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© The Rockefeller University Press,
0021-9525/2000//591 $5.00
The Journal of Cell Biology, Volume 149, Number 3,
, 2000 591-602
Original Article |
The Interaction of the Chaperonin Tailless Complex Polypeptide 1 (Tcp1) Ring Complex (Tric) with Ribosome-Bound Nascent Chains Examined Using Photo-Cross-Linking
jfrydman{at}leland.stanford.edu
The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) (also called chaperonin containing TCP1 [CCT]) is a hetero-oligomeric complex that facilitates the proper folding of many cellular proteins. To better understand the manner in which TRiC interacts with newly translated polypeptides, we examined its association with nascent chains using a photo-cross-linking approach. To this end, a series of ribosome-bound nascent chains of defined lengths was prepared using truncated mRNAs. Photoactivatable probes were incorporated into these 35S- labeled nascent chains during translation. Upon photolysis, TRiC was cross-linked to ribosome-bound polypeptides exposing at least 50–90 amino acids outside the ribosomal exit channel, indicating that the chaperonin associates with much shorter nascent chains than indicated by previous studies. Cross-links were observed for nascent chains of the cytosolic proteins actin, luciferase, and enolase, but not to ribosome-bound preprolactin. The pattern of cross-links became more complex as the nascent chain increased in length. These results suggest a chain length–dependent increase in the number of TRiC subunits involved in the interaction that is consistent with the idea that the substrate participates in subunit-specific contacts with the chaperonin. Both ribosome isolation by centrifugation through sucrose cushions and immunoprecipitation with anti-puromycin antibodies demonstrated that the photoadducts form on ribosome-bound polypeptides. Our results indicate that TRiC/CCT associates with the translating polypeptide shortly after it emerges from the ribosome and suggest a close association between the chaperonin and the translational apparatus.
Key Words: protein folding • actin • luciferase • translation • chaperonin
© 2000 The Rockefeller University Press
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Introduction |
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The Hsc70 class of molecular chaperones has been the focus of extensive studies (Hartl 1996; Bukau and Horwich 1998). In contrast, little is known about the mechanism and binding determinants of the chaperonins found in eukaryotic cells (reviewed in Willison and Horwich 1996; Gutsche et al. 1999). The chaperonin TRiC/CCT is a ring-shaped complex that consists of eight different, yet homologous, subunits ranging between 50 and 60 kD (Frydman et al. 1992; Gao et al. 1992; Lewis et al. 1992). The substrate binds in the central cavity and is folded by the chaperonin in an ATP-dependent manner. Unlike Hsc70, TRiC does not interact with short extended peptides or proteins (Frydman et al. 1994; Frydman and Hartl 1996; Rommelaere et al. 1999). The mode of interaction between TRiC and its substrates remains to be determined. TRiC was originally proposed to be a chaperone specialized for the folding of actin and tubulin (Lewis et al. 1996), but recent experiments suggest a broader substrate spectrum in vivo (Farr et al. 1997; Won et al. 1998; Srikakulam and Winkelmann 1999; Thulasiraman et al. 1999).
Whereas the mechanism of chaperone-mediated folding was classically studied using full-length denatured protein substrates, in the cell, proteins enter the cytosol vectorially during translation. The vectorial nature of the translation process constrains the folding of the nascent chain, as the NH2 terminus enters the cytosol first and the initial folding attempts may be localized at the NH2-terminal end of the polypeptide (Frydman et al. 1994; Netzer and Hartl 1997; Nicola et al. 1999). However, the cooperative nature of the interactions that stabilize folded structures makes it necessary that a complete folding domain (
50–300 amino acids) is available for productive folding into a native tertiary structure (Jaenicke 1991). Recent studies showing that the process of translation influences the folding pathway (Frydman et al. 1999) underscore the importance of elucidating the mechanism of protein folding in the context of translation. Clearly, this will require a better understanding of how chaperones interact with polypeptides as they emerge from the ribosome. A number of studies indicate that the molecular chaperones Hsc70 and TRiC bind to ribosome-associated polypeptides during translation (Frydman et al. 1994; Hansen et al. 1994; Dobrzynski et al. 1996; Frydman and Hartl 1996; James et al. 1997; Pfund et al. 1998; Yan et al. 1998). However, it has been proposed recently that TRiC interacts posttranslationally with newly made polypeptides, whereas the nascent chains interact cotranslationally with a novel chaperone complex named genes involved in microtubule biogenesis complex (GIMc) prefoldin (Hansen et al. 1999). Many of these studies have relied primarily on techniques such as immunoprecipitation and nondenaturing PAGE, where chaperone-bound ligands (i.e., nascent chains) are separated at some point from free ligands. In such experiments, complexes are detected only if their dissociation rates are slow compared with the rate of separation of free and bound ligands. These techniques therefore favor the detection of high-affinity interactions.
To clarify the controversial interaction of TRiC with nascent chains, we have used another approach, photo-cross-linking, that can detect highly dynamic and transient nascent chain–chaperone interactions. As described herein, this approach reveals that TRiC interacts with ribosome-bound nascent chains. The interaction begins at a much earlier stage than we had previously detected using other techniques. Furthermore, these short nascent chains cross-link to TRiC even before they can form stable high-affinity complexes with the chaperonin, suggesting that TRiC is positioned in close proximity to the site on the ribosome from which the nascent chain emerges. Our results provide further support for the notion that the chaperone machinery is functionally coupled to translation and may even interact directly with the ribosome.
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Materials and Methods |
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mAb (23C) was obtained from StressGen Biotechnologies (Lewis et al. 1992). Dog pancreas signal recognition particle (SRP) was purified as described (Walter and Blobel 1983). All other reagents were of the highest quality available.
Preparation of mRNA
pGEM-mouse β-actin was linearized in the coding region by digestion with BglII, SnaBI, Asp718, ScaI, or ApaLI, and pGEM-luciferase was linearized by digestion with HnfI, BslI, BbvI, AflIII, EcoRI, or BspEI restriction endonucleases (New England Biolabs, Inc.). Truncated mRNAs coding for nascent actin polypeptides were generated by RNA transcription of these linearized plasmids in vitro, using SP6 RNA polymerase as before (Krieg et al. 1989; Frydman and Hartl 1996). The nascent chains thus generated for actin contained 84, 133, 220, 301, 337, or 371 total amino acids and contained 5, 7, 10, 13, 17, or 18 lysine residues, respectively. The nascent luciferase polypeptides contained 77, 92, 125, 164, 197, or 232 amino acids and 6, 6, 6, 13, 14, or 15 lysines, respectively. pBSK-enolase (p-eno46; Holland et al. 1981) was linearized by digestion with DdeI, EcoRI, or BglI to yield polypeptides of 137, 251, or 375 amino acids (and 12, 22, and 34 lysines, respectively) by in vitro RNA transcription with T3 polymerase. A preprolactin (pPL) nascent chain of 86 amino acids (four lysines) was generated as described previously (Krieg et al. 1989).
Translations, Photolysis, and Analysis
Yeast tRNALys was purified and aminoacylated as described elsewhere (Crowley et al. 1993) and then modified as before (Krieg et al. 1986) to yield photoreactive N
-(5-azido-2-nitrobenzoyl)-Lys-tRNALys (ANB-Lys-tRNALys) (750–800 pmol Lys/A260 unit of tRNA; 75–80% -labeled). Nuclease-treated rabbit reticulocyte lysate was pretreated to remove endogenous lysine using a Sephadex G-25 spin column at 4°C (typically 1 ml lysate was placed on a 5-ml column of Sephadex G-25 prespun in water and centrifuged at 1,750 g for 1 min). Translations contained 50% (vol/vol) nuclease-treated and desalted lysate, 80 mM KOAc, pH 7.5, 1 mM Mg(OAc)2, 50 µM hemin hydrochloride, 2 µCi/µL [35S]methionine, 0.6 µM ANB-Lys-tRNA, and an energy generating system containing all amino acids except lysine and methionine as described elsewhere (Crowley et al. 1993). After translation in the dark for 40 min at 26°C, the ATP present in the lysate was depleted by addition of apyrase to a final concentration of 0.1 U/µL and incubation for 5 min at 26°C. When indicated, DTT (20 mM final) was added to the lysate to inactivate the cross-linker, either before the translation reaction or after photolysis. The presence or absence of 2 mM cycloheximide during photolysis did not affect the outcome of the experiments. In some experiments, puromycin was added to a final concentration of 2 mM and incubated for 20 min at 26°C, either before or immediately after photolysis. The effect of ATP on the cross-linking reaction was assessed by either omitting the apyrase treatment before photolysis (which left the endogenous ATP regenerating system functional) or by supplementing with additional ATP (1 mM) and Mg(OAc)2 (2 mM) before photolysis. Both conditions yielded similar results. In either case apyrase was added after photolysis.
After photolysis for 10 min at 0°C as before (Do et al. 1996), unlabeled methionine (2 mM) and DTT were added to the reactions. Samples (4 µl for direct analysis, 50 µl for immunoprecipitation) were directly analyzed by SDS-PAGE or immunoprecipitated by rocking overnight at 4°C with 2 µl anti-TCP1 antibody (1 mg/ml) in 680 µl of buffer A (20 mM Hepes, pH 7.5, 100 mM KOAc, 5 mM Mg[OAc]2, 5% [vol/vol] glycerol) and 15 µl of BSA-saturated protein A–Sepharose beads. Immunoprecipitated material was separated by SDS-PAGE, and radioactivity in dried gels was detected using a Bio-Rad GS-250 PhosphorImager.
To separate ribosome–nascent chain complexes from the translation mixture, 25 µl of translation mixture was layered over 100 µl of sucrose cushion (0.5 M sucrose, 25 mM Hepes, pH 7.5, 80 mM KOAc, 1 mM Mg[OAc]2) and centrifuged in a TL100 rotor at 100,000 rpm for 4 min at 4°C. The ribosomal pellets were washed with 25 mM Hepes, pH 7.5, 80 mM KOAc, 1 mM Mg(OAc)2, and resuspended in SDS sample buffer or in buffer A for immunoprecipitation.
To confirm that the photo-cross-links originated from nascent chains attached to ribosomes, samples were incubated with 2 mM puromycin after photolysis, as above. Excess puromycin was removed by gel filtration over Sephadex G-25, then samples were immunoprecipitated with 2 µl anti-puromycin antiserum (a generous gift of Dr. Peter Walter, University of California San Francisco, San Francisco, CA) in buffer A with protein A–Sepharose as above.
Nondenaturing gel electrophoresis (16 h, 4°C, 120 V) was performed using 4–10% polyacrylamide gels (native PAGE) in 80 mM MOPS-KOH, pH 7.0, 1 mM MgCl2 as described (Frydman et al. 1994).
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A homogeneous population of nascent chains can be achieved by exploiting the fact that translation products of truncated mRNAs lacking a stop codon remain ribosome-bound as peptidyl-tRNAs (e.g., Krieg et al. 1989). Translation in the presence of excess truncated mRNA will limit ribosomal initiation to one event per mRNA, thereby resulting in a population of ribosome-bound nascent chains whose length is dictated by the length of the truncated mRNA. This approach yields samples that are homogeneous in terms of the length of the nascent chain, and hence are at a particular state of nascent chain folding and processing. Importantly, these stable translation intermediates are effective tools for the dissection of chaperone interactions with the elongating polypeptide, particularly considering that the kinetics of translation in eukaryotic cells are already much slower (on the order of minutes) than the rate of binding of chaperones to substrate polypeptides, which appears to be diffusion-limited (Corrales and Fersht 1995; Fekkes et al. 1995). Consequently, the time of association between chaperones and ribosome-bound polypeptides, and hence the possibility of detecting these complexes, is primarily dictated by their dissociation rates both in vitro and in vivo, where the crowded conditions prevalent in the cytosol increase the association constants by several orders of magnitude (Ellis 1997; van Den Berg et al. 1999).
Actin mRNAs truncated at different positions within the coding region of the message were translated to generate a set of ribosome-bound nascent chains of defined length (Fig. 1). These translation reactions produced polypeptides of the expected molecular weight (Fig. 1 a), and were thus used to examine how the length of the nascent chains affects their interaction with the chaperonin.
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200 amino acids do not form a stable complex with TRiC, consistent with previous observations (Frydman et al. 1994; Dobrzynski et al. 1996; Frydman and Hartl 1996).
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Photoreactive Nascent Actin and Luciferase Chains Are Cross-linked to the Cytoplasmic Chaperonin TRiC
The second constraint noted above, i.e., the dynamic nature of nascent chain–chaperone interactions, can be circumvented by incorporating photoactivatable cross-linkers into a homogeneous population of nascent chains. When ribosome-bound nascent chains containing photoreactive probes are photolyzed, chaperones bound to the nascent chain may become covalently attached to the nascent chain if located close to a photoreactive probe at the time of its activation. This approach makes possible the biochemical analysis of the interactions of nascent chains by stabilizing short-range interactions between ribosome-bound polypeptides and associated proteins. Here we have employed this approach to examine the interactions of actin nascent chains with the cytoplasmic chaperonin TRiC.
To incorporate a photoactivatable azido moiety into newly translated actin chains, ANB-Lys-tRNA (Krieg et al. 1986) with a photoreactive aryl azide covalently attached to the -amino group of the lysine (Fig. 2 a) was added to the in vitro translation reactions. Since the ANB-Lys-tRNA must compete with endogenous Lys-tRNA, only a fraction (25%; Krieg et al. 1989) of the regularly spaced lysine residues in each actin nascent chain is replaced with a photoactivatable probe. Translation intermediates containing nascent actin chains of a specific length were prepared by translation of a particular truncated actin mRNA lacking a stop codon in reticulocyte lysate containing [35S]methionine and/or ANB-[14C]Lys-tRNA. Incorporation of the photoreactive probe in the nascent chains was confirmed by measuring the 14C content of nascent chains after translation in the absence of [35S]methionine (data not shown).
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Interestingly, cross-links were observed with actin chains as short as 133 amino acids (Fig. 2 c, lane 2). This suggests that TRiC can interact with nascent chains at an earlier stage than previously observed by coimmunoprecipitation and native gel analysis (Frydman and Hartl 1996; see also Fig. 1 b, lanes 2 and 8, and Fig. 4, lane 2). However, a nascent chain of 84 amino acids, which only exposes 50 amino acids to the cytosol (Malkin and Rich 1967; Blobel and Sabatini 1970), was not cross-linked to TRiC (Fig. 2 c, lane 1).
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The change in cross-linking pattern observed for nascent chains of increasing length is intriguing. Previous experiments analyzing photoadducts generated by nascent chains bearing a single photoprobe adjacent to different sites in a target protein have not shown significant variation in photoadduct mobilities in SDS-PAGE (e.g., High et al. 1993; Mothes et al. 1994; Do et al. 1996), though one such change has been observed recently by Plath et al. 1998. It therefore seems unlikely that the photoadducts with different mobilities in Fig. 2c and Fig. d arise from changes in the intramolecular location of nascent chain cross-links to the same TRiC subunit. The most probable explanation for the various photoadducts is that the nascent chain is cross-linking to different subunits in the complex. If so, this result may bear on the question of how TRiC recognizes its substrates. If all the subunits in TRiC possess substrate-binding sites of the same or similar specificity, the photoreactive probes would have an equal chance to react with all the subunits. Since the increase in nascent chain length is accompanied by an increase in the number of cross-linker–bearing lysines, and consequently in the probability of productive cross-linking events, the cross-links observed for longer chains should display an increase in intensity rather than the observed change in pattern. Our results are thus not consistent with a model where all TRiC subunits have equivalent substrate specificities. Instead, they suggest that emerging regions in the elongating polypeptide engage in subunit-specific interactions with different components of the ring complex.
The Specificity of TRiC Interactions with Substrates
Our finding that short actin and luciferase chains unexpectedly cross-linked to TRiC raised the possibility that TRiC has a broader range of interacting substrates than previously recognized using standard techniques. This led us to examine the pattern of cross-links of enolase, a 40-kD β-barrel protein that does not interact stably with TRiC (Fig. 3 a). After translation in reticulocyte lysate, neither enolase nor its nascent chains were associated with TRiC as determined by coimmunoprecipitation (Fig. 3 a) and native gel electrophoresis (data not shown). Surprisingly, these enolase nascent chains of 137, 251, and 375 amino acids were cross-linked to TRiC with great efficiency (Fig. 3 b). Moreover, the photoadducts with TRiC were the major products in the total cross-linking reaction (Fig. 3 b, lanes 1–3). Thus, although the interaction of TRiC with enolase is too weak to be detected by coimmunoprecipitation, the chaperone contacts the nascent chain during translation.
Although the photo-cross-linking data therefore reveal that the specificity of TRiC is broader than previously thought, not all polypeptides interact with TRiC cotranslationally. We next examined whether TRiC could cross-link to ribosome-bound nascent chains of the secretory protein pPL (Fig. 3 c). Previous studies indicated that an 86–amino acid pPL nascent chain translated in yeast extracts was cross-linked to the Hsc70 homologue SSB (Pfund et al. 1998). However, after translation in reticulocyte lysate, this ribosome-bound pPL chain cross-linked very inefficiently to TRiC subunits (Fig. 3 c, lane 3) but very efficiently to endogenous SRP54 (Fig. 3 c, lanes 1 and 2), as reported previously (Krieg et al. 1986). Moreover, the weak cross-links to TRiC were further diminished by addition of purified SRP (to 64 nM final concentration) to the lysate (Fig. 3 c, lane 4). Since the concentration of TRiC in the lysate is 0.4 µM (Frydman et al. 1994), this result suggests that SRP favorably competes with TRiC for binding to pPL, effectively blocking its interaction with TRiC. However, we did observe cross-links to TRiC after release of the pPL nascent chain from the ribosome (data not shown). Thus, TRiC is in principle capable of interacting with the pPL 86mer, but such an interaction most likely does not occur in vivo because SRP binding will first target the ribosome to the ER membrane and translocation into the ER will proceed cotranslationally.
The TRiC–Nascent Chain Interaction Occurs Cotranslationally
We next determined whether the cross-links between the chaperone and the actin nascent chains indeed occurred while the polypeptides were ribosome-bound. This question was addressed by two independent criteria. First, ribosome–nascent chain–TRiC complexes containing the actin 133mer were purified after photolysis by centrifugation through a dense sucrose cushion. As shown in Fig. 4 a, lane 1, the ribosomal pellet contained most of the TRiC–nascent chain photoadducts. In contrast, if the nascent chains were released from the ribosome by incubation with puromycin after photolysis and before sedimentation, the TRiC cross-links were no longer associated with the ribosomes and were instead found in the supernatant of the ultracentrifugation (Fig. 4 a, lane 4). Thus, the TRiC–nascent chain photoadducts were released from the ribosomes in a puromycin-dependent manner, indicating that TRiC was cross-linked to ribosome-bound peptidyl-tRNAs.
The cotranslational nature of the cross-links between nascent chains and TRiC was tested directly by taking advantage of the chemistry of puromycin-mediated release from the ribosome (Fig. 4 b, upper panel). Puromycin mimics an aminoacyl-tRNA and reacts covalently with a peptidyl-tRNA in the ribosomal P site, a reaction that transfers the growing nascent chain from the tRNA to the puromycin. The chain thus released carries a COOH-terminal puromycin tag. To examine whether the nascent chains associate with TRiC while ribosome-bound, translation intermediates were therefore photolyzed as above, treated with DTT to eliminate unreacted photoprobes, and then incubated with puromycin. Any nascent chains that had reacted with puromycin were then immunoprecipitated with an anti-puromycin antibody. Since the puromycin reaction must be catalyzed by the peptidyltransferase center of the ribosome, only nascent chains bound functionally to ribosomes at the time puromycin was added will become covalently attached to puromycin. Importantly, all photoprobes were inactivated, either by photolysis or by DTT, before the puromycin treatment. Consequently, the puromycin-specific antibody only immunoprecipitates photoadducts that were generated while the nascent chains were bound to ribosomes. As shown in Fig. 4 b, SDS-PAGE of these immunoprecipitations confirmed that for both actin and luciferase, the nascent chains had cross-linked to TRiC before the addition of puromycin (Fig. 4 b). Notably, the cross-links to TRiC were the predominant bands observed when using puromycin-specific antibodies. Thus, TRiC interacts with nascent chains that are associated with functional, translating ribosomes.
ATP Dependence of Cross-links to TRiC
The above photo-cross-linking data reveal that TRiC is positioned in close proximity to actin and other nascent chains, even if the ribosome-bound polypeptides are too short to form complexes with TRiC that survive immunoprecipitation. To gain further insight into the TRiC–nascent chain interaction, we examined their sensitivity to ATP. Incubation with ATP reduces the affinity of TRiC for its substrates, and thus results in their release from the chaperonin (Frydman et al. 1992). We thus compared the effect of performing the photolysis reaction in the presence or absence of ATP (Fig. 5 a). Removal of ATP from the lysate by incubation with apyrase should stabilize the interactions with the chaperonin. In contrast, the presence of ATP during photolysis should promote dissociation and hence diminish the amount of cross-linked product. As expected, incubation with ATP greatly reduced the extent of photo-cross-linking between TRiC and the longer nascent chains of luciferase and actin (Fig. 5 a, lanes 3, 4, 7, and 8). Surprisingly, ATP did not reduce the cross-links between TRiC and short nascent chains. Instead, these cross-links were enhanced by the presence of ATP (Fig. 5 a, lanes 1, 2, 5, and 6).
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The molecular basis for the ATP-dependent increase in cross-linking to short nascent chains remains unclear, but this result emphasizes the fact that the ATP dependence of TRiC function has yet to be characterized in detail. It is clear from the results presented here that TRiC binds differently to short and long nascent chains in the presence of ATP. There are several mechanisms that could account for this observation. For instance, cross-linking to TRiC may first require the ATP-dependent release of the nascent chain from an upstream cofactor, such as Hsc70. Alternatively, an ATP-mediated conformational change in TRiC may help position the chaperone in the vicinity of the ribosomal exit site, and thus facilitate binding to short nascent chains. It is also possible that individual subunits of TRiC interact differently with substrate and ATP. Future experiments addressing these possibilities may clarify the interplay between molecular chaperones and the translational machinery.
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Discussion |
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The Photo-Cross-linking Approach
The association of translating polypeptides with molecular chaperones plays a critical role in the folding process. However, the transient and dynamic nature of these associations presents a problem for the molecular analysis of this process. Here, we have generated translation intermediates consisting of nascent chains of defined lengths carrying photoreactive probes evenly located at multiple sites along the entire length of the polypeptide. Photolysis generates covalent links between the nascent chain and associated protein(s), thereby stabilizing these labile interactions for further analysis.
This approach allows the identification of endogenous proteins that bind to the nascent chain as it is being synthesized. Equally important, the interaction between the nascent chain and a particular protein will be stabilized and detected even if the affinity of the interaction is insufficient to maintain the protein–protein complex during conventional analysis, as might be the case for very short nascent chains. In addition, in the photo-cross-linking approach, the nascent chain is not released from the ribosome until after the assay (i.e., photolysis) has been completed, whereas complexes are analyzed by coimmunoprecipitation and native gel electrophoresis only after the nascent chain has been released from the ribosome. Each of these advantages was borne out when the photo-cross-linking approach was applied to actin translation intermediates.
Association of TRiC with Ribosome-bound Nascent Chains
The major cross-linked products observed for ribosome-bound actin nascent chains corresponded to photoadducts with TRiC. Two experimental approaches demonstrated that TRiC cross-linked to ribosome-bound nascent chains. First, the TRiC photoadducts sedimented with the ribosomal fraction after ultracentrifugation through a sucrose cushion. This association was not observed if the nascent chains were released from the ribosome by puromycin treatment before the ultracentrifugation step. Furthermore, to distinguish between photoadducts formed by ribosome-bound actin and by actin that had been released from the ribosome, we added puromycin to samples after photolysis had been completed. Only nascent chains functionally bound to ribosomes could react with puromycin. Consequently, immunoprecipitation with anti-puromycin antibodies selected only those photoadducts whose nascent chains are elongation-competent at the time of cross-linking. As seen in Fig. 4 b, this approach conclusively demonstrates that the photoadducts with TRiC were generated on ribosome-bound nascent chains. These results are consistent with a number of studies indicating that TRiC interacts cotranslationally with nascent chains (Frydman et al. 1994; Dobrzynski et al. 1996; Frydman and Hartl 1996), and do not support the alternative proposal that the interaction of TRiC with its substrates is strictly posttranslational (Hansen et al. 1999).
The cotranslational nature of TRiC/CCT binding to nascent chains is also supported by experiments indicating that this chaperonin associates with ribosomal fractions. Comigration of the chaperonin with ribosomes upon size fractionation of cell extracts has been observed in vitro in reticulocyte lysate (Frydman et al. 1994), and in vivo in P19 embryonic carcinoma cells, where a significant fraction (5–20%) of the cellular TRiC appeared to be ribosome-associated (Roobol and Carden 1999). Furthermore, the TRiC–ribosome interaction was confirmed by coimmunoprecipitation of ribosomes with the chaperonin (Roobol and Carden 1999).
An analysis of the chain length dependence of cross-link formation yielded an unexpected result. TRiC association with short nascent polypeptides has not been detected previously using other techniques. Yet cross-links to TRiC were detected for actin nascent chains as short as 133 amino acids, which expose only 90–100 amino acids outside the peptide channel. Similarly, we detected cross-links to luciferase nascent chains as short as 77 amino acids. These results indicate that chaperonins can interact with nascent chains very soon after they emerge from the ribosome. It is therefore conceivable that TRiC is already located in close proximity to the nascent chain, perhaps as a result of a specific recruitment mechanism.
Consistent with such a possibility, the study of protein targeting into organelles has produced several examples where chaperone components are physically recruited to the translocation machinery to bind to the incoming polypeptide (Brodsky and Schekman 1993; Kessler and Blobel 1996; Voos et al. 1996). Likewise, experiments in Saccharomyces cerevisiae have shown that the Hsp70 protein SSB binds to translating ribosomes through specific interactions with the translational machinery (James et al. 1997; Pfund et al. 1998). At present, there is no evidence for a direct physical interaction between TRiC and components of the basic translational machinery. However, TRiC binding to newly translated polypeptides could be facilitated by other components of the folding machinery, such as the Hsp–Hsc70 system (Frydman et al. 1994), the nascent chain–associated complex (NAC, Wang et al. 1995), and/or the recently described GIMc or prefoldin complex (Geissler et al. 1998; Vainberg et al. 1998; Hansen et al. 1999). Interestingly, incubation with ATP enhanced the cross-links between TRiC and the short nascent chains (Fig. 5), indicating that TRiC recruitment may be ATP-mediated, as expected if binding of nascent chains to the chaperonin is promoted by Hsc70. This is consistent with previous experiments showing that the interaction of TRiC with luciferase nascent chains requires the action of Hsc70 (Frydman et al. 1994). It has been suggested recently that prefoldin binds nascent chains and delivers them to TRiC (Vainberg et al. 1998; Hansen et al. 1999). However, both biochemical and genetic analyses indicate that prefoldin is not required for substrate binding to TRiC (Rommelaere et al. 1999; Siegers et al. 1999).
The specific recruitment of chaperones to bind to translating polypeptides would provide a mechanistic explanation for the observed coupling between translation and folding observed in intact eukaryotic cells, which probably contributes to the formation of a protected folding environment for nascent chains (Siegers et al. 1999; Thulasiraman et al. 1999). The mechanism for TRiC recruitment to bind nascent chains requires further investigation. We believe that the cross-linking approach described here for TRiC will permit the identification of both upstream and downstream cofactors of TRiC. Although the focus of the experiments presented here was to characterize the interaction of nascent chains with TRiC, they also revealed cross-links between nascent chains and as-yet unidentified components. Based on their molecular weight, some of the cross-links observed for longer chains might correspond to subunits of the GIMc complex (see Fig. 2 a and Fig. 3). In addition, we also observed cross-links with Hsc70 in the case of short luciferase nascent chains (77mer and 94mer; data not shown). Since the chaperone interactions detected using this approach are critically dependent on the proximity of the photoreactive probes to the chaperone binding site, elucidating the role of Hsc70 and GIMc prefoldin in the folding of newly translated proteins may require the introduction of the photoreactive group at different positions within the nascent chain.
Determinants for Substrate Binding to TRiC
Unlike its bacterial homologue, GroEL, the eukaryotic chaperonin TRiC is composed of different subunits. Most of the subunit heterogeneity resides in the putative substrate-binding site (Kim et al. 1994). However, the physiological significance of this diversity has been unclear, because little is known about what determines polypeptide binding to TRiC.
The analysis of the chain length dependence of cross-links between TRiC and both actin and luciferase nascent chains indicated that short chains appeared to contact predominantly one TRiC subunit, whereas longer chains were cross-linked to several subunits. Interestingly, enolase nascent chains are efficiently cross-linked to TRiC, but the cross-links appear to be predominantly to one TRiC subunit despite the higher proportion of lysines in the enolase nascent chains. Notably, there is a striking correlation between the extent to which the nascent chain is cross-linked to multiple TRiC subunits and the stability of the TRiC–nascent chain complexes to immunoprecipitation, supporting the idea that the frequency and number of different photoadducts indeed reflects subunit-specific interactions with different binding sites within the nascent chains. Our data are consistent with a model where stable interactions between a folding polypeptide and TRiC arise from a polyvalent set of weak interactions between defined substrate motifs and individual chaperonin subunits. Interestingly, this interpretation agrees with two recent studies on the interaction of actin with TRiC. First, deletion analysis of actin suggested that stable chaperonin binding requires at least three discrete regions in the polypeptide (Rommelaere et al. 1999). In addition, a structural analysis of the chaperonin–actin complex using immuno-EM supports the idea that the polypeptide interacts with specific subunits in the chaperonin (Llorca et al. 1999).
The possibility that each TRiC subunit contributes to the recognition of specific motifs may help explain how chaperonin substrates are selected in vivo. Identification of the chaperonin subunits that are cross-linked to specific nascent chains will provide important insights into the principles that govern substrate binding to TRiC.
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Acknowledgments |
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This work was supported by National Institutes of Health grants GM56433 (to J. Frydman) and GM26494 (to A.E. Johnson), and by the Robert A. Welch Foundation (A.E. Johnson).
Submitted: 3 January 2000
Revised: 23 February 2000
Accepted: 3 March 2000
Dr. McCallum's present address is Merck & Co. Inc., Rahway, NJ 07065.
ANB, N-(5-azido-2-nitrobenzoyl); CCT, chaperonin containing TCP1; GIMc, genes involved in microtubule biogenesis complex; Hsc70, 70-kD heat shock protein cognate; pPL, preprolactin; SRP, signal recognition particle; TCP1, tailless complex polypeptide 1; TRiC, TCP1-ring complex.
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References |
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