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Functional Divergence between Co-chaperones of Hsc70

Functional Divergence between Co-chaperones of Hsc70 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 40, pp. 27100 –27109, October 3, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Received for publication, May 22, 2008, and in revised form, July 28, 2008 Published, JBC Papers in Press, August 6, 2008, DOI 10.1074/jbc.M803923200 Stefan Tzankov, Michael J. H. Wong, Kun Shi, Christina Nassif, and Jason C. Young From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada The ATPase cycle of the chaperone Hsc70 is regulated by contain J domains that stimulate ATP hydrolysis by Hsp70, and co-chaperones; Hsp40/DnaJ-related proteins stimulate ATP consequently substrate binding. Nucleotide exchange factors hydrolysis by Hsc70 and can bind unfolded polypeptides them- (NEFs), such as GrpE in E. coli, trigger the dissociation of selves. Conversely, various nucleotide exchange factors (NEFs) bound ADP from Hsp70 to allow the binding of ATP, resetting stimulate ADP-ATP exchange by Hsc70. We analyzed the puri- the cycle. The principles of this mechanism appear to be con- fied Hsp40-related co-chaperones DJA1 (Hdj2) and DJA2 served in Hsp70 chaperones, including the major cytosolic (Hdj3) and found that they had a distinct pattern of binding to a form in humans, Hsc70 (HSPA8) (1, 2). range of polypeptides. DJA2 alone could stimulate Hsc70-medi- The DnaJ-related co-chaperones are also conserved between ated refolding of luciferase in the absence of NEF, whereas DJA1 species. Type 1 J domain co-chaperones are homologous to was much less active. The addition of the Bag1 NEF increased DnaJ throughout their sequence and have the same domain refolding by Hsc70 and DJA2, as did the newly characterized architecture. Following their N-terminal J domains, they con- NEF Hsp110, but each NEF had a different optimal concentra- tain a linker sequence, zinc finger and central regions, and a tion ratio to Hsc70. Notably, the NEF HspBP1 could not C-terminal homodimerization region. Unfolded polypeptides increase refolding by Hsc70 and DJA2 at any concentration, and are thought to be bound by the central region of these proteins; none of the NEFs improved the refolding activity with DJA1. substrates are transferred to the Hsp70 partner upon J domain Instead, DJA1 was inhibitory of refolding with DJA2 and Hsc70. activation of ATP hydrolysis by the Hsp70 (1, 3). Although DnaJ All combinations of DJA1 or DJA2 with the three NEFs stimu- is the only J domain co-chaperone of E. coli DnaK, humans have lated the Hsc70 ATPase rate, although Hsp110 became less three cytosolic type 1 co-chaperones: DJA1 (DNAJA1, Hdj2, effective with increasing concentrations. A chimeric DJA2 hav- HSDJ), DJA2 (DNAJA2, Hdj3, HIRIP4), and DJA4 (DNAJA4, ing its Hsc70-stimulatory J domain replaced with that of DJA1 Hdj4). Comparison with the type 1 co-chaperone Ydj1 of Sac- was functional for polypeptide binding and ATPase stimulation charomyces cerevisiae suggests that the human DJAs will have of Hsc70. However, it could not support efficient Hsc70-medi- similar overall structures. DJA1 and DJA2 are constitutively ated refolding and also inhibited refolding with DJA2 and expressed, whereas DJA4 is less highly expressed and may be Hsc70. These results suggest a more complex model of Hsc70 specialized. In addition, eukaryotic type 2 J domain co-chaper- mechanism than has been previously thought, with notable ones are known that diverge from DnaJ in the substrate-binding functional divergence between Hsc70 co-chaperones. and C-terminal regions. The human type 2 member DJB1 (Hsp40, Hdj1) is expressed at low levels under normal condi- tions and is mainly expressed in the heat shock stress response. DJB1 also binds substrate more weakly than type 1 co-chaper- The Hsp70 family of proteins are ATP-dependent molecular ones. Thus, the DJA1 and DJA2 co-chaperones are thought to chaperones that assist the folding of polypeptides. Hsp70 chap- erones have a typical structure divided into ATPase and sub- be the major partners of human Hsc70 (3–10). The distinction between the human DJAs remains largely unexplored. Our strate-binding domains that work in an ATP-driven substrate recent work proposed a partial specialization between the binding cycle. The mechanism of Hsp70 proteins has been well human type 1 co-chaperones, perhaps to allow assistance of a established in studies of the Escherichia coli homolog DnaK. In the ATP-bound state, an Hsp70 chaperone has low affinity for wider range of substrates (11). Interestingly, the NEF co-chaperones are divergent between unfolded polypeptide. After hydrolysis of ATP, Hsp70 in the E. coli and humans. The three types of human NEFs: Bag ADP-bound state binds substrate with high affinity. Exchange domain proteins, HspBP1, and Hsp110, are structurally unre- of ADP for ATP then reverts Hsp70 to its low polypeptide affin- ity state. Conversion of an Hsp70 between these two nucleotide lated to each other and to the single E. coli NEF GrpE. The C-terminal domain of Bag1 (C-Bag) was the first shown to states is controlled by different co-chaperone proteins. The have NEF activity for Hsc70, and homologous Bag domains Hsp40/DnaJ-related co-chaperones, including E. coli DnaJ, have since been identified in several other human proteins. The mechanical action of C-Bag on Hsc70 appears to be equivalent * This work was supported by a Canadian Institutes of Health Research oper- to that of GrpE on DnaK, despite the difference in NEF struc- ating grant and the Canadian Foundation for Innovation. The costs of pub- lication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: NEF, nucleotide exchange factor; ANT, adenine Canada Research Chair in Molecular Chaperones. To whom correspond- nucleotide translocator; CiC, citrate carrier; ER, estrogen receptor ; GR, ence should be addressed: Dept. of Biochemistry, McGill University, glucocorticoid receptor  isoform; MR, mineralocorticoid receptor; OGC, 3655 Promenade Sir William Osler, Montreal, PQ H3G 1Y6, Canada. oxaloglutarate carrier; PiC, inorganic phosphate carrier; PR, progesterone E-mail: [email protected]. receptor; RL, reticulocyte lysate. 27100 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 40 •OCTOBER 3, 2008 This is an Open Access article under the CC BY license. Function of Hsc70 Co-chaperones ture (12–16). HspBP1 was subsequently found to have NEF 500 mM NaCl, 20 mM imidazole, and 20 mM KH PO , pH 7.5, 2 4 activity. The structural effect of HspBP1 on Hsc70 is distinct and eluted with 300 mM imidazole and 20 mM KH PO , pH 7.5, 2 4 from that of C-Bag (17–19). Most recently, Hsp110 (HSPH1, and then on a Mono Q 5/50 GL (GE Healthcare) equilibrated in Hsp105) was shown to be an NEF for Hsc70. Hsp110 repre- 50 mM NaCl and 20 mM KH PO , pH 7.5, and eluted with a 50 to 2 4 sents a subclass of proteins structurally related to Hsp70; how- 600 mM NaCl gradient. HspBP1 was dialyzed into buffer G, and ever, its ATPase activity and substrate binding are minimal. Hsp110 was further purified on a Superdex 200 Hi-Load 16/60 The Bag domain and HspBP1 NEFs are also thought to not bind column (GE Healthcare) equilibrated in buffer G. DNA encod- substrate. A recent structural study suggests a large conforma- ing the chimeric mutant DJA1–2 containing amino acids 1–95 tion shift within Hsp110 may be involved in its NEF function of DJA1 and 96–412 of DJA2 was constructed by overlapping (20–27). Although the biochemical mechanisms of these NEFs PCR and inserted into pPROEXHTa (Invitrogen), and the pro- have been recently revealed, little is known of the biological tein was purified similarly to DJA2. Where indicated, His tags difference between them. were removed by digestion with His-tagged TEV protease 4 °C The important question of how Hsc70 mechanically assists overnight followed by repurification on nickel-Sepharose. polypeptide folding is still under investigation. Studies of the Polypeptide Binding—Binding of various polypeptides to E. coli DnaK-DnaJ-GrpE system have provided the outline of a DJA1, DJA2, and DJA4 was tested as described (11). Plasmids model (1, 28). Increases in the steady-state ATPase rate of encoding bovine phosphate carrier A (PiC) and mouse adenine DnaK require both DnaJ and GrpE to promote the ATP hydrol- nucleotide carrier 2 (ANT) were as described (29); those for rat ysis and nucleotide exchange steps. However, refolding of the citrate carrier (CiC) and bovine oxaloglutarate carrier (OGC) model polypeptide luciferase is optimal at a particular ratio of were from Vincenzo Zara (Lecce, Italy); those for human glu- DnaK to DnaJ and GrpE and, by implication, at the ATPase rate cocorticoid receptor (GR), estrogen receptor  (ER), proges- supported by that ratio. At this rate, the balance between terone receptor (PR), and mineralocorticoid receptor (MR) nucleotide-dependent polypeptide binding and release by were from Theo Rein (Munich, Germany); and those for human Hsc70 appears to be optimal for substrate folding. Other factors cytochrome b and synaptobrevin 2 were from Stephen High may be the binding of polypeptide by DnaJ and its transfer to (Manchester, UK). Purified DJA1, DJA2, and DJA4 were pre- DnaK. bound on nickel-Sepharose in buffer containing 500 mM NaCl, The multiple DJAs and NEFs of human Hsc70 provide a fresh 20 mM Hepes-KOH, pH 7.5, and 5 mM MgOAc for 30 min at opportunity to examine this model. In the simplest formula- 4 °C. Cell-free translations of the various polypeptides were tion, an optimal Hsc70 ATPase rate for refolding should be performed with the TNT-coupled RL system with SP6 or T7 supported by different co-chaperone combinations. The func- polymerase (Promega) supplemented with [ S]methionine tions of DJA1 and DJA2 have been compared together with (GE Healthcare and PerkinElmer Life Sciences), diluted 1:20 Hsc70 and the Bag1 NEF (6) but not with HspBP1 or Hsp110. into buffer G containing 20 mM imidazole, 0.1% Triton X-100 We have reported that DJA1 was significantly less active than and 2 mg/ml ovalbumin, and added to the immobilized DJA DJA2 in promoting the Hsc70- and C-Bag-mediated refolding proteins. The final reactions contained 5 M wild-type DJA of luciferase, although both had polypeptide binding and protein and 5% translation mixture. After 5 min at room tem- ATPase stimulatory properties (11), but differences between perature, the binding reactions were terminated by the addition DJA1 and DJA2 with regard to the other NEFs are not known. of 0.1 unit/l apyrase. The protein complexes were recovered at Conversely, HspBP1 and Hsp110 have only been analyzed in 4 °C for 30 min and washed with buffer G containing 20 mM combination with the biologically less relevant DJB1/Hsp40, imidazole and 0.1% Triton X-100. The protein complexes were like most data on Bag1 (13, 15, 18, 19, 22, 23). We therefore analyzed by SDS-PAGE and phosphorimaging quantitation. carried out a systematic analysis of the major DJA1 and DJA2 Hsc70 Activation—Hsc70 activation by the co-chaperones co-chaperones with Hsc70 and the Bag1, HspBP1, and Hsp110 was tested as described (11). To assay polypeptide refolding, NEFs. firefly luciferase (Sigma) was denatured in buffer G containing 6 M guanidinium-HCl and 1 mM dithiothreitol for 10 min at room EXPERIMENTAL PROCEDURES temperature. Refolding reactions were preassembled on ice, Reagents and Proteins—Unless stated otherwise, all chemical containing 4 M Hsc70 and the indicated amounts of purified reagents were from Sigma or BioShop Canada Inc. (Missis- co-chaperones in buffer G supplemented with 39 mM NaCl and sauga, Canada). Restriction enzymes and other recombinant 2mM ATP. Luciferase was diluted 1:100 into reactions to a final DNA reagents were from New England Biolabs, Invitrogen, and concentration of 5.4 nM and incubated at 30 °C. Control reac- Stratagene. Untreated rabbit reticulocyte lysate (RL) was from tions contained 70% RL in the same buffer or treated with 0.1 Green Hectares (Oregon, WI) and desalted into buffer G (100 unit/l apyrase as a negative control. At the 60 min or indicated mM KOAc, 20 mM Hepes-KOH, pH 7.5, and 5 mM MgOAc )on time point, the aliquots were diluted 2:25 into luciferase assay a Hi-Prep 26/10 Fast Desalting column (GE Healthcare). reagent (Promega), and luciferase activity was measured in an His-tagged human DJA1, DJA2, DJA4, Hsc70, and C-Bag EG&G Berthold Lumat LB 9507 luminometer. were expressed in Rosetta 2 E. coli cells (Novagen) and purified To test the ATPase activity of Hsc70, the reactions were pre- as described (11). His-tagged human HspBP1 and Hsp110 (19, assembled on ice as described for the refolding assays, except 23) were expressed in Rosetta 2 E. coli cells for2hat37°Cand that they were supplemented with 5 Ci/ml [- P]ATP (Per- for4hat30 °C, respectively. They were purified by chromatog- kin Elmer) in addition to 2 mM ATP, and the reactions were raphy on nickel-Sepharose HP (GE Healthcare) equilibrated in initiated by incubation at 30 °C. At various time points, the OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27101 Function of Hsc70 Co-chaperones aliquots were removed and terminated with 37.5 mM EDTA. The samples were separated by thin layer chromatography on polyethylene-imine cellulose (Mallinckrodt Baker) developed in 0.5 M LiCl and 0.5 M formic acid. The ADP produced was determined by phosphorimaging quantitation, and the linear enzymatic rates (V ) were calculated by regression analysis. max RESULTS Polypeptide Binding by DJAs—DJA1 and DJA2, as well as DJA4, are homologous to each other in their J domains and their central polypeptide-binding regions. However, sequence FIGURE 1. Polypeptide binding by DJAs. The indicated polypeptides were radiolabeled by cell-free translation and co-precipitated either with nickel- divergence between DJA1 and DJA2 (55% identity, 69% similar- Sepharose alone or with purified His-tagged DJA1, DJA2, or DJA4 and nickel- ity) is almost as high as between each of them and Ydj1 (46– Sepharose. Bound polypeptide was quantified by SDS-PAGE and phospho- 47% identity, 63–65% similarity), which would suggest func- rimaging analysis. In these and all experiments, the error bars represent the standard deviations from the mean of at least three independent trials. The tional differences between DJA1 and DJA2. These proteins amount of polypeptide bound by negative control beads or by DJA1, DJA2, or were previously compared in their binding of two mitochon- DJA4 was plotted as a percentage of input translated material. cytb5, cyto- chrome b ; syb2, synaptobrevin 2. drial preproteins, which depend on the Hsc70-Hsp90 chaper- one system for their targeting (11, 29). DJA1 bound the PiC and ANT 2-fold better than DJA2, and DJA4 bound ANT better widely: 16% relative to input for PR, 25% for GR, 49% for ER, than PiC. and 66% for MR. The tail-anchored proteins cytochrome b and To obtain a more complete picture of DJA substrate binding synaptobrevin 2 were bound poorly by DJA1, at less than 6% of characteristics, a wider range of polypeptides, which were input and close to background binding by the nickel-Sepharose potential chaperone substrates, was examined. The mitochon- control. So, the interaction of the mitochondrial preproteins drial CiC and OGC, like PiC and ANT, belong to the structural and steroid hormone receptors with DJA1 seems to vary family of metabolite transporters of the mitochondrial inner between the different proteins, whereas the tail-anchored pro- membrane. They all have six transmembrane domains and teins are bound poorly by DJA1. remain unfolded before insertion into the membrane (30). CiC Polypeptide binding by DJA2 and DJA4 was next analyzed. and OGC preproteins also show involvement of the chaperone- As observed before, PiC binding by DJA2 was approximately Tom70 pathway in their mitochondrial import (31, 32). In con- half that by DJA1, and binding of CiC and OGC by DJA2 was trast to the mitochondrial preproteins, the steroid hormone now also observed at approximately half relative to DJA1 bind- receptors are cytosolic and nuclear. Several of these proteins, ing, respectively (Fig. 1). DJA2 binding of GR and ER was more including GR, ER, PR and MR, respectively, are known to similar to DJA1. Binding of PR by DJA2 was quite low, whereas depend on the Hsc70-Hsp90 system to maintain them in a hor- binding of MR was essentially identical to DJA1. Very little mone-activable state (33, 34). Their hormone-binding domains cytochrome b and synaptobrevin 2 was bound by DJA2. In appear to be the main chaperone binding sites and may be con- comparison, DJA4 binding also showed variation in the same formationally unstable in the absence of hormone. Certain range. Binding by DJA4 was comparable with DJA1 for CiC and transmembrane tail-anchored proteins such as synaptobrevin 2 OGC and progressively weaker for MR and ER. GR and PR are also assisted by Hsc70 during their insertion into the endo- were bound most weakly by DJA4 at approximately one-fourth plasmic reticulum membrane. Other tail-anchored proteins of DJA1 binding. Thus, the substrate binding profiles of DJA1 such as cytochrome b appear to be less dependent on chaper- and DJA2 are clearly different. Overall, DJA1 shows moderately ones for insertion (35). The binding of the above polypeptides stronger binding of these polypeptides, but with exceptions (PR by the purified DJAs was therefore tested with our previously and MR). DJA4 may be more similar to DJA1 in its profile, established method (11). although exceptions are also observed (GR and PR). The differ- The various polypeptides were radiolabeled by cell-free ences in binding are significant but mostly range within an translation in rabbit RL, a standard model cytosol for chaper- order of magnitude. This is consistent with our proposed idea one experiments. The labeled polypeptides were then co-pre- of partial specialization between the DJA co-chaperones. cipitated with purified His-tagged DJA1, DJA2, or DJA4 and Polypeptide Refolding by Hsc70, DJA2, and NEFs—We nickel-Sepharose. The existing chaperones in the RL compete recently reported that DJA2 was quite active in supporting the for substrate binding with the DJAs but also help maintain sol- refolding of chemically denatured luciferase by purified Hsc70, ubility of the substrates; the radiolabeling allows accurate quan- whereas DJA1 had very poor activity (11). Those experiments titation of the bound polypeptides by phosphorimaging analy- were performed using 4 M Hsc70, 4 M of either DJA1 or sis. The amount of bound polypeptide relative to input labeled DJA2, and 0.5 M of the Bag1 NEF domain (C-Bag) (13, 14). The material was determined, and the binding between the 8:1 ratio of Hsc70 to NEF had been thought to be optimal for co-chaperones was compared. Substrate binding by DJA1, Hsc70-mediated refolding, as well as for the E. coli DnaK-DnaJ- DJA2, and DJA4 relative to input is shown in Fig. 1. More CiC GrpE system (6). To compare DJA refolding functions with dif- and OGC were bound by DJA1 than the established preprotein ferent NEFs, we first tested refolding in the absence of any NEF. substrate PiC, over 80% of input in the case of OGC (Fig. 1). The The model cytosol RL was used as a positive control. Luciferase amounts of steroid hormone receptors bound by DJA1 varied was denatured in guanidine and rapidly diluted 1:100 to 5.4 nM 27102 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 40 •OCTOBER 3, 2008 Function of Hsc70 Co-chaperones above, but with increasing concen- trations of the C-Bag, HspBP1 and Hsp110 NEFs. The levels of refold- ing after 60 min relative to that of the Hsc70 and DJA2 control reac- tion without NEF was plotted (Fig. 2, B–D). Increasing amounts of C-Bag produced a moderate but sig- nificant increase in refolding that peaked at 1.24-fold above Hsc70 and DJA2 alone (Fig. 2B). The opti- mal C-Bag concentration for refold- ing was at 4 M, equimolar to Hsc70 and DJA2. 21 M inhibited refolding to 0.4 of the Hsc70-DJA2 control. HspBP1 had a very different behavior. Increasing HspBP1 amounts did not increase refolding by Hsc70-DJA2 at all, but progres- sively inhibited refolding to 0.6 of the control (Fig. 2C). For Hsp110, the result was different again. 1 M Hsp110 significantly raised refold- ing to 1.20-fold of the control (Fig. 2D), similar to the maximum effect of C-Bag. Higher levels of Hsp110 clearly inhibited refolding, to less than 0.5 of the control at 8 M and above. To confirm these results, the time course of luciferase refolding at optimal concentrations of C-Bag FIGURE 2. Polypeptide refolding by Hsc70, DJA2, and NEFs. Luciferase was denatured in 6 M guanidine and (4 M) and Hsp110 (1 M) with diluted 1:100 into refolding reactions containing RL or the indicated combinations of Hsc70 and co-chaper- Hsc70 and DJA2 was examined. The ones. Refolding at 30 °C was monitored by luciferase activity. A, activity upon refolding in RL or with 4M Hsc70 increase in refolding compared with alone or with 4M DJA1 or DJA2, was monitored over time and plotted as a percentage of the activity at 60 min of the RL positive control. B–D, refolding activity after 60 min with 4 M Hsc70, 4 M DJA2, and the indicated Hsc70-DJA2 alone was evident amounts of C-Bag, HspBP1, and Hsp110 was plotted relative to that with Hsc70 and DJA2 alone. The y axis throughout the time course and scales are identical. In this and all subsequent experiments, refolding after 60 min with 4 M Hsc70 and 4 M DJA2 was used as control reactions, with the amount of refolded luciferase activity set to 1. E, refolding activity particularly at the early (5 min and over time with 4 M Hsc70, 4 M DJA2, and either 4 M C-Bag or 1 M Hsp110 was plotted relative to Hsc70 and 15 min) time points (Fig. 2E). So, DJA2 alone at 60 min. For comparison, the data for Hsc70 and DJA2 alone from A is replotted here. both the Bag1 and Hsp110 NEF activities can boost refolding by in RL or reactions containing purified proteins, supplemented Hsc70 and DJA2, although specific concentration ratios of with 2 mM ATP, and refolding at 30 °C was monitored by the these NEFs to Hsc70 seem to be required. As seen for HspBP1, luciferase enzymatic activity (11). As expected, RL provided NEF activity does not necessarily promote Hsc70-mediated efficient refolding of luciferase, whereas 4 M Hsc70 alone refolding. could not (Fig. 2A). Remarkably, 4 M Hsc70 and 4 M DJA2 DJA1 Negatively Effects Hsc70-mediated Polypeptide Refold- was relatively effective at refolding, to above 70% of the RL ing—It was possible that one or more of the NEFs might have control by 60 min. This suggested that unlike for DnaK, an NEF a greater effect on Hsc70-mediated refolding with DJA1, may not be stringently required for Hsc70 function when an which had poor activity by itself. The refolding of chemically appropriate DJA partner is available. Consistent with earlier denatured luciferase was thus tested as above but with 4 M results, an equivalent amount of Hsc70 and DJA1 was inactive each of Hsc70 and DJA1 and increasing amounts of C-Bag, in refolding. HspBP1, and Hsp110. The levels of refolding after 60 min Although an NEF may not be absolutely required for the relative to that of Hsc70 and DJA2 control reactions were Hsc70-DJA2 pair, it may still improve the efficiency of the plotted. No amount of any of the NEFs could activate refold- refolding reaction. An increase in refolding of thermally ing by Hsc70 and DJA1 above 0.25 that of the Hsc70-DJA2 denatured and in some cases chemically denatured luciferase control (Fig. 3A). This clear difference between DJA1 and had been reported for Bag1 and Hsp110 together with Hsc70 DJA2 suggests some mechanistic divergence, despite their and DJB1/Hsp40 (13, 23). Therefore, chemically denatured luciferase refolding was tested with 4 M Hsc70 and DJA2 as sequence homology. OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27103 Function of Hsc70 Co-chaperones tions having different amounts of DJA1 and DJA2 were exam- ined. Reactions with 4 M Hsc70 and a constant 4 M total DJA proteins were analyzed (Fig. 3C), because optimization tests with 4 M Hsc70 had previously shown maximum refolding at 4 M DJA2 (Fig. 3B), whereas different concentrations of DJA1 in this range were equally ineffective with Hsc70 (not shown). In fact, we observed that DJA1 inhibited refolding when it was present. For example, reactions with 4 M Hsc70 and 2 M DJA2 alone produced significant refolding activity, 0.82 of the control, whereas further addition of 2 M DJA1 reduced refold- ing to 0.25. The addition of C-Bag or Hsp110 NEF did not restore the refolding activity inhibited by DJA1. The addition of optimal concentrations of C-Bag (4 M) and Hsp110 (1 M) further increased refolding by Hsc70 and DJA2 alone (Fig. 3B), but the presence of DJA1 still blocked refolding (Fig. 3C). Indeed, refolding in the presence of 2 M DJA1 was as poor when C-Bag or Hsp110 were added, as when the NEFs were absent. Thus, DJA1 is not only inactive in the Hsc70-mediated refolding tested here but appears to inhibit this particular func- tion of DJA2 and Hsc70 and perhaps other functions. We fur- ther ruled out effects of the His tags on the inhibition of refold- ing. Hsc70 with its His tag removed behaved similarly to tagged Hsc70. It was activated to refold luciferase by DJA2, but not DJA1. C-Bag and Hsp110 further increased its refolding with DJA2, but not HspBP1. HspBP1, after removal of the His tag, did not enhance refolding by Hsc70-DJA2. Similarly, DJA1, after removal of the His tag, still could not activate Hsc70 to refold luciferase and was inhibitory of the Hsc70-DJA2 refold- ing reaction (not shown). Nontagged DJA2 was not isolated in sufficient amounts to be tested and may be more active than tagged DJA2. NEFs Diverge in ATPase Stimulation of Hsc70—The results in Figs. 2 and 3 indicated that both C-Bag and Hsp110 were able to increase the refolding function of Hsc70 and DJA2 but not that with DJA1. With either of the DJA co-chaperones, the expected effect of an NEF would be to increase the overall ATPase rate of Hsc70. Earlier work found that the ADP-ATP exchange step can be rate-limiting for the steady-state Hsc70 (and DnaK) ATPase and that both an NEF and a DnaJ-related co-chaperone were required for ATPase stimulation (1, 36, 37). It was possible that increased refolding activity correlated with a specific ATPase rate of Hsc70; inability to reach this rate might explain the weaker refolding function of HspBP1 or, less probably, of DJA1. We therefore investigated the relationship between the Hsc70 ATPase rate and refolding activity at differ- FIGURE 3. DJA1 inhibits polypeptide refolding. Luciferase refolding was monitored as in Fig. 2, with the indicated combinations of Hsc70 and co- ent concentrations of each NEF. As established (11), the reac- chaperones. A, refolding activity after 60 min with 4M Hsc70, 4M DJA1, and tions were set up similar to the refolding reactions but lacking the indicated amounts of C-Bag, HspBP1, and Hsp110 was plotted relative to the Hsc70 and DJA2 control reactions. B, refolding activity after 60 min with denatured luciferase and containing [- P]ATP to monitor 4 M Hsc70, the indicated amounts of DJA2, and either no addition or 4 M ADP production over time at 30 °C (Fig. 4A). Linear (steady- C-Bag or 1 M Hsp110 was plotted relative to the Hsc70 and DJA2 control state) enzymatic rates were calculated, and representative rates reactions. C, refolding activity after 60 min with 4 M Hsc70, the indicated amounts of DJA2 and DJA1, and either 4 M C-Bag or 1 M Hsp110 was plot- are presented in Table 1. ted relative to the Hsc70 and DJA2 control reactions. As expected, 4 M Hsc70 with 4 M of either DJA1 or DJA2 had a low enzymatic rate in the absence of NEF, 1.1 and 1.0 The effect of DJA1 on an Hsc70-mediated refolding reaction min , respectively (Table 1 and Fig. 4B). Low background might be neutral or inhibitory. In the first case, a refolding reac- rates were also observed for the co-chaperones in the absence of tion with Hsc70 and DJA2 would remain unaffected by the Hsc70, and the addition of DJA1 or DJA2 and C-Bag stimulated addition of DJA1. In the latter case, DJA1 would interfere with the Hsc70 ATPase activity. In the absence of Hsc70, the back- the reaction. To address this question, luciferase refolding reac- ground ATPase rate of Hsp110 was not increased by either 27104 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 40 •OCTOBER 3, 2008 Function of Hsc70 Co-chaperones increased steadily with C-Bag con- centration until 10 M C-Bag, with higher concentrations causing only small increases in rate (Fig. 4C). A maximum rate of 11 min could be reached at high C-Bag lev- els. The C-Bag effect on Hsc70 with DJA2 was essentially the same, with a similar high rate observed above 10 M C-Bag (Fig. 4D). HspBP1 also raised the Hsc70 ATPase rate with increasing concentration but was markedly less efficient than C-Bag. With DJA1, 10 M HspBP1 was needed to achieve a rate of 6 min , compared with 4 M of C-Bag (Fig. 4C). At 18 M HspBP1, the rate observed for DJA1 and Hsc70 was 10 min , whereas the maximum for the comparable DJA2 reaction was 8 min . Surprisingly, the behavior of Hsp110 was alto- gether different. As noted above, low concentrations of Hsp110 were strongly stimulatory of the Hsc70 ATPase, with DJA1 or DJA2. How- ever, the maximum rate observed for DJA1-stimulated Hsc70 was just FIGURE 4. NEFs diverge in Hsc70 ATPase stimulation. ADP production in 30 °C reactions containing the over 8 min at 4 M Hsp110, and indicated combinations of Hsc70 and co-chaperones was monitored by thin layer chromatography separation of radiolabeled ADP from ATP and phosphorimaging analysis. A, examples of ADP production over time in higher amounts of the NEF caused a reactions with 4 M Hsc70, 4 M DJA2, and either 4 M C-Bag, 4 M HspBP1, or 1 M Hsp110. Steady-state moderate reduction in the ATPase (linear) ATPase rates in Table 1 are based on similar experiments. B, Hsc70 ATPase rates were measured for rate instead of a further increase reactions with the indicated combinations of 4 M Hsc70, 4 M DJA1 or DJA2, 4 M C-Bag, 4 M HspBP1, or 0.5 M Hsp110. C and D, ATPase rates were measured with 4 M Hsc70, 4 M of either DJA1 or DJA2, and the (Fig. 4C). The effect with DJA2 was indicated amounts of C-Bag, HspBP1, and Hsp110. also clear; a peak rate of less than 7 min was attained at 1 to 2 M TABLE 1 Hsp110, and higher concentrations reduced ATPase rates Steady-state ATPase rates of Hsc70 in combination with the below this level (Fig. 4D). So, the three NEFs have quite differ- indicated co-chaperones ent characteristics in their regulation of the Hsc70 ATPase. Average enzymatic rates (min ) and standard deviations from the mean are shown. In the refolding assays, the highest Hsc70-DJA2 function was NEF 4 M DJA1 4 M DJA2 4 M DJA1–2 observed between 2 and 8 M C-Bag, with the highest point at 4 4 M Hsc70 1.1  0.1 1.0  0.2 1.2  0.1 M C-Bag (Fig. 2B). These conditions corresponded to DJA2- 4 M Hsc70 4 M C-Bag 6.1  0.3 4.8  0.2 5.5  0.6 4 M Hsc70 4 M HspBP1 2.0  0.2 1.7  0.2 2.2  0.1 stimulated Hsc70 ATPase rates between 3 and 9 min , with 4 M Hsc70 1 M Hsp110 8.0  1.1 6.7  0.3 6.6  0.2 the peak refolding activity 5 min (Fig. 4D). In comparison, refolding was stimulated by Hsp110 between a narrow concen- DJA1 or DJA2 (not shown). At 4 M C-Bag and 4 M DJA2, tration range of 0.5–2 M, with maximum activity at 1 M. which provided maximum refolding function in our conditions, The ATPase rates supported by these amounts of Hsp110 were 1 1 the Hsc70 ATPase rate was 4.8 min . In parallel Hsc70-DJA2 between 4 and 8 min , and peak refolding corresponded to 1 1 reactions, 4 M HspBP1 produced less stimulation of the Hsc70 just under 7 min . An Hsc70 ATPase rate 6 min could ATPase rate to 1.7 min , but 1M Hsp110 raised the rate more thus be the optimal rate for its refolding activity, regardless of strongly to 6.7 min . The Hsc70 ATPase rates with the differ- the NEF being used. However, HspBP1 was also able to raise the ent NEFs were slightly higher with DJA1 than with DJA2 (Fig. Hsc70 rate with DJA2 to this range, at higher concentrations 4B and Table 1). We previously observed that DJA1 was a some- of the NEF14 M but was still unable to increase the Hsc70 what better activator of the Hsc70 ATPase under saturating refolding function. Furthermore, 8 M Hsp110 was strongly C-Bag concentrations (11), and our data now confirm this inhibitory of the refolding function, although the corre- under conditions closer to that of the cell. sponding ATPase rate was close to 5 min and not far from We next tested the Hsc70 ATPase rate at the same fixed the proposed optimal rate. Finally, DJA1 appears generally concentrations of DJA2 and DJA1, over different concentra- similar to DJA2 in ATPase stimulation together with the tions of each NEF. The DJA1-stimulated Hsc70 ATPase rate NEFs despite its poor refolding function under all condi- OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27105 Function of Hsc70 Co-chaperones between its different domains, could be better adapted for this refolding reaction than in DJA1. To address these questions, we con- structed a chimeric DJA that had the N-terminal J domain and linker of DJA1 (residues 1–95) and the substrate-binding zinc finger, cen- tral, and C-terminal regions of DJA2 (residues 96–412) (Fig. 5A). We reasoned that the chimeric DJA1–2 should have the same substrate binding properties of DJA2 and should stimulate the Hsc70 ATPase like DJA1. However, if DJA1 and DJA2 have different interactions between their respective N- and C-terminal regions, these interac- tions will be mismatched in DJA1–2. On gel filtration chroma- tography, purified DJA1–2 had a profile similar to those of DJA1 and DJA2, consistent with native homodimers (not shown). DJA1–2 was therefore tested for the proper- ties of substrate binding, stimula- tion of the Hsc70 ATPase, and Hsc70-mediated refolding. Binding of the representative substrates PiC, ANT, GR, and MR to DJA1–2 was assayed as described in Fig. 1 and quantified relative to DJA2 binding (Fig. 5B). As expected, the levels of polypeptide binding were similar. Slightly less GR was bound by DJA1–2 than by DJA2, but it is not certain that this difference is significant. Next, the activity of the FIGURE 5. Activity of a DJA1-DJA2 chimera. A, upper panel, schematic of the domain architecture of DJA1, Hsc70 ATPase was tested as in Fig. DJA2 and the DJA1–2 mutant. The positions of the J domain, linker, middle domain with zinc fingers, and 4. At 4 M Hsc70 and 4 M DJA1–2, C-terminal domain are marked. DJA1–2 contains residues 1–95 of DJA1 and residues 96 – 412 of DJA2. Lower panel, CLUSTALW2 alignment of the N-terminal regions of DJA1 and DJA2. The linker sequences are in italics, a rate of 1.2 min was observed, and the sequence of DJA1–2 is underlined. B, binding of radiolabeled polypeptides to DJA2 and DJA1–2 was essentially the same as that with tested as in Fig. 1, and the amount bound by DJA1–2 was plotted relative to the amount bound by DJA2. C, DJA1 and DJA2 (Table 1). The addi- Hsc70 ATPase rates were measured as in Fig. 4 for reactions with 4 M Hsc70, 4 M DJA1–2, and the indicated amounts of C-Bag, HspBP1, and Hsp110. D and E, luciferase refolding was monitored as in Fig. 2, with the tion of various amounts of C-Bag, indicated combinations of Hsc70 and co-chaperones. D, refolding activity after 60 min with 4 M Hsc70, 4 M HspBP1, and Hsp110 NEF to the DJA1–2, and the indicated amounts of C-Bag, HspBP1, and Hsp110, was plotted relative to the Hsc70 and DJA2 control reactions. E, refolding activity after 60 min with 4 M Hsc70 and the indicated amounts of DJA2 and DJA1–2 and Hsc70 reaction DJA1–2 was plotted relative to the Hsc70 and DJA2 control reactions. boosted the ATPase rates (Fig. 5C), with a pattern closest to that of wild- tions. Therefore, for the DJA and the NEF co-chaperones, type DJA1. The ATPase rates rose progressively with higher the ability to activate Hsc70 enzymatically to a certain level C-Bag concentrations, reaching 7.9 min at 8 M C-Bag. As may be necessary for refolding function but is not in itself observed with the wild-type DJAs, HspBP1 was less effective at sufficient. stimulating the ATPase rates, with 8 M HspBP1 supporting a Activity of a DJA1-DJA2 Chimera—The unfavorable or neg- rate of 3.0 min . Hsp110 stimulated the ATPase activity ative effect of DJA1 on Hsc70-mediated refolding could not be strongly at low concentrations, but peak ATPase rates of 8 explained by lack of ATPase stimulation (Fig. 4). Because DJA1 min were observed at 2 M and 4 M Hsp110. This was sim- bound substrates somewhat more strongly than DJA2, it was ilar to the effect of Hsp110 on the DJA1 and Hsc70 reaction, possible that the stronger binding interfered with the refolding which also peaked at 4 M Hsp110 (Fig. 4C). For DJA2, maxi- reaction, or some other feature of DJA2, such as coordination mum stimulation was observed at 1 M Hsp110, and higher 27106 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 40 •OCTOBER 3, 2008 Function of Hsc70 Co-chaperones concentrations led to a decline in rate. Thus, DJA1–2 resembles functional difference, as observed in the refolding with Hsc70 of DJA2 in its polypeptide binding characteristics and DJA1 in its guanidine-denatured luciferase, but seems insufficient to fully ATPase stimulation in concert with NEFs. explain the difference. There may be an Hsc70 ATPase rate that DJA1–2 was next tested in the Hsc70-mediated refolding of is optimal for refolding when stimulated by DJA2 and either luciferase. Reactions contained 4 M Hsc70 and 4 M DJA1–2 Bag1 or Hsp110, but DJA1 with the NEFs can also stimulate and as above were compared with control reactions containing Hsc70 to the same extent. Because none of the NEFs can sup- 4 M Hsc70 and DJA2 after 60 min of refolding. Interestingly, port refolding by DJA1 and Hsc70, the difference in refolding is reactions with DJA1–2 showed little refolded protein, less than not due to the preference of the DJAs for a specific NEF. The 0.25 of the DJA2-Hsc70 control (Fig. 5D). The addition of possibility that stronger polypeptide binding by DJA1 com- C-Bag NEF over a range of concentrations improved refolding pared with DJA2 is the major hindrance to Hsc70-mediated with DJA1–2 only marginally; HspBP1 had no effect, whereas refolding is argued against by the poor refolding ability of the Hsp110 at higher concentrations appeared to reduce even the chimeric DJA1–2 mutant, which has the substrate-binding basal level of refolding. Thus, although the majority of DJA1–2 domain of DJA2. So, there are likely to be other properties of the was derived from DJA2 and its J domain was clearly active, the DJAs that are less well established but that contribute to their mechanism required for the refolding reaction was disrupted. functional difference. To determine whether DJA1–2 had a neutral or negative One such property may be coordination between the J effect on refolding, reactions with a constant level of 4 M domain, linker, and central regions of the DJAs. Such coordi- Hsc70 and different amounts of DJA1–2 and DJA2 were tested. nation may be important for the transfer of polypeptide from As was done for DJA1 and DJA2 (Fig. 3C), DJA1–2 ranged from the DJA to Hsc70. This idea agrees with our results and may 4to0M, and DJA2 from 0 to 4M, with the total concentration have a structural basis in the linker segment or domain surfaces of DJA remaining constant. Like DJA1, the DJA1–2 mutant of the DJAs. We hypothesize that the interdomain coordination appeared to inhibit DJA2-stimulated refolding (Fig. 5E), of DJA1 is somewhat different from that of DJA2, such that the although not quite as strongly as DJA1. For example, 1M DJA1 Hsc70-mediated refolding activity examined here is not sup- inhibited refolding reactions with 3 M DJA2 to less than 0.30 of ported. The ability of DJA1 to bind substrate, but not to transfer the control (Fig. 3, B and C), and 1 M DJA1–2 under the same substrate productively to Hsc70, could explain the inhibition of conditions reduced refolding to 0.52. Overall, the results with refolding observed in our mixed DJA refolding experiments. the chimeric DJA1–2 mutant suggest that the complete refold- This idea could also explain our observation that the DJA1–2 ing activity of DJA2 with Hsc70 requires more than the inde- mutant inhibited the refolding mediated by Hsc70 and DJA2. pendent function of its domains, but also another feature such Given that the DJA1–2 mutant has the J domain and linker of as interdomain coordination. The J domain and linker of DJA1 and central region from DJA2, we might expect there to DJA1–2, derived from DJA1, may be unable to properly interact be a lack of coordination between the domains and hence with the rest of the protein. Furthermore, the coordination impaired transfer of substrate to Hsc70. Nevertheless, the between the domains of wild-type DJA1 may be different domain coordination in wild-type DJA1 might be arranged for enough from that within DJA2 that it cannot function well in the chaperoning of a range of substrates or polypeptide confor- the refolding reaction studied here. NEFs can improve the func- mations different from that of DJA2. Indeed, we found that tion of DJA2 with Hsc70 but cannot overcome the deficiency of DJA1 may be more effective in the co-translational folding of DJA1 or DJA1–2. luciferase (11), which is distinct from the refolding of mature protein studied here. DISCUSSION We expect that interdomain coordination involves spe- The systematic investigation of the Hsc70 co-chaperones cific structures within the DJAs. One possibility would be the suggested three conclusions. First, DJA1 and DJA2 are func- Gly/Phe-rich linker between the J domain and central tionally different. This difference cannot be simply explained by region, including an Asp-Ile-Phe tripeptide motif. Work their polypeptide binding or Hsc70 stimulatory properties or with E. coli DnaJ and S. cerevisiae Ydj1 suggested that the preference for an NEF partner. Second, the Bag1 and Hsp110 Asp-Ile-Phe motif was important for the function of both NEFs are also functionally distinct from HspBP1. The appro- co-chaperones, potentially for substrate transfer to the part- priate ratios between Hsc70 and its co-chaperones may corre- ner Hsp70s (38, 39). The linker in the DJAs is conserved only late with an optimal ATPase rate of Hsc70, but cycling at this in the type 1 co-chaperones, suggesting a mechanism unique rate is not sufficient for complete chaperone function. Third, to this class and not found in type 2 proteins such as DJB1. our results suggest a more complex model of human Hsc70 Another possibility for interdomain coordination is contact function than is readily apparent in the E. coli DnaK system. between the J domains and the zinc finger regions of the DJA1 and DJA2 are distinct in the characteristics of their DJAs. These structures are close together in the quaternary separate domains: the stimulation of the Hsc70 ATPase by structure of the DJA1 homodimer (8) and presumably also in their J domains and the binding of a range of polypeptides by DJA2. J domains are thought to contact Hsc70 through a their central regions. These differences extend our earlier highly conserved surface (40), but other surfaces of the observation of variations between the DJAs, further supporting domain may form other interactions. our proposal of partial specialization between the DJAs. How- The Hsc70 NEFs are structurally very different from each ever, the variation in domain properties, although significant, is other, so mechanistic differences were less unexpected. We within a limited range. This variation may contribute to their propose that their biochemical properties observed here OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27107 Function of Hsc70 Co-chaperones 3. Qiu, X. B., Shao, Y. M., Miao, S., and Wang, L. (2006) Cell Mol. Life Sci. 63, agree with their apparent biological roles. The Bag1 NEF is 2560–2570 effective in promoting refolding, but at concentrations 4. Minami, Y., Hohfeld, J., Ohtsuka, K., and Hartl, F. U. (1996) J. Biol. Chem. equimolar to Hsc70. In cells, Bag domains are typically found 271, 19617–19624 in combination with other functional domains, and these 5. Terada, K., Kanazawa, M., Bukau, B., and Mori, M. (1997) J. Cell Biol. 139, proteins seem to have more specialized roles. Bag1 and Bag2 1089–1095 are involved in regulating protein degradation, whereas Bag4 6. Terada, K., and Mori, M. (2000) J. Biol. Chem. 275, 24728–24734 and Bag5 work in apoptotic signaling pathways (13, 16, 7. Hafizur, R. M., Yano, M., Gotoh, T., Mori, M., and Terada, K. (2004) J. Biochem. (Tokyo) 135, 193–200 41– 43). Such specialized proteins may be expressed at lower 8. Borges, J. C., Fischer, H., Craievich, A. F., and Ramos, C. H. (2005) J. Biol. levels and would only reach concentrations equal to Hsc70 in Chem. 280, 13671–13681 localized complexes. HspBP1 is the least efficient as an NEF 9. Li, J., Qian, X., and Sha, B. (2003) Structure 11, 1475–1483 and is inhibitory of Hsc70 refolding function under all con- 10. Wu, Y., Li, J., Jin, Z., Fu, Z., and Sha, B. (2005) J. Mol. Biol. 346, 1005–1011 ditions tested. Biologically, HspBP1 promotes the degrada- 11. Bhangoo, M. K., Tzankov, S., Fan, A. C., Dejgaard, K., Thomas, D. Y., and tion of proteins and blocks the anti-apoptotic function of Young, J. C. (2007) Mol. Biol. Cell 18, 3414–3428 12. Hohfeld, J., and Jentsch, S. (1997) EMBO J. 16, 6209–6216 stress-induced Hsp70 (44, 45). These functions are consist- 13. Luders, J., Demand, J., Papp, O., and Hohfeld, J. (2000) J. Biol. Chem. 275, ent with HspBP1 acting as an inhibitor of Hsc70. Such inhi- 14817–14823 bition of Hsc70 may be useful for the cell when degradation 14. Sondermann, H., Scheufler, C., Schneider, C., Hohfeld, J., Hartl, F. U., and as opposed to refolding becomes a better survival strategy. Moarefi, I. (2001) Science 291, 1553–1557 At low concentrations of NEF, Hsp110 is the strongest acti- 15. Gassler, C. S., Wiederkehr, T., Brehmer, D., Bukau, B., and Mayer, M. P. vator of the Hsc70 ATPase and promotes refolding at these (2001) J. Biol. Chem. 276, 32538–32544 16. Takayama, S., and Reed, J. C. (2001) Nat. Cell Biol. 3, E237–E241 same low concentrations. Thus, Hsp110 may be the general 17. 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(2007) Cell 131, 106–120 the cytosol. The Hsp110 orthologs Sse1 and Sse2 seem the 27. Andreasson, C., Fiaux, J., Rampelt, H., Mayer, M. P., and Bukau, B. (2008) most important biologically, and their combined deletion is J. Biol. Chem. 283, 8877–8884 28. Szabo, A., Langer, T., Schroder, H., Flanagan, J., Bukau, B., and Hartl, F. U. lethal (23, 24). The single Bag-related protein Snl1 is mem- (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10345–10349 brane-anchored and seems to have a specialized function in 29. Fan, A. C., Bhangoo, M. K., and Young, J. C. (2006) J. Biol. Chem. 281, the endoplasmic reticulum or nuclear membranes (41). The 33313–33324 HspBP1 ortholog Fes1 is moderately important; its deletion 30. de Marcos-Lousa, C., Sideris, D. P., and Tokatlidis, K. (2006) Trends Bio- causes temperature-sensitive growth, and it cannot fully chem. Sci. 31, 259–267 substitute for Sse1/Sse2 deletion (17, 19, 24). These observa- 31. 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M., and Ulrich Hartl, F. (2003) Trends Biochem. Sci. 44. Alberti, S., Bohse, K., Arndt, V., Schmitz, A., and Hohfeld, J. (2004) Mol. 28, 541–547 Biol. Cell 15, 4003–4010 42. Arndt, V., Daniel, C., Nastainczyk, W., Alberti, S., and Hohfeld, J. (2005) 45. Tanimura, S., Hirano, A. I., Hashizume, J., Yasunaga, M., Kawabata, T., Mol. Biol. Cell 16, 5891–5900 Ozaki, K., and Kohno, M. (2007) J. Biol. Chem. 282, 35430–35439 OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27109 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Functional Divergence between Co-chaperones of Hsc70

Tzankov, Stefan; Wong, Michael J.H.; Shi, Kun; Nassif, Christina; Young, Jason C.
Journal of Biological Chemistry – Oct 1, 2008
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 40, pp. 27100 –27109, October 3, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Received for publication, May 22, 2008, and in revised form, July 28, 2008 Published, JBC Papers in Press, August 6, 2008, DOI 10.1074/jbc.M803923200 Stefan Tzankov, Michael J. H. Wong, Kun Shi, Christina Nassif, and Jason C. Young From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada The ATPase cycle of the chaperone Hsc70 is regulated by contain J domains that stimulate ATP hydrolysis by Hsp70, and co-chaperones; Hsp40/DnaJ-related proteins stimulate ATP consequently substrate binding. Nucleotide exchange factors hydrolysis by Hsc70 and can bind unfolded polypeptides them- (NEFs), such as GrpE in E. coli, trigger the dissociation of selves. Conversely, various nucleotide exchange factors (NEFs) bound ADP from Hsp70 to allow the binding of ATP, resetting stimulate ADP-ATP exchange by Hsc70. We analyzed the puri- the cycle. The principles of this mechanism appear to be con- fied Hsp40-related co-chaperones DJA1 (Hdj2) and DJA2 served in Hsp70 chaperones, including the major cytosolic (Hdj3) and found that they had a distinct pattern of binding to a form in humans, Hsc70 (HSPA8) (1, 2). range of polypeptides. DJA2 alone could stimulate Hsc70-medi- The DnaJ-related co-chaperones are also conserved between ated refolding of luciferase in the absence of NEF, whereas DJA1 species. Type 1 J domain co-chaperones are homologous to was much less active. The addition of the Bag1 NEF increased DnaJ throughout their sequence and have the same domain refolding by Hsc70 and DJA2, as did the newly characterized architecture. Following their N-terminal J domains, they con- NEF Hsp110, but each NEF had a different optimal concentra- tain a linker sequence, zinc finger and central regions, and a tion ratio to Hsc70. Notably, the NEF HspBP1 could not C-terminal homodimerization region. Unfolded polypeptides increase refolding by Hsc70 and DJA2 at any concentration, and are thought to be bound by the central region of these proteins; none of the NEFs improved the refolding activity with DJA1. substrates are transferred to the Hsp70 partner upon J domain Instead, DJA1 was inhibitory of refolding with DJA2 and Hsc70. activation of ATP hydrolysis by the Hsp70 (1, 3). Although DnaJ All combinations of DJA1 or DJA2 with the three NEFs stimu- is the only J domain co-chaperone of E. coli DnaK, humans have lated the Hsc70 ATPase rate, although Hsp110 became less three cytosolic type 1 co-chaperones: DJA1 (DNAJA1, Hdj2, effective with increasing concentrations. A chimeric DJA2 hav- HSDJ), DJA2 (DNAJA2, Hdj3, HIRIP4), and DJA4 (DNAJA4, ing its Hsc70-stimulatory J domain replaced with that of DJA1 Hdj4). Comparison with the type 1 co-chaperone Ydj1 of Sac- was functional for polypeptide binding and ATPase stimulation charomyces cerevisiae suggests that the human DJAs will have of Hsc70. However, it could not support efficient Hsc70-medi- similar overall structures. DJA1 and DJA2 are constitutively ated refolding and also inhibited refolding with DJA2 and expressed, whereas DJA4 is less highly expressed and may be Hsc70. These results suggest a more complex model of Hsc70 specialized. In addition, eukaryotic type 2 J domain co-chaper- mechanism than has been previously thought, with notable ones are known that diverge from DnaJ in the substrate-binding functional divergence between Hsc70 co-chaperones. and C-terminal regions. The human type 2 member DJB1 (Hsp40, Hdj1) is expressed at low levels under normal condi- tions and is mainly expressed in the heat shock stress response. DJB1 also binds substrate more weakly than type 1 co-chaper- The Hsp70 family of proteins are ATP-dependent molecular ones. Thus, the DJA1 and DJA2 co-chaperones are thought to chaperones that assist the folding of polypeptides. Hsp70 chap- erones have a typical structure divided into ATPase and sub- be the major partners of human Hsc70 (3–10). The distinction between the human DJAs remains largely unexplored. Our strate-binding domains that work in an ATP-driven substrate recent work proposed a partial specialization between the binding cycle. The mechanism of Hsp70 proteins has been well human type 1 co-chaperones, perhaps to allow assistance of a established in studies of the Escherichia coli homolog DnaK. In the ATP-bound state, an Hsp70 chaperone has low affinity for wider range of substrates (11). Interestingly, the NEF co-chaperones are divergent between unfolded polypeptide. After hydrolysis of ATP, Hsp70 in the E. coli and humans. The three types of human NEFs: Bag ADP-bound state binds substrate with high affinity. Exchange domain proteins, HspBP1, and Hsp110, are structurally unre- of ADP for ATP then reverts Hsp70 to its low polypeptide affin- ity state. Conversion of an Hsp70 between these two nucleotide lated to each other and to the single E. coli NEF GrpE. The C-terminal domain of Bag1 (C-Bag) was the first shown to states is controlled by different co-chaperone proteins. The have NEF activity for Hsc70, and homologous Bag domains Hsp40/DnaJ-related co-chaperones, including E. coli DnaJ, have since been identified in several other human proteins. The mechanical action of C-Bag on Hsc70 appears to be equivalent * This work was supported by a Canadian Institutes of Health Research oper- to that of GrpE on DnaK, despite the difference in NEF struc- ating grant and the Canadian Foundation for Innovation. The costs of pub- lication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: NEF, nucleotide exchange factor; ANT, adenine Canada Research Chair in Molecular Chaperones. To whom correspond- nucleotide translocator; CiC, citrate carrier; ER, estrogen receptor ; GR, ence should be addressed: Dept. of Biochemistry, McGill University, glucocorticoid receptor  isoform; MR, mineralocorticoid receptor; OGC, 3655 Promenade Sir William Osler, Montreal, PQ H3G 1Y6, Canada. oxaloglutarate carrier; PiC, inorganic phosphate carrier; PR, progesterone E-mail: [email protected]. receptor; RL, reticulocyte lysate. 27100 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 40 •OCTOBER 3, 2008 This is an Open Access article under the CC BY license. Function of Hsc70 Co-chaperones ture (12–16). HspBP1 was subsequently found to have NEF 500 mM NaCl, 20 mM imidazole, and 20 mM KH PO , pH 7.5, 2 4 activity. The structural effect of HspBP1 on Hsc70 is distinct and eluted with 300 mM imidazole and 20 mM KH PO , pH 7.5, 2 4 from that of C-Bag (17–19). Most recently, Hsp110 (HSPH1, and then on a Mono Q 5/50 GL (GE Healthcare) equilibrated in Hsp105) was shown to be an NEF for Hsc70. Hsp110 repre- 50 mM NaCl and 20 mM KH PO , pH 7.5, and eluted with a 50 to 2 4 sents a subclass of proteins structurally related to Hsp70; how- 600 mM NaCl gradient. HspBP1 was dialyzed into buffer G, and ever, its ATPase activity and substrate binding are minimal. Hsp110 was further purified on a Superdex 200 Hi-Load 16/60 The Bag domain and HspBP1 NEFs are also thought to not bind column (GE Healthcare) equilibrated in buffer G. DNA encod- substrate. A recent structural study suggests a large conforma- ing the chimeric mutant DJA1–2 containing amino acids 1–95 tion shift within Hsp110 may be involved in its NEF function of DJA1 and 96–412 of DJA2 was constructed by overlapping (20–27). Although the biochemical mechanisms of these NEFs PCR and inserted into pPROEXHTa (Invitrogen), and the pro- have been recently revealed, little is known of the biological tein was purified similarly to DJA2. Where indicated, His tags difference between them. were removed by digestion with His-tagged TEV protease 4 °C The important question of how Hsc70 mechanically assists overnight followed by repurification on nickel-Sepharose. polypeptide folding is still under investigation. Studies of the Polypeptide Binding—Binding of various polypeptides to E. coli DnaK-DnaJ-GrpE system have provided the outline of a DJA1, DJA2, and DJA4 was tested as described (11). Plasmids model (1, 28). Increases in the steady-state ATPase rate of encoding bovine phosphate carrier A (PiC) and mouse adenine DnaK require both DnaJ and GrpE to promote the ATP hydrol- nucleotide carrier 2 (ANT) were as described (29); those for rat ysis and nucleotide exchange steps. However, refolding of the citrate carrier (CiC) and bovine oxaloglutarate carrier (OGC) model polypeptide luciferase is optimal at a particular ratio of were from Vincenzo Zara (Lecce, Italy); those for human glu- DnaK to DnaJ and GrpE and, by implication, at the ATPase rate cocorticoid receptor (GR), estrogen receptor  (ER), proges- supported by that ratio. At this rate, the balance between terone receptor (PR), and mineralocorticoid receptor (MR) nucleotide-dependent polypeptide binding and release by were from Theo Rein (Munich, Germany); and those for human Hsc70 appears to be optimal for substrate folding. Other factors cytochrome b and synaptobrevin 2 were from Stephen High may be the binding of polypeptide by DnaJ and its transfer to (Manchester, UK). Purified DJA1, DJA2, and DJA4 were pre- DnaK. bound on nickel-Sepharose in buffer containing 500 mM NaCl, The multiple DJAs and NEFs of human Hsc70 provide a fresh 20 mM Hepes-KOH, pH 7.5, and 5 mM MgOAc for 30 min at opportunity to examine this model. In the simplest formula- 4 °C. Cell-free translations of the various polypeptides were tion, an optimal Hsc70 ATPase rate for refolding should be performed with the TNT-coupled RL system with SP6 or T7 supported by different co-chaperone combinations. The func- polymerase (Promega) supplemented with [ S]methionine tions of DJA1 and DJA2 have been compared together with (GE Healthcare and PerkinElmer Life Sciences), diluted 1:20 Hsc70 and the Bag1 NEF (6) but not with HspBP1 or Hsp110. into buffer G containing 20 mM imidazole, 0.1% Triton X-100 We have reported that DJA1 was significantly less active than and 2 mg/ml ovalbumin, and added to the immobilized DJA DJA2 in promoting the Hsc70- and C-Bag-mediated refolding proteins. The final reactions contained 5 M wild-type DJA of luciferase, although both had polypeptide binding and protein and 5% translation mixture. After 5 min at room tem- ATPase stimulatory properties (11), but differences between perature, the binding reactions were terminated by the addition DJA1 and DJA2 with regard to the other NEFs are not known. of 0.1 unit/l apyrase. The protein complexes were recovered at Conversely, HspBP1 and Hsp110 have only been analyzed in 4 °C for 30 min and washed with buffer G containing 20 mM combination with the biologically less relevant DJB1/Hsp40, imidazole and 0.1% Triton X-100. The protein complexes were like most data on Bag1 (13, 15, 18, 19, 22, 23). We therefore analyzed by SDS-PAGE and phosphorimaging quantitation. carried out a systematic analysis of the major DJA1 and DJA2 Hsc70 Activation—Hsc70 activation by the co-chaperones co-chaperones with Hsc70 and the Bag1, HspBP1, and Hsp110 was tested as described (11). To assay polypeptide refolding, NEFs. firefly luciferase (Sigma) was denatured in buffer G containing 6 M guanidinium-HCl and 1 mM dithiothreitol for 10 min at room EXPERIMENTAL PROCEDURES temperature. Refolding reactions were preassembled on ice, Reagents and Proteins—Unless stated otherwise, all chemical containing 4 M Hsc70 and the indicated amounts of purified reagents were from Sigma or BioShop Canada Inc. (Missis- co-chaperones in buffer G supplemented with 39 mM NaCl and sauga, Canada). Restriction enzymes and other recombinant 2mM ATP. Luciferase was diluted 1:100 into reactions to a final DNA reagents were from New England Biolabs, Invitrogen, and concentration of 5.4 nM and incubated at 30 °C. Control reac- Stratagene. Untreated rabbit reticulocyte lysate (RL) was from tions contained 70% RL in the same buffer or treated with 0.1 Green Hectares (Oregon, WI) and desalted into buffer G (100 unit/l apyrase as a negative control. At the 60 min or indicated mM KOAc, 20 mM Hepes-KOH, pH 7.5, and 5 mM MgOAc )on time point, the aliquots were diluted 2:25 into luciferase assay a Hi-Prep 26/10 Fast Desalting column (GE Healthcare). reagent (Promega), and luciferase activity was measured in an His-tagged human DJA1, DJA2, DJA4, Hsc70, and C-Bag EG&G Berthold Lumat LB 9507 luminometer. were expressed in Rosetta 2 E. coli cells (Novagen) and purified To test the ATPase activity of Hsc70, the reactions were pre- as described (11). His-tagged human HspBP1 and Hsp110 (19, assembled on ice as described for the refolding assays, except 23) were expressed in Rosetta 2 E. coli cells for2hat37°Cand that they were supplemented with 5 Ci/ml [- P]ATP (Per- for4hat30 °C, respectively. They were purified by chromatog- kin Elmer) in addition to 2 mM ATP, and the reactions were raphy on nickel-Sepharose HP (GE Healthcare) equilibrated in initiated by incubation at 30 °C. At various time points, the OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27101 Function of Hsc70 Co-chaperones aliquots were removed and terminated with 37.5 mM EDTA. The samples were separated by thin layer chromatography on polyethylene-imine cellulose (Mallinckrodt Baker) developed in 0.5 M LiCl and 0.5 M formic acid. The ADP produced was determined by phosphorimaging quantitation, and the linear enzymatic rates (V ) were calculated by regression analysis. max RESULTS Polypeptide Binding by DJAs—DJA1 and DJA2, as well as DJA4, are homologous to each other in their J domains and their central polypeptide-binding regions. However, sequence FIGURE 1. Polypeptide binding by DJAs. The indicated polypeptides were radiolabeled by cell-free translation and co-precipitated either with nickel- divergence between DJA1 and DJA2 (55% identity, 69% similar- Sepharose alone or with purified His-tagged DJA1, DJA2, or DJA4 and nickel- ity) is almost as high as between each of them and Ydj1 (46– Sepharose. Bound polypeptide was quantified by SDS-PAGE and phospho- 47% identity, 63–65% similarity), which would suggest func- rimaging analysis. In these and all experiments, the error bars represent the standard deviations from the mean of at least three independent trials. The tional differences between DJA1 and DJA2. These proteins amount of polypeptide bound by negative control beads or by DJA1, DJA2, or were previously compared in their binding of two mitochon- DJA4 was plotted as a percentage of input translated material. cytb5, cyto- chrome b ; syb2, synaptobrevin 2. drial preproteins, which depend on the Hsc70-Hsp90 chaper- one system for their targeting (11, 29). DJA1 bound the PiC and ANT 2-fold better than DJA2, and DJA4 bound ANT better widely: 16% relative to input for PR, 25% for GR, 49% for ER, than PiC. and 66% for MR. The tail-anchored proteins cytochrome b and To obtain a more complete picture of DJA substrate binding synaptobrevin 2 were bound poorly by DJA1, at less than 6% of characteristics, a wider range of polypeptides, which were input and close to background binding by the nickel-Sepharose potential chaperone substrates, was examined. The mitochon- control. So, the interaction of the mitochondrial preproteins drial CiC and OGC, like PiC and ANT, belong to the structural and steroid hormone receptors with DJA1 seems to vary family of metabolite transporters of the mitochondrial inner between the different proteins, whereas the tail-anchored pro- membrane. They all have six transmembrane domains and teins are bound poorly by DJA1. remain unfolded before insertion into the membrane (30). CiC Polypeptide binding by DJA2 and DJA4 was next analyzed. and OGC preproteins also show involvement of the chaperone- As observed before, PiC binding by DJA2 was approximately Tom70 pathway in their mitochondrial import (31, 32). In con- half that by DJA1, and binding of CiC and OGC by DJA2 was trast to the mitochondrial preproteins, the steroid hormone now also observed at approximately half relative to DJA1 bind- receptors are cytosolic and nuclear. Several of these proteins, ing, respectively (Fig. 1). DJA2 binding of GR and ER was more including GR, ER, PR and MR, respectively, are known to similar to DJA1. Binding of PR by DJA2 was quite low, whereas depend on the Hsc70-Hsp90 system to maintain them in a hor- binding of MR was essentially identical to DJA1. Very little mone-activable state (33, 34). Their hormone-binding domains cytochrome b and synaptobrevin 2 was bound by DJA2. In appear to be the main chaperone binding sites and may be con- comparison, DJA4 binding also showed variation in the same formationally unstable in the absence of hormone. Certain range. Binding by DJA4 was comparable with DJA1 for CiC and transmembrane tail-anchored proteins such as synaptobrevin 2 OGC and progressively weaker for MR and ER. GR and PR are also assisted by Hsc70 during their insertion into the endo- were bound most weakly by DJA4 at approximately one-fourth plasmic reticulum membrane. Other tail-anchored proteins of DJA1 binding. Thus, the substrate binding profiles of DJA1 such as cytochrome b appear to be less dependent on chaper- and DJA2 are clearly different. Overall, DJA1 shows moderately ones for insertion (35). The binding of the above polypeptides stronger binding of these polypeptides, but with exceptions (PR by the purified DJAs was therefore tested with our previously and MR). DJA4 may be more similar to DJA1 in its profile, established method (11). although exceptions are also observed (GR and PR). The differ- The various polypeptides were radiolabeled by cell-free ences in binding are significant but mostly range within an translation in rabbit RL, a standard model cytosol for chaper- order of magnitude. This is consistent with our proposed idea one experiments. The labeled polypeptides were then co-pre- of partial specialization between the DJA co-chaperones. cipitated with purified His-tagged DJA1, DJA2, or DJA4 and Polypeptide Refolding by Hsc70, DJA2, and NEFs—We nickel-Sepharose. The existing chaperones in the RL compete recently reported that DJA2 was quite active in supporting the for substrate binding with the DJAs but also help maintain sol- refolding of chemically denatured luciferase by purified Hsc70, ubility of the substrates; the radiolabeling allows accurate quan- whereas DJA1 had very poor activity (11). Those experiments titation of the bound polypeptides by phosphorimaging analy- were performed using 4 M Hsc70, 4 M of either DJA1 or sis. The amount of bound polypeptide relative to input labeled DJA2, and 0.5 M of the Bag1 NEF domain (C-Bag) (13, 14). The material was determined, and the binding between the 8:1 ratio of Hsc70 to NEF had been thought to be optimal for co-chaperones was compared. Substrate binding by DJA1, Hsc70-mediated refolding, as well as for the E. coli DnaK-DnaJ- DJA2, and DJA4 relative to input is shown in Fig. 1. More CiC GrpE system (6). To compare DJA refolding functions with dif- and OGC were bound by DJA1 than the established preprotein ferent NEFs, we first tested refolding in the absence of any NEF. substrate PiC, over 80% of input in the case of OGC (Fig. 1). The The model cytosol RL was used as a positive control. Luciferase amounts of steroid hormone receptors bound by DJA1 varied was denatured in guanidine and rapidly diluted 1:100 to 5.4 nM 27102 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 40 •OCTOBER 3, 2008 Function of Hsc70 Co-chaperones above, but with increasing concen- trations of the C-Bag, HspBP1 and Hsp110 NEFs. The levels of refold- ing after 60 min relative to that of the Hsc70 and DJA2 control reac- tion without NEF was plotted (Fig. 2, B–D). Increasing amounts of C-Bag produced a moderate but sig- nificant increase in refolding that peaked at 1.24-fold above Hsc70 and DJA2 alone (Fig. 2B). The opti- mal C-Bag concentration for refold- ing was at 4 M, equimolar to Hsc70 and DJA2. 21 M inhibited refolding to 0.4 of the Hsc70-DJA2 control. HspBP1 had a very different behavior. Increasing HspBP1 amounts did not increase refolding by Hsc70-DJA2 at all, but progres- sively inhibited refolding to 0.6 of the control (Fig. 2C). For Hsp110, the result was different again. 1 M Hsp110 significantly raised refold- ing to 1.20-fold of the control (Fig. 2D), similar to the maximum effect of C-Bag. Higher levels of Hsp110 clearly inhibited refolding, to less than 0.5 of the control at 8 M and above. To confirm these results, the time course of luciferase refolding at optimal concentrations of C-Bag FIGURE 2. Polypeptide refolding by Hsc70, DJA2, and NEFs. Luciferase was denatured in 6 M guanidine and (4 M) and Hsp110 (1 M) with diluted 1:100 into refolding reactions containing RL or the indicated combinations of Hsc70 and co-chaper- Hsc70 and DJA2 was examined. The ones. Refolding at 30 °C was monitored by luciferase activity. A, activity upon refolding in RL or with 4M Hsc70 increase in refolding compared with alone or with 4M DJA1 or DJA2, was monitored over time and plotted as a percentage of the activity at 60 min of the RL positive control. B–D, refolding activity after 60 min with 4 M Hsc70, 4 M DJA2, and the indicated Hsc70-DJA2 alone was evident amounts of C-Bag, HspBP1, and Hsp110 was plotted relative to that with Hsc70 and DJA2 alone. The y axis throughout the time course and scales are identical. In this and all subsequent experiments, refolding after 60 min with 4 M Hsc70 and 4 M DJA2 was used as control reactions, with the amount of refolded luciferase activity set to 1. E, refolding activity particularly at the early (5 min and over time with 4 M Hsc70, 4 M DJA2, and either 4 M C-Bag or 1 M Hsp110 was plotted relative to Hsc70 and 15 min) time points (Fig. 2E). So, DJA2 alone at 60 min. For comparison, the data for Hsc70 and DJA2 alone from A is replotted here. both the Bag1 and Hsp110 NEF activities can boost refolding by in RL or reactions containing purified proteins, supplemented Hsc70 and DJA2, although specific concentration ratios of with 2 mM ATP, and refolding at 30 °C was monitored by the these NEFs to Hsc70 seem to be required. As seen for HspBP1, luciferase enzymatic activity (11). As expected, RL provided NEF activity does not necessarily promote Hsc70-mediated efficient refolding of luciferase, whereas 4 M Hsc70 alone refolding. could not (Fig. 2A). Remarkably, 4 M Hsc70 and 4 M DJA2 DJA1 Negatively Effects Hsc70-mediated Polypeptide Refold- was relatively effective at refolding, to above 70% of the RL ing—It was possible that one or more of the NEFs might have control by 60 min. This suggested that unlike for DnaK, an NEF a greater effect on Hsc70-mediated refolding with DJA1, may not be stringently required for Hsc70 function when an which had poor activity by itself. The refolding of chemically appropriate DJA partner is available. Consistent with earlier denatured luciferase was thus tested as above but with 4 M results, an equivalent amount of Hsc70 and DJA1 was inactive each of Hsc70 and DJA1 and increasing amounts of C-Bag, in refolding. HspBP1, and Hsp110. The levels of refolding after 60 min Although an NEF may not be absolutely required for the relative to that of Hsc70 and DJA2 control reactions were Hsc70-DJA2 pair, it may still improve the efficiency of the plotted. No amount of any of the NEFs could activate refold- refolding reaction. An increase in refolding of thermally ing by Hsc70 and DJA1 above 0.25 that of the Hsc70-DJA2 denatured and in some cases chemically denatured luciferase control (Fig. 3A). This clear difference between DJA1 and had been reported for Bag1 and Hsp110 together with Hsc70 DJA2 suggests some mechanistic divergence, despite their and DJB1/Hsp40 (13, 23). Therefore, chemically denatured luciferase refolding was tested with 4 M Hsc70 and DJA2 as sequence homology. OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27103 Function of Hsc70 Co-chaperones tions having different amounts of DJA1 and DJA2 were exam- ined. Reactions with 4 M Hsc70 and a constant 4 M total DJA proteins were analyzed (Fig. 3C), because optimization tests with 4 M Hsc70 had previously shown maximum refolding at 4 M DJA2 (Fig. 3B), whereas different concentrations of DJA1 in this range were equally ineffective with Hsc70 (not shown). In fact, we observed that DJA1 inhibited refolding when it was present. For example, reactions with 4 M Hsc70 and 2 M DJA2 alone produced significant refolding activity, 0.82 of the control, whereas further addition of 2 M DJA1 reduced refold- ing to 0.25. The addition of C-Bag or Hsp110 NEF did not restore the refolding activity inhibited by DJA1. The addition of optimal concentrations of C-Bag (4 M) and Hsp110 (1 M) further increased refolding by Hsc70 and DJA2 alone (Fig. 3B), but the presence of DJA1 still blocked refolding (Fig. 3C). Indeed, refolding in the presence of 2 M DJA1 was as poor when C-Bag or Hsp110 were added, as when the NEFs were absent. Thus, DJA1 is not only inactive in the Hsc70-mediated refolding tested here but appears to inhibit this particular func- tion of DJA2 and Hsc70 and perhaps other functions. We fur- ther ruled out effects of the His tags on the inhibition of refold- ing. Hsc70 with its His tag removed behaved similarly to tagged Hsc70. It was activated to refold luciferase by DJA2, but not DJA1. C-Bag and Hsp110 further increased its refolding with DJA2, but not HspBP1. HspBP1, after removal of the His tag, did not enhance refolding by Hsc70-DJA2. Similarly, DJA1, after removal of the His tag, still could not activate Hsc70 to refold luciferase and was inhibitory of the Hsc70-DJA2 refold- ing reaction (not shown). Nontagged DJA2 was not isolated in sufficient amounts to be tested and may be more active than tagged DJA2. NEFs Diverge in ATPase Stimulation of Hsc70—The results in Figs. 2 and 3 indicated that both C-Bag and Hsp110 were able to increase the refolding function of Hsc70 and DJA2 but not that with DJA1. With either of the DJA co-chaperones, the expected effect of an NEF would be to increase the overall ATPase rate of Hsc70. Earlier work found that the ADP-ATP exchange step can be rate-limiting for the steady-state Hsc70 (and DnaK) ATPase and that both an NEF and a DnaJ-related co-chaperone were required for ATPase stimulation (1, 36, 37). It was possible that increased refolding activity correlated with a specific ATPase rate of Hsc70; inability to reach this rate might explain the weaker refolding function of HspBP1 or, less probably, of DJA1. We therefore investigated the relationship between the Hsc70 ATPase rate and refolding activity at differ- FIGURE 3. DJA1 inhibits polypeptide refolding. Luciferase refolding was monitored as in Fig. 2, with the indicated combinations of Hsc70 and co- ent concentrations of each NEF. As established (11), the reac- chaperones. A, refolding activity after 60 min with 4M Hsc70, 4M DJA1, and tions were set up similar to the refolding reactions but lacking the indicated amounts of C-Bag, HspBP1, and Hsp110 was plotted relative to the Hsc70 and DJA2 control reactions. B, refolding activity after 60 min with denatured luciferase and containing [- P]ATP to monitor 4 M Hsc70, the indicated amounts of DJA2, and either no addition or 4 M ADP production over time at 30 °C (Fig. 4A). Linear (steady- C-Bag or 1 M Hsp110 was plotted relative to the Hsc70 and DJA2 control state) enzymatic rates were calculated, and representative rates reactions. C, refolding activity after 60 min with 4 M Hsc70, the indicated amounts of DJA2 and DJA1, and either 4 M C-Bag or 1 M Hsp110 was plot- are presented in Table 1. ted relative to the Hsc70 and DJA2 control reactions. As expected, 4 M Hsc70 with 4 M of either DJA1 or DJA2 had a low enzymatic rate in the absence of NEF, 1.1 and 1.0 The effect of DJA1 on an Hsc70-mediated refolding reaction min , respectively (Table 1 and Fig. 4B). Low background might be neutral or inhibitory. In the first case, a refolding reac- rates were also observed for the co-chaperones in the absence of tion with Hsc70 and DJA2 would remain unaffected by the Hsc70, and the addition of DJA1 or DJA2 and C-Bag stimulated addition of DJA1. In the latter case, DJA1 would interfere with the Hsc70 ATPase activity. In the absence of Hsc70, the back- the reaction. To address this question, luciferase refolding reac- ground ATPase rate of Hsp110 was not increased by either 27104 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 40 •OCTOBER 3, 2008 Function of Hsc70 Co-chaperones increased steadily with C-Bag con- centration until 10 M C-Bag, with higher concentrations causing only small increases in rate (Fig. 4C). A maximum rate of 11 min could be reached at high C-Bag lev- els. The C-Bag effect on Hsc70 with DJA2 was essentially the same, with a similar high rate observed above 10 M C-Bag (Fig. 4D). HspBP1 also raised the Hsc70 ATPase rate with increasing concentration but was markedly less efficient than C-Bag. With DJA1, 10 M HspBP1 was needed to achieve a rate of 6 min , compared with 4 M of C-Bag (Fig. 4C). At 18 M HspBP1, the rate observed for DJA1 and Hsc70 was 10 min , whereas the maximum for the comparable DJA2 reaction was 8 min . Surprisingly, the behavior of Hsp110 was alto- gether different. As noted above, low concentrations of Hsp110 were strongly stimulatory of the Hsc70 ATPase, with DJA1 or DJA2. How- ever, the maximum rate observed for DJA1-stimulated Hsc70 was just FIGURE 4. NEFs diverge in Hsc70 ATPase stimulation. ADP production in 30 °C reactions containing the over 8 min at 4 M Hsp110, and indicated combinations of Hsc70 and co-chaperones was monitored by thin layer chromatography separation of radiolabeled ADP from ATP and phosphorimaging analysis. A, examples of ADP production over time in higher amounts of the NEF caused a reactions with 4 M Hsc70, 4 M DJA2, and either 4 M C-Bag, 4 M HspBP1, or 1 M Hsp110. Steady-state moderate reduction in the ATPase (linear) ATPase rates in Table 1 are based on similar experiments. B, Hsc70 ATPase rates were measured for rate instead of a further increase reactions with the indicated combinations of 4 M Hsc70, 4 M DJA1 or DJA2, 4 M C-Bag, 4 M HspBP1, or 0.5 M Hsp110. C and D, ATPase rates were measured with 4 M Hsc70, 4 M of either DJA1 or DJA2, and the (Fig. 4C). The effect with DJA2 was indicated amounts of C-Bag, HspBP1, and Hsp110. also clear; a peak rate of less than 7 min was attained at 1 to 2 M TABLE 1 Hsp110, and higher concentrations reduced ATPase rates Steady-state ATPase rates of Hsc70 in combination with the below this level (Fig. 4D). So, the three NEFs have quite differ- indicated co-chaperones ent characteristics in their regulation of the Hsc70 ATPase. Average enzymatic rates (min ) and standard deviations from the mean are shown. In the refolding assays, the highest Hsc70-DJA2 function was NEF 4 M DJA1 4 M DJA2 4 M DJA1–2 observed between 2 and 8 M C-Bag, with the highest point at 4 4 M Hsc70 1.1  0.1 1.0  0.2 1.2  0.1 M C-Bag (Fig. 2B). These conditions corresponded to DJA2- 4 M Hsc70 4 M C-Bag 6.1  0.3 4.8  0.2 5.5  0.6 4 M Hsc70 4 M HspBP1 2.0  0.2 1.7  0.2 2.2  0.1 stimulated Hsc70 ATPase rates between 3 and 9 min , with 4 M Hsc70 1 M Hsp110 8.0  1.1 6.7  0.3 6.6  0.2 the peak refolding activity 5 min (Fig. 4D). In comparison, refolding was stimulated by Hsp110 between a narrow concen- DJA1 or DJA2 (not shown). At 4 M C-Bag and 4 M DJA2, tration range of 0.5–2 M, with maximum activity at 1 M. which provided maximum refolding function in our conditions, The ATPase rates supported by these amounts of Hsp110 were 1 1 the Hsc70 ATPase rate was 4.8 min . In parallel Hsc70-DJA2 between 4 and 8 min , and peak refolding corresponded to 1 1 reactions, 4 M HspBP1 produced less stimulation of the Hsc70 just under 7 min . An Hsc70 ATPase rate 6 min could ATPase rate to 1.7 min , but 1M Hsp110 raised the rate more thus be the optimal rate for its refolding activity, regardless of strongly to 6.7 min . The Hsc70 ATPase rates with the differ- the NEF being used. However, HspBP1 was also able to raise the ent NEFs were slightly higher with DJA1 than with DJA2 (Fig. Hsc70 rate with DJA2 to this range, at higher concentrations 4B and Table 1). We previously observed that DJA1 was a some- of the NEF14 M but was still unable to increase the Hsc70 what better activator of the Hsc70 ATPase under saturating refolding function. Furthermore, 8 M Hsp110 was strongly C-Bag concentrations (11), and our data now confirm this inhibitory of the refolding function, although the corre- under conditions closer to that of the cell. sponding ATPase rate was close to 5 min and not far from We next tested the Hsc70 ATPase rate at the same fixed the proposed optimal rate. Finally, DJA1 appears generally concentrations of DJA2 and DJA1, over different concentra- similar to DJA2 in ATPase stimulation together with the tions of each NEF. The DJA1-stimulated Hsc70 ATPase rate NEFs despite its poor refolding function under all condi- OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27105 Function of Hsc70 Co-chaperones between its different domains, could be better adapted for this refolding reaction than in DJA1. To address these questions, we con- structed a chimeric DJA that had the N-terminal J domain and linker of DJA1 (residues 1–95) and the substrate-binding zinc finger, cen- tral, and C-terminal regions of DJA2 (residues 96–412) (Fig. 5A). We reasoned that the chimeric DJA1–2 should have the same substrate binding properties of DJA2 and should stimulate the Hsc70 ATPase like DJA1. However, if DJA1 and DJA2 have different interactions between their respective N- and C-terminal regions, these interac- tions will be mismatched in DJA1–2. On gel filtration chroma- tography, purified DJA1–2 had a profile similar to those of DJA1 and DJA2, consistent with native homodimers (not shown). DJA1–2 was therefore tested for the proper- ties of substrate binding, stimula- tion of the Hsc70 ATPase, and Hsc70-mediated refolding. Binding of the representative substrates PiC, ANT, GR, and MR to DJA1–2 was assayed as described in Fig. 1 and quantified relative to DJA2 binding (Fig. 5B). As expected, the levels of polypeptide binding were similar. Slightly less GR was bound by DJA1–2 than by DJA2, but it is not certain that this difference is significant. Next, the activity of the FIGURE 5. Activity of a DJA1-DJA2 chimera. A, upper panel, schematic of the domain architecture of DJA1, Hsc70 ATPase was tested as in Fig. DJA2 and the DJA1–2 mutant. The positions of the J domain, linker, middle domain with zinc fingers, and 4. At 4 M Hsc70 and 4 M DJA1–2, C-terminal domain are marked. DJA1–2 contains residues 1–95 of DJA1 and residues 96 – 412 of DJA2. Lower panel, CLUSTALW2 alignment of the N-terminal regions of DJA1 and DJA2. The linker sequences are in italics, a rate of 1.2 min was observed, and the sequence of DJA1–2 is underlined. B, binding of radiolabeled polypeptides to DJA2 and DJA1–2 was essentially the same as that with tested as in Fig. 1, and the amount bound by DJA1–2 was plotted relative to the amount bound by DJA2. C, DJA1 and DJA2 (Table 1). The addi- Hsc70 ATPase rates were measured as in Fig. 4 for reactions with 4 M Hsc70, 4 M DJA1–2, and the indicated amounts of C-Bag, HspBP1, and Hsp110. D and E, luciferase refolding was monitored as in Fig. 2, with the tion of various amounts of C-Bag, indicated combinations of Hsc70 and co-chaperones. D, refolding activity after 60 min with 4 M Hsc70, 4 M HspBP1, and Hsp110 NEF to the DJA1–2, and the indicated amounts of C-Bag, HspBP1, and Hsp110, was plotted relative to the Hsc70 and DJA2 control reactions. E, refolding activity after 60 min with 4 M Hsc70 and the indicated amounts of DJA2 and DJA1–2 and Hsc70 reaction DJA1–2 was plotted relative to the Hsc70 and DJA2 control reactions. boosted the ATPase rates (Fig. 5C), with a pattern closest to that of wild- tions. Therefore, for the DJA and the NEF co-chaperones, type DJA1. The ATPase rates rose progressively with higher the ability to activate Hsc70 enzymatically to a certain level C-Bag concentrations, reaching 7.9 min at 8 M C-Bag. As may be necessary for refolding function but is not in itself observed with the wild-type DJAs, HspBP1 was less effective at sufficient. stimulating the ATPase rates, with 8 M HspBP1 supporting a Activity of a DJA1-DJA2 Chimera—The unfavorable or neg- rate of 3.0 min . Hsp110 stimulated the ATPase activity ative effect of DJA1 on Hsc70-mediated refolding could not be strongly at low concentrations, but peak ATPase rates of 8 explained by lack of ATPase stimulation (Fig. 4). Because DJA1 min were observed at 2 M and 4 M Hsp110. This was sim- bound substrates somewhat more strongly than DJA2, it was ilar to the effect of Hsp110 on the DJA1 and Hsc70 reaction, possible that the stronger binding interfered with the refolding which also peaked at 4 M Hsp110 (Fig. 4C). For DJA2, maxi- reaction, or some other feature of DJA2, such as coordination mum stimulation was observed at 1 M Hsp110, and higher 27106 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 40 •OCTOBER 3, 2008 Function of Hsc70 Co-chaperones concentrations led to a decline in rate. Thus, DJA1–2 resembles functional difference, as observed in the refolding with Hsc70 of DJA2 in its polypeptide binding characteristics and DJA1 in its guanidine-denatured luciferase, but seems insufficient to fully ATPase stimulation in concert with NEFs. explain the difference. There may be an Hsc70 ATPase rate that DJA1–2 was next tested in the Hsc70-mediated refolding of is optimal for refolding when stimulated by DJA2 and either luciferase. Reactions contained 4 M Hsc70 and 4 M DJA1–2 Bag1 or Hsp110, but DJA1 with the NEFs can also stimulate and as above were compared with control reactions containing Hsc70 to the same extent. Because none of the NEFs can sup- 4 M Hsc70 and DJA2 after 60 min of refolding. Interestingly, port refolding by DJA1 and Hsc70, the difference in refolding is reactions with DJA1–2 showed little refolded protein, less than not due to the preference of the DJAs for a specific NEF. The 0.25 of the DJA2-Hsc70 control (Fig. 5D). The addition of possibility that stronger polypeptide binding by DJA1 com- C-Bag NEF over a range of concentrations improved refolding pared with DJA2 is the major hindrance to Hsc70-mediated with DJA1–2 only marginally; HspBP1 had no effect, whereas refolding is argued against by the poor refolding ability of the Hsp110 at higher concentrations appeared to reduce even the chimeric DJA1–2 mutant, which has the substrate-binding basal level of refolding. Thus, although the majority of DJA1–2 domain of DJA2. So, there are likely to be other properties of the was derived from DJA2 and its J domain was clearly active, the DJAs that are less well established but that contribute to their mechanism required for the refolding reaction was disrupted. functional difference. To determine whether DJA1–2 had a neutral or negative One such property may be coordination between the J effect on refolding, reactions with a constant level of 4 M domain, linker, and central regions of the DJAs. Such coordi- Hsc70 and different amounts of DJA1–2 and DJA2 were tested. nation may be important for the transfer of polypeptide from As was done for DJA1 and DJA2 (Fig. 3C), DJA1–2 ranged from the DJA to Hsc70. This idea agrees with our results and may 4to0M, and DJA2 from 0 to 4M, with the total concentration have a structural basis in the linker segment or domain surfaces of DJA remaining constant. Like DJA1, the DJA1–2 mutant of the DJAs. We hypothesize that the interdomain coordination appeared to inhibit DJA2-stimulated refolding (Fig. 5E), of DJA1 is somewhat different from that of DJA2, such that the although not quite as strongly as DJA1. For example, 1M DJA1 Hsc70-mediated refolding activity examined here is not sup- inhibited refolding reactions with 3 M DJA2 to less than 0.30 of ported. The ability of DJA1 to bind substrate, but not to transfer the control (Fig. 3, B and C), and 1 M DJA1–2 under the same substrate productively to Hsc70, could explain the inhibition of conditions reduced refolding to 0.52. Overall, the results with refolding observed in our mixed DJA refolding experiments. the chimeric DJA1–2 mutant suggest that the complete refold- This idea could also explain our observation that the DJA1–2 ing activity of DJA2 with Hsc70 requires more than the inde- mutant inhibited the refolding mediated by Hsc70 and DJA2. pendent function of its domains, but also another feature such Given that the DJA1–2 mutant has the J domain and linker of as interdomain coordination. The J domain and linker of DJA1 and central region from DJA2, we might expect there to DJA1–2, derived from DJA1, may be unable to properly interact be a lack of coordination between the domains and hence with the rest of the protein. Furthermore, the coordination impaired transfer of substrate to Hsc70. Nevertheless, the between the domains of wild-type DJA1 may be different domain coordination in wild-type DJA1 might be arranged for enough from that within DJA2 that it cannot function well in the chaperoning of a range of substrates or polypeptide confor- the refolding reaction studied here. NEFs can improve the func- mations different from that of DJA2. Indeed, we found that tion of DJA2 with Hsc70 but cannot overcome the deficiency of DJA1 may be more effective in the co-translational folding of DJA1 or DJA1–2. luciferase (11), which is distinct from the refolding of mature protein studied here. DISCUSSION We expect that interdomain coordination involves spe- The systematic investigation of the Hsc70 co-chaperones cific structures within the DJAs. One possibility would be the suggested three conclusions. First, DJA1 and DJA2 are func- Gly/Phe-rich linker between the J domain and central tionally different. This difference cannot be simply explained by region, including an Asp-Ile-Phe tripeptide motif. Work their polypeptide binding or Hsc70 stimulatory properties or with E. coli DnaJ and S. cerevisiae Ydj1 suggested that the preference for an NEF partner. Second, the Bag1 and Hsp110 Asp-Ile-Phe motif was important for the function of both NEFs are also functionally distinct from HspBP1. The appro- co-chaperones, potentially for substrate transfer to the part- priate ratios between Hsc70 and its co-chaperones may corre- ner Hsp70s (38, 39). The linker in the DJAs is conserved only late with an optimal ATPase rate of Hsc70, but cycling at this in the type 1 co-chaperones, suggesting a mechanism unique rate is not sufficient for complete chaperone function. Third, to this class and not found in type 2 proteins such as DJB1. our results suggest a more complex model of human Hsc70 Another possibility for interdomain coordination is contact function than is readily apparent in the E. coli DnaK system. between the J domains and the zinc finger regions of the DJA1 and DJA2 are distinct in the characteristics of their DJAs. These structures are close together in the quaternary separate domains: the stimulation of the Hsc70 ATPase by structure of the DJA1 homodimer (8) and presumably also in their J domains and the binding of a range of polypeptides by DJA2. J domains are thought to contact Hsc70 through a their central regions. These differences extend our earlier highly conserved surface (40), but other surfaces of the observation of variations between the DJAs, further supporting domain may form other interactions. our proposal of partial specialization between the DJAs. How- The Hsc70 NEFs are structurally very different from each ever, the variation in domain properties, although significant, is other, so mechanistic differences were less unexpected. We within a limited range. This variation may contribute to their propose that their biochemical properties observed here OCTOBER 3, 2008• VOLUME 283 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 27107 Function of Hsc70 Co-chaperones 3. Qiu, X. B., Shao, Y. M., Miao, S., and Wang, L. (2006) Cell Mol. Life Sci. 63, agree with their apparent biological roles. The Bag1 NEF is 2560–2570 effective in promoting refolding, but at concentrations 4. Minami, Y., Hohfeld, J., Ohtsuka, K., and Hartl, F. U. (1996) J. Biol. Chem. equimolar to Hsc70. In cells, Bag domains are typically found 271, 19617–19624 in combination with other functional domains, and these 5. Terada, K., Kanazawa, M., Bukau, B., and Mori, M. (1997) J. Cell Biol. 139, proteins seem to have more specialized roles. Bag1 and Bag2 1089–1095 are involved in regulating protein degradation, whereas Bag4 6. Terada, K., and Mori, M. (2000) J. Biol. Chem. 275, 24728–24734 and Bag5 work in apoptotic signaling pathways (13, 16, 7. Hafizur, R. M., Yano, M., Gotoh, T., Mori, M., and Terada, K. (2004) J. Biochem. (Tokyo) 135, 193–200 41– 43). Such specialized proteins may be expressed at lower 8. Borges, J. C., Fischer, H., Craievich, A. F., and Ramos, C. H. (2005) J. Biol. levels and would only reach concentrations equal to Hsc70 in Chem. 280, 13671–13681 localized complexes. HspBP1 is the least efficient as an NEF 9. Li, J., Qian, X., and Sha, B. (2003) Structure 11, 1475–1483 and is inhibitory of Hsc70 refolding function under all con- 10. Wu, Y., Li, J., Jin, Z., Fu, Z., and Sha, B. (2005) J. Mol. Biol. 346, 1005–1011 ditions tested. Biologically, HspBP1 promotes the degrada- 11. Bhangoo, M. K., Tzankov, S., Fan, A. C., Dejgaard, K., Thomas, D. Y., and tion of proteins and blocks the anti-apoptotic function of Young, J. C. (2007) Mol. Biol. Cell 18, 3414–3428 12. Hohfeld, J., and Jentsch, S. (1997) EMBO J. 16, 6209–6216 stress-induced Hsp70 (44, 45). These functions are consist- 13. Luders, J., Demand, J., Papp, O., and Hohfeld, J. (2000) J. Biol. Chem. 275, ent with HspBP1 acting as an inhibitor of Hsc70. Such inhi- 14817–14823 bition of Hsc70 may be useful for the cell when degradation 14. Sondermann, H., Scheufler, C., Schneider, C., Hohfeld, J., Hartl, F. U., and as opposed to refolding becomes a better survival strategy. Moarefi, I. (2001) Science 291, 1553–1557 At low concentrations of NEF, Hsp110 is the strongest acti- 15. Gassler, C. S., Wiederkehr, T., Brehmer, D., Bukau, B., and Mayer, M. P. vator of the Hsc70 ATPase and promotes refolding at these (2001) J. Biol. Chem. 276, 32538–32544 16. Takayama, S., and Reed, J. C. (2001) Nat. Cell Biol. 3, E237–E241 same low concentrations. Thus, Hsp110 may be the general 17. Kabani, M., McLellan, C., Raynes, D. A., Guerriero, V., and Brodsky, J. L. NEF for Hsc70 chaperoning function but with its expression (2002) FEBS Lett. 531, 339–342 levels tightly regulated. Hsp110 is inhibitory of both the 18. Raynes, D. A., and Guerriero, V., Jr. (1998) J. Biol. Chem. 273, refolding and ATPase activities of Hsc70 at higher concen- 32883–32888 trations. It is possible that Hsp110 at high concentrations 19. Shomura, Y., Dragovic, Z., Chang, H. C., Tzvetkov, N., Young, J. C., Brod- binds substrates through its C-terminal region, competing sky, J. L., Guerriero, V., Hartl, F. U., and Bracher, A. (2005) Mol. Cell 17, 367–379 with Hsc70. Overexpression of Hsp110 may be another 20. Oh, H. J., Chen, X., and Subjeck, J. R. (1997) J. Biol. Chem. 272, method for the cell to switch Hsc70 from a folding to a deg- 31636–31640 radation strategy. 21. Yamagishi, N., Nishihori, H., Ishihara, K., Ohtsuka, K., and Hatayama, T. 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(2007) Cell 131, 106–120 the cytosol. The Hsp110 orthologs Sse1 and Sse2 seem the 27. Andreasson, C., Fiaux, J., Rampelt, H., Mayer, M. P., and Bukau, B. (2008) most important biologically, and their combined deletion is J. Biol. Chem. 283, 8877–8884 28. Szabo, A., Langer, T., Schroder, H., Flanagan, J., Bukau, B., and Hartl, F. U. lethal (23, 24). The single Bag-related protein Snl1 is mem- (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10345–10349 brane-anchored and seems to have a specialized function in 29. Fan, A. C., Bhangoo, M. K., and Young, J. C. (2006) J. Biol. Chem. 281, the endoplasmic reticulum or nuclear membranes (41). The 33313–33324 HspBP1 ortholog Fes1 is moderately important; its deletion 30. de Marcos-Lousa, C., Sideris, D. P., and Tokatlidis, K. (2006) Trends Bio- causes temperature-sensitive growth, and it cannot fully chem. Sci. 31, 259–267 substitute for Sse1/Sse2 deletion (17, 19, 24). These observa- 31. Palmisano, A., Zara, V., Honlinger, A., Vozza, A., Dekker, P. J., Pfanner, N., and Palmieri, F. (1998) Biochem. J. 333, 151–158 tions are consistent with the divergence between human 32. Zara, V., Ferramosca, A., Palmisano, I., Palmieri, F., and Rassow, J. (2003) Hsp110 as a general NEF, Bag proteins as functionally spe- J. Mol. Biol. 325, 399–408 cialized, and HspBP1 as a distinct inhibitory factor. The 33. Pratt, W. B., and Toft, D. O. (2003) Exp. Biol. Med. (Maywood) 228, human DJAs also seem to have diverged in their exact 111–133 mechanisms. 34. Picard, D. (2006) Trends Endocrinol. Metab. 17, 229–235 35. Abell, B. M., Rabu, C., Leznicki, P., Young, J. C., and High, S. (2007) J. Cell Acknowledgments—We thank Melanie Bhangoo and Anna Fan for Sci. 120, 1743–1751 technical assistance. 36. Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2874–2878 37. 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