2-3.

11. Dolinski KJ, Cardenas ME, Heitman J. 1998. CNS1 encodes an essentialp60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin40 mutations and interacts with Hsp90. Mol Cell Biol 18:7344 –7352.

12. Marsh JA, Kalton HM, Gaber RF. 1998. Cns1 is an essential proteinassociated with the hsp90 chaperone complex in Saccharomyces cerevisiaethat can restore cyclophilin 40-dependent functions in cpr7Delta cells.Mol Cell Biol 18:7353–7359.

13. Zuehlke AD, Johnson JL. 2012. Chaperoning the chaperone: a role for theco-chaperone Cpr7 in modulating Hsp90 function in Saccharomycescerevisiae. Genetics 191:805– 814. http://dx.doi.org/10.1534/genetics.112.140319.

14. Zuehlke AD, Wren N, Tenge V, Johnson JL. 2013. Interaction of heatshock protein 90 and the co-chaperone Cpr6 with Ura2, a bifunctional

Hsp90 Cochaperones Interact with the Intact Ribosome

January 2015 Volume 14 Number 1 ec.asm.org 61Eukaryotic Cell

on April 15, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

enzyme required for pyrimidine biosynthesis. J Biol Chem 288:27406 –27414. http://dx.doi.org/10.1074/jbc.M113.504142.

15. Mayr C, Richter K, Lilie H, Buchner J. 2000. Cpr6 and Cpr7, two closelyrelated Hsp90-associated immunophilins from Saccharomyces cerevisiae,differ in their functional properties. J Biol Chem 275:34140 –34146. http://dx.doi.org/10.1074/jbc.M005251200.

16. Li J, Richter K, Reinstein J, Buchner J. 2013. Integration of the acceler-ator Aha1 in the Hsp90 co-chaperone cycle. Nat Struct Mol Biol 20:326 –331. http://dx.doi.org/10.1038/nsmb.2502.

17. Tesic M, Marsh JA, Cullinan SB, Gaber RF. 2003. Functional interac-tions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharo-myces cerevisiae. J Biol Chem 278:32692–32701. http://dx.doi.org/10.1074/jbc.M304315200.

18. Johnson JL, Zuehlke AD, Tenge VR, Langworthy JC. 2014. Mutation ofessential Hsp90 co-chaperones SGT1 or CNS1 renders yeast hypersensi-tive to overexpression of other co-chaperones. Curr Genet 60:265–276.http://dx.doi.org/10.1007/s00294-014-0432-3.

19. Yan W, Schilke B, Pfund C, Walter W, Kim S, Craig EA. 1998. Zuotin,a ribosome-associated DnaJ molecular chaperone. EMBO J 17:4809 –4817. http://dx.doi.org/10.1093/emboj/17.16.4809.

20. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, De-phoure N, O’Shea EK, Weissman JS. 2003. Global analysis of proteinexpression in yeast. Nature 425:737–741. http://dx.doi.org/10.1038/nature02046.

21. Johnson JL, Halas A, Flom G. 2007. Nucleotide-dependent interaction ofSaccharomyces cerevisiae Hsp90 with the cochaperone proteins Sti1,Cpr6, and Sba1. Mol Cell Biol 27:768 –776. http://dx.doi.org/10.1128/MCB.01034-06.

22. Flom G, Behal RH, Rosen L, Cole DG, Johnson JL. 2007. Definition ofthe minimal fragments of Sti1 required for dimerization, interaction withHsp70 and Hsp90 and in vivo functions. Biochem J 404:159 –167. http://dx.doi.org/10.1042/BJ20070084.

23. Scheufler C, Brinker A, Bourenkov G, Pegoraro S, Moroder L, BartunikH, Hartl FU, Moarefi I. 2000. Structure of TPR domain-peptide com-plexes: critical elements in the assembly of the Hsp70-Hsp90 multichap-erone machine. Cell 101:199 –210. http://dx.doi.org/10.1016/S0092-8674(00)80830-2.

24. Taylor P, Dornan J, Carrello A, Minchin RF, Ratajczak T, WalkinshawMD. 2001. Two structures of cyclophilin 40: folding and fidelity in theTPR domains. Structure 9:431– 438. http://dx.doi.org/10.1016/S0969-2126(01)00603-7.

25. Ward BK, Allan RK, Mok D, Temple SE, Taylor P, Dornan J, MarkPJ, Shaw DJ, Kumar P, Walkinshaw MD, Ratajczak T. 2002. Astructure-based mutational analysis of cyclophilin 40 identifies keyresidues in the core tetratricopeptide repeat domain that mediatebinding to Hsp90. J Biol Chem 277:40799 – 40809. http://dx.doi.org/10.1074/jbc.M207097200.

26. Carrello A, Allan RK, Morgan SL, Owen BA, Mok D, Ward BK,Minchin RF, Toft DO, Ratajczak T. 2004. Interaction of the Hsp90cochaperone cyclophilin 40 with Hsc70. Cell Stress Chaperones 9:167–181. http://dx.doi.org/10.1379/CSC-26R.1.

27. Leidig C, Bange G, Kopp J, Amlacher S, Aravind A, Wickles S, Witte G,Hurt E, Beckmann R, Sinning I. 2012. Structural characterization of aeukaryotic chaperone—the ribosome-associated complex. Nat Struct MolBiol 20:23–28. http://dx.doi.org/10.1038/nsmb.2447.

28. Louvion JF, Warth R, Picard D. 1996. Two eukaryote-specific regions ofHsp82 are dispensable for its viability and signal transduction functions inyeast. Proc Natl Acad Sci U S A 93:13937–13942. http://dx.doi.org/10.1073/pnas.93.24.13937.

29. Preissler S, Deuerling E. 2012. Ribosome-associated chaperones as keyplayers in proteostasis. Trends Biochem Sci 37:274 –283. http://dx.doi.org/10.1016/j.tibs.2012.03.002.

30. Tesic M. 2003. Functional analysis of CNS1, an essential co-chaperone ofthe heat shock protein 90 in Saccharomyces cerevisiae. Ph.D. thesis,Northwestern University, Chicago, IL.

31. Nelson RJ, Ziegelhoffer T, Nicolet C, Werner-Washburne M, Craig EA.1992. The translation machinery and 70 kd heat shock protein cooperatein protein synthesis. Cell 71:97–105. http://dx.doi.org/10.1016/0092-8674(92)90269-I.

32. Duina AA, Marsh JA, Kurtz RB, Chang HC, Lindquist S, Gaber RF.1998. The peptidyl-prolyl isomerase domain of the CyP-40 cyclophilinhomolog Cpr7 is not required to support growth or glucocorticoid recep-

tor activity in Saccharomyces cerevisiae. J Biol Chem 273:10819 –10822.http://dx.doi.org/10.1074/jbc.273.18.10819.

33. Hundley H, Eisenman H, Walter W, Evans T, Hotokezaka Y, Wied-mann M, Craig E. 2002. The in vivo function of the ribosome-associatedHsp70, Ssz1, does not require its putative peptide-binding domain. ProcNatl Acad Sci U S A 99:4203– 4208. http://dx.doi.org/10.1073/pnas.062048399.

34. Walton-Diaz A, Khan S, Bourboulia D, Trepel JB, Neckers L, Mol-lapour M. 2013. Contributions of co-chaperones and post-translationalmodifications towards Hsp90 drug sensitivity. Future Med Chem 5:1059 –1071. http://dx.doi.org/10.4155/fmc.13.88.

35. Caplan AJ, Mandal AK, Theodoraki MA. 2007. Molecular chaperonesand protein kinase quality control. Trends Cell Biol 17:87–92. http://dx.doi.org/10.1016/j.tcb.2006.12.002.

36. Stuttmann J, Parker JE, Noel LD. 2008. Staying in the fold: the SGT1/chaperone machinery in maintenance and evolution of leucine-rich re-peat proteins. Plant Signal Behav 3:283–285. http://dx.doi.org/10.4161/psb.3.5.5576.

37. Anderson LJ, Lin K, Compton T, Wiedmann B. 2011. Inhibition ofcyclophilins alters lipid trafficking and blocks hepatitis C virus secretion.Virol J 8:329. http://dx.doi.org/10.1186/1743-422X-8-329.

38. Lin JY, Mendu V, Pogany J, Qin J, Nagy PD. 2012. The TPR domain inthe host Cyp40-like cyclophilin binds to the viral replication protein andinhibits the assembly of the tombusviral replicase. PLoS Pathog8:e1002491. http://dx.doi.org/10.1371/journal.ppat.1002491.

39. Iki T, Yoshikawa M, Meshi T, Ishikawa M. 2012. Cyclophilin 40 facili-tates HSP90-mediated RISC assembly in plants. EMBO J 31:267–278.http://dx.doi.org/10.1038/emboj.2011.395.

40. Jimenez B, Ugwu F, Zhao R, Orti L, Makhnevych T, Pineda-Lucena A,Houry WA. 2012. Structure of minimal tetratricopeptide repeat domainprotein Tah1 reveals mechanism of its interaction with Pih1 and Hsp90. JBiol Chem 287:5698 –5709. http://dx.doi.org/10.1074/jbc.M111.287458.

41. Catlett MG, Kaplan KB. 2006. Sgt1p is a unique co-chaperone that acts asa client-adaptor to link Hsp90 to Skp1p. J Biol Chem 281:33739 –33748.http://dx.doi.org/10.1074/jbc.M603847200.

42. Morgan RM, Hernandez-Ramirez LC, Trivellin G, Zhou L, Roe SM,Korbonits M, Prodromou C. 2012. Structure of the TPR domain of AIP:lack of client protein interaction with the C-terminal alpha-7 helix of theTPR domain of AIP is sufficient for pituitary adenoma predisposition.PLoS One 7:e53339. http://dx.doi.org/10.1371/journal.pone.0053339.

43. Kramer G, Patzelt H, Rauch T, Kurz TA, Vorderwulbecke S, Bukau B,Deuerling E. 2004. Trigger factor peptidyl-prolyl cis/trans isomerase ac-tivity is not essential for the folding of cytosolic proteins in Escherichiacoli. J Biol Chem 279:14165–14170. http://dx.doi.org/10.1074/jbc.M313635200.

44. Liu CP, Zhou QM, Fan DJ, Zhou JM. 2010. PPIase domain of triggerfactor acts as auxiliary chaperone site to assist the folding of protein sub-strates bound to the crevice of trigger factor. Int J Biochem Cell Biol 42:890 –901. http://dx.doi.org/10.1016/j.biocel.2010.01.019.

45. Saio T, Guan X, Rossi P, Economou A, Kalodimos CG. 2014. Structuralbasis for protein antiaggregation activity of the trigger factor chaperone.Science 344:1250494. http://dx.doi.org/10.1126/science.1250494.

46. Miller CA. 2002. Two tetratricopeptide repeat proteins facilitate humanaryl hydrocarbon receptor signalling in yeast. Cell Signal 14:615– 623.http://dx.doi.org/10.1016/S0898-6568(02)00002-5.

47. Nathan DF, Vos MH, Lindquist S. 1999. Identification of SSF1, CNS1,and HCH1 as multicopy suppressors of a Saccharomyces cerevisiae Hsp90loss-of-function mutation. Proc Natl Acad Sci U S A 96:1409 –1414. http://dx.doi.org/10.1073/pnas.96.4.1409.

48. Moir RD, Willis IM. 2004. Tetratricopeptide repeats of Tfc4 and a lim-iting step in the assembly of the initiation factor TFIIIB. Adv ProteinChem 67:93–121. http://dx.doi.org/10.1016/S0065-3233(04)67004-5.

49. Dmitriev RI, Korneenko TV, Bessonov AA, Shakhparonov MI, Mody-anov NN, Pestov NB. 2007. Characterization of hampin/MSL1 as a nodein the nuclear interactome. Biochem Biophys Res Commun 355:1051–1057. http://dx.doi.org/10.1016/j.bbrc.2007.02.073.

50. Crevel G, Bates H, Huikeshoven H, Cotterill S. 2001. The DrosophilaDpit47 protein is a nuclear Hsp90 co-chaperone that interacts with DNApolymerase alpha. J Cell Sci 114:2015–2025.

51. Crevel G, Bennett D, Cotterill S. 2008. The human TPR protein TTC4 isa putative Hsp90 co-chaperone which interacts with CDC6 and showsalterations in transformed Cells. PLoS One 3:e0001737. http://dx.doi.org/10.1371/journal.pone.0001737.

Tenge et al.

62 ec.asm.org January 2015 Volume 14 Number 1Eukaryotic Cell

on April 15, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

52. Dmitriev RI, Okkelman IA, Abdulin RA, Shakhparonov MI, Pestov NB.2009. Nuclear transport of protein TTC4 depends on the cell cycle. CellTissue Res 336:521–527. http://dx.doi.org/10.1007/s00441-009-0785-y.

53. Genest O, Reidy M, Street TO, Hoskins JR, Camberg JL, Agard DA,Masison DC, Wickner S. 2013. Uncovering a region of heat shock protein90 important for client binding in E. coli and chaperone function in yeast.Mol Cell 49:464 – 473. http://dx.doi.org/10.1016/j.molcel.2012.11.017.

54. Hainzl O, Wegele H, Richter K, Buchner J. 2004. Cns1 is an activator ofthe Ssa1 ATPase activity. J Biol Chem 279:23267–23273. http://dx.doi.org/10.1074/jbc.M402189200.

55. Verghese J, Morano KA. 2012. A lysine-rich region within fungal BAGdomain-containing proteins mediates a novel association with ribosomes.Eukaryot Cell 11:1003–1011. http://dx.doi.org/10.1128/EC.00146-12.

56. Abbas-Terki T, Donze O, Briand PA, Picard D. 2001. Hsp104 interactswith Hsp90 cochaperones in respiring yeast. Mol Cell Biol 21:7569 –7575.http://dx.doi.org/10.1128/MCB.21.22.7569-7575.2001.

57. Hibbs MA, Hess DC, Myers CL, Huttenhower C, Li K, TroyanskayaOG. 2007. Exploring the functional landscape of gene expression: directedsearch of large microarray compendia. Bioinformatics 23:2692–2699.http://dx.doi.org/10.1093/bioinformatics/btm403.

58. Liu B, Han Y, Qian SB. 2013. Cotranslational response to proteotoxic

stress by elongation pausing of ribosomes. Mol Cell 49:453– 463. http://dx.doi.org/10.1016/j.molcel.2012.12.001.

59. Shalgi R, Hurt JA, Krykbaeva I, Taipale M, Lindquist S, Burge CB. 2013.Widespread regulation of translation by elongation pausing in heat shock.Mol Cell 49:439 – 452. http://dx.doi.org/10.1016/j.molcel.2012.11.028.

60. Echtenkamp FJ, Zelin E, Oxelmark E, Woo JI, Andrews BJ, GarabedianM, Freeman BC. 2011. Global functional map of the p23 molecular chap-erone reveals an extensive cellular network. Mol Cell 43:229 –241. http://dx.doi.org/10.1016/j.molcel.2011.05.029.

61. Gavin AC, Aloy P, Grandi P, Krause R, Boesche M, Marzioch M, RauC, Jensen LJ, Bastuck S, Dumpelfeld B, Edelmann A, Heurtier MA,Hoffman V, Hoefert C, Klein K, Hudak M, Michon AM, Schelder M,Schirle M, Remor M, Rudi T, Hooper S, Bauer A, Bouwmeester T,Casari G, Drewes G, Neubauer G, Rick JM, Kuster B, Bork P, RussellRB, Superti-Furga G. 2006. Proteome survey reveals modularity of theyeast cell machinery. Nature 440:631– 636. http://dx.doi.org/10.1038/nature04532.

62. Li J, Richter K, Buchner J. 2011. Mixed Hsp90-cochaperone complexesare important for the progression of the reaction cycle. Nat Struct Mol Biol18:61– 66. http://dx.doi.org/10.1038/nsmb.1965.

Hsp90 Cochaperones Interact with the Intact Ribosome

January 2015 Volume 14 Number 1 ec.asm.org 63Eukaryotic Cell

on April 15, 2021 by guest

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Dow

nloaded from