Correspondence to: Tom A. Rapoport, Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115-6091. Tel:617-432-0637 Fax:617-432-1190
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Abstract |
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In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptideassociated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.
Key Words: cytosolic chaperones, endoplasmic reticulum, posttranslational protein translocation, Sec complex, yeast
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Introduction |
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It is generally believed that proteins are transported through a protein-conducting channel of the ER membrane in an unfolded conformation. In cotranslational translocation, an unfolded state is maintained simply by the fact that the nascent polypeptide chain is transported while being synthesized on a ribosome that is bound to the membrane channel. In contrast, for posttranslationally transported proteins there must be a mechanism that prevents their aggregation or premature folding into a stable structure before they are transported through the channel. Cytosolic chaperones are likely involved to keep a translocation substrate in an unfolded or loosely folded conformation before it enters the channel (
Posttranslational translocation occurs both in yeast and in mammalian cells, although in the latter case only substrates smaller than 70 amino acids are translocated and the mechanism of transport is not well understood ( factor (pp
F, 165 amino acids), Kar2p can function as a molecular ratchet to move the polypeptide chain across the membrane (
Cytosolic chaperones implicated in posttranslational protein translocation include Hsp70 and its cofactor, the J protein Ydj1p (F and can stimulate its posttranslational translocation in vitro (
While it is conceivable that a posttranslational translocation substrate interacts with the same cytosolic chaperones as a polypeptide chain remaining in the cytosol, some differences may exist. For polypeptides lacking signal sequences, Hsp70 and Hsp40 (a mammalian cytosolic J protein) have been found to associate with ribosome-bound nascent chains (
Here, we have used a systematic photo-cross-linking approach to probe interactions of cytosolic proteins with translocation substrates during early steps of their posttranslational translocation in yeast, until their binding to the Sec complex. Our results indicate that a posttranslational substrate synthesized in the reticulocyte lysate system interacts with SRP and NAC as long as it is associated with the ribosome. After termination of translation, it interacts with several cytosolic chaperones, including Hsp70 and TRiC/CCT, and has thus likely the same interaction partners as a polypeptide lacking a signal sequence. In fact, no specific cytosolic receptor for signal sequences of posttranslational substrates could be detected. Release of the cytosolic proteins from the translocation substrate occurs spontaneously, and the Sec complex plays no active role. Once bound to the Sec complex, the polypeptide chain is not associated with any cytosolic protein, explaining how its subsequent translocation through the membrane channel can occur without interference of cytosolic proteins.
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Materials and Methods |
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Constructs
cDNA coding for wild-type (wt) ppF was cloned into the vector pAlter (Promega). All lysine codons in wt pp
F were altered to arginine codons (76, 84, 96, 103, 117, 124, 138, 145, and 159), and single lysines were introduced using appropriate oligonucleotides (
pp
F and M2 pp
F are signal sequence mutants of K5 and wt pp
F, respectively, with a deletion of amino acids 1014 or a substitution of alanine for proline at position 13. The signal sequence of pp
F (165 amino acids) comprises amino acids 122.
Transcription, Translation, and PhotoCross-linking
mRNAs coding for the different full-length ppF polypeptides and proOmpA were generated as described (
F molecules were produced after linearization of the plasmid with NciI by in vitro transcription with T7 RNA polymerase. In vitro translation was carried out in a reticulocyte lysate system for 25 min at 30°C in the presence of [35S]methionine and trifluoromethyl-diazirino-benzoic acid (TDBA)-lysyl tRNA (
Immunoprecipitations of Cytosolic Cross-linked Products
For immunoprecipitation (IP) after denaturation, SDS was added to the irradiated sample to a final concentration of 2%. The mixture was incubated for 15 min at 65°C, and the SDS concentration was adjusted to 0.1% by dilution with IP buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Triton X-100) containing 1% BSA. Antibodies to Hsp70 (SPA 815 from StressGen Biotechnologies, or K19 [sc-1059] from Santa Cruz Biotechnology, Inc.), to TCP1 (CTA123 from StressGen Biotechnologies; CTA-191, CTA-122, and CTA-184 gave similar results), to Hsp70/Hsp90 organizing protein (p60/Hop) (SRA1500 from StressGen Biotechnologies), to SRP54, and to the
and ß subunits of NAC (
Sucrose Gradient Centrifugation and Translocation Assays
30 µl of the irradiated translation mixture containing full-length ppF or proOmpA was diluted twofold with buffer A, layered on top of a 540-µl sucrose gradient (0.31.0 M sucrose in buffer A), and centrifuged for 6 h at 48,000 rpm in a Beckman SW55 rotor. After centrifugation, 50-µl fractions were collected starting from the top. To analyze the translocation competence of different pp
F complexes, a translation mixture containing wt pp
F without photoreactive probes was separated in a sucrose gradient. The translocation assays with different fractions of the sucrose gradients were performed with reconstituted proteoliposomes containing purified yeast Sec complex in the membrane and recombinant Kar2p and ATP in the lumen, as described (
Dissociation of Cytosolic Complexes
After in vitro translation of full-length ppF and sedimentation of ribosomes, 10 µl of the translation mixture was added to 100 µl of buffer A containing 2 mM ATP, 40 mM creatine phosphate, and 1 mg/ml creatine kinase. Where indicated, 1.5 µmol GroEL trap (D87K; provided by F.U. Hartl and his laboratory, Max-Planck-Institute for Biochemistry, Martinsried, Germany), or reconstituted proteoliposomes containing Sec complex were present. After mixing, 10 µl was immediately removed and placed on dry ice to stop the reaction (0.5-min time point). The remainder of the mixture was incubated at 30°C. 10-µl samples were removed at the indicated time points and immediately frozen on dry ice. At the end, all samples were irradiated with UV light for 15 min on dry ice.
To test whether the dissociation of cytosolic complexes leads to a reduction of translocation competence, in vitrosynthesized full-length ppF was incubated for 30 min at 30°C or 0°C. Subsequently, half of the sample was irradiated with UV light for 15 min on ice, and the other half was incubated with reconstituted proteoliposomes containing Sec complex for binding of pp
F to Sec complex.
IP of ppF Bound to Sec Complex
Binding of ppF to reconstituted proteoliposomes containing Sec complex was done essentially as described (
F translation mixture was either first irradiated for 15 min on ice and subsequently incubated with 30 µl of reconstituted proteoliposomes for 15 min at 30°C, or first incubated with proteoliposomes and then irradiated. The samples were subsequently solubilized in digitonin, and the Sec complex was immunoprecipitated with antibodies to Sec62p.
Electrophoresis
Analysis of all samples was performed by SDS-PAGE with 7.517.5% linear acrylamide gels, followed by autoradiography or analysis with a Fuji PhosphorImager BAS1000.
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Results |
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Interactions of Ribosome-bound ppF in the Cytosol
We first used photo-cross-linking to investigate interactions of ribosome-bound nascent ppF with cytosolic proteins. A truncated mRNA coding for a pp
F polypeptide chain missing only the last five amino acids was translated in vitro in a reticulocyte lysate system in the presence of [35S]methionine. When the ribosome reaches the end of the mRNA, the radioactively labeled nascent chain remains bound to the ribosome as peptidyl tRNA because there is no stop codon to effect termination of translation. The translation system also contained a modified lysyl tRNA that carries a carbene-generating probe in the side chain of the amino acid (
F. The cross-linked products were analyzed by SDS-PAGE and autoradiography. With wt pp
F, containing all of its nine lysines in the COOH-terminal half of the polypeptide chain, cross-links occurred mostly to the two subunits of NAC (Fig 1, lane 6, circles), as demonstrated by IP with antibodies to both NAC subunits after SDS denaturation (data not shown). To identify interaction partners of the signal sequence, we substituted all the lysines of wt pp
F with arginines and introduced a single lysine residue at position 5 within the signal sequence. As previously observed with cotranslational translocation substrates (
F is cross-linked to different regions of SRP54, resulting in cross-linked products with different mobilities in SDS gels, as observed with cross-linking of pp
F to Sec61p (
F interacts mainly with SRP and NAC. It should be noted that nascent pp
F interacts with SRP even when the chain is almost full length, in contrast to the cotranslational translocation substrate preprolactin, which only interacts with SRP when the nascent chain is short (
F may have folding characteristics that keep the signal sequence exposed.
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Next, we tested whether the cross-linking pattern changes when ppF carries a defective signal sequence (K5
and M2 pp
F mutants). M2 and wt pp
F, which contain the photoreactive probes at the same positions in the COOH-terminal domain, showed identical cross-linking patterns (Fig 1, compare lane 8 with lane 6). However, the K5
mutant, containing a single probe at position 5 of its defective signal sequence, showed drastically reduced SRP54 cross-links but no other changes compared with K5 pp
F, containing an intact signal sequence (Fig 1, compare lane 4 with lane 2). These data demonstrate that the interaction of pp
F with SRP, in contrast to that with NAC, requires a functional signal sequence, confirming previous results obtained with preprolactin (
Release of SRP and NAC upon Termination of Translation
Next, we studied whether the interaction of ppF with cytosolic proteins changes upon termination of translation. To generate ribosome-released full-length pp
F, in vitro translation in a reticulocyte lysate system was performed with mRNA containing the natural stop codon, and the ribosomes were removed by centrifugation. In these experiments, we employed a pp
F mutant that carries a single photoreactive probe at position 10 of the signal sequence (K10 pp
F). Ribosome-associated, truncated K10 pp
F of 160 residues gave the same cross-linking pattern as nascent K5 pp
F studied before (Fig 2, lane 3; compare with Fig 1, lane 2); both SRP and NAC cross-links could be verified by IP with specific antibodies (Fig 2, lanes 6 and 7). In contrast, ribosome-released full-length K10 pp
F of 165 residues could not be cross-linked to either SRP or NAC (Fig 2, lane 1; IPs in lanes 9 and 10), and several new cross-linked products appeared instead (Fig 2, lane 1). Similar results were obtained when the truncated nascent chain of 160 residues was released from the ribosome with EDTA (Fig 2, compare lane 4 with lane 3). The resulting cross-linking pattern was similar to that of full-length K10 pp
F (Fig 2, compare lane 4 with lane 2). These results demonstrate that both SRP and NAC are released from the translocation substrate pp
F upon termination of translation.
Interactions of Full-Length ppF in the Cytosol
To analyze interactions of full-length ppF with cytosolic proteins in more detail, we generated 37 pp
F mutants that each contain a single lysine within either the signal sequence or the mature part of the protein. These mutants can be used to scan the environment of distinct regions of the translocation substrate in a systematic manner. All mutant proteins containing photoreactive probes were efficiently bound to the Sec complex and translocated across yeast ER membranes (data not shown; see also
Each of the single lysine mutants gave cross-links to several cytosolic proteins present in reticulocyte lysate (Fig 3). In each case, the cross-links were dependent on the presence of photoreactive probes in the polypeptide chain (data not shown). The cross-linking pattern was remarkably similar for all positions probed throughout the polypeptide chain. The major cross-linking partners had molecular masses of 200, 180, 70, 62, 60, 50, and 20 kD (in each case, the molecular mass of pp
F [20 kD] was subtracted from the size of the cross-linked product). Some differences between regions were noted. Cross-links to the 70- and 50-kD proteins were more prominent with photoreactive probes at positions within the central part of the signal sequence (positions 515) than with probes at all following positions. At position 40, the cross-link to the 200-kD protein disappeared while the band containing the 180-kD protein became more prominent. Beyond position 95, a strong cross-link to a protein of
55 kD was observed. The fact that a single position of pp
F could be cross-linked to several proteins suggests that there are different populations of pp
F molecules that contact either different sets of proteins or the same set with different orientations.
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With wt ppF, which contains nine lysine residues in the COOH-terminal half, a complex cross-linking pattern was seen. Several cross-linked products corresponded to those seen with single-lysine mutants (e.g., the cross-links to proteins of
180, 62, 60, 55, and 20 kD). The many bands seen without irradiation are caused by extensive ubiquitination of pp
F at the lysine residues (data not shown). Taken together, these results show that both the signal sequence and the mature part of newly synthesized full-length pp
F interact with several proteins of the reticulocyte lysate, and that there are relatively small differences in the interaction patterns probed at various positions of the polypeptide chain.
Next, we tested whether the cross-linking pattern changes when ppF carries a defective signal sequence. Regardless of whether the photoreactive probes were located in the signal sequence (K5
pp
F) or in the COOH-terminal region of the polypeptide chain (M2 pp
F), the cross-linking pattern of the signal sequence mutant was indistinguishable from that of the corresponding protein with a functional signal sequence (Fig 3, compare K5
with K5 pp
F, and M2 with wt pp
F; first and last four lanes). These data suggest that there is no cytosolic protein that specifically interacts with the signal sequence of the posttranslational translocation substrate. In addition, they suggest that completed polypeptides with and without signal sequence interact with the same set of cytosolic proteins.
We also observed a cross-linked product that migrates in SDS gels slightly faster than ppF itself (Fig 3, arrow). This product is probably generated by internal cross-linking within the pp
F molecule, resulting in a more compact structure with higher mobility in SDS gels. Internal cross-links occurred with some variation in intensity throughout the entire polypeptide chain, suggesting that pp
F may be in a collapsed conformation.
Full-Length ppF Interacts with Cytosolic Chaperones
To identify the cytosolic cross-linking partners of full-length ppF, we performed IP experiments with antibodies directed against cytosolic chaperones and their cofactors. First, cross-linked products obtained with pp
F containing the photoreactive probe at position 10 of the signal sequence were analyzed (Fig 4 A). After denaturation of the irradiated sample in SDS, antibodies to Hsp70 and TCP1
, a subunit of TRiC/CCT, immunoprecipitated cross-linked products of the expected size (Fig 4 A, lanes 4 and 5, p70 and p62, respectively). Minor cross-links to p60/Hop, a chaperone cofactor that is known to interact with TRiC/CCT, Hsp70, and Hsp90 (
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To test whether Hsp70 and TCP1 interact with pp
F alone or in conjunction with other proteins, we performed IPs under native conditions. The efficiency of IP was significantly higher than under denaturing conditions. With both Hsp70 and TCP1
antibodies, the cross-linked product of the expected size corresponded to a major band among the total products (Fig 4 A, lanes 7 [p70] and 8 [p62]; compare with lane 2), indicating that both Hsp70 and TCP1
are major interaction partners of the signal sequence of pp
F. Hsp70 antibodies also precipitated the prominent cross-linked products containing the proteins of
50 and 20 kD (Fig 4 A, lane 7). TCP1
antibodies coprecipitated cross-links to a protein slightly smaller than TCP1
itself (p60) as well as products containing the 20-kD protein (Fig 4 A, lane 8). The native IPs with Hsp70 and TCP1
antibodies together account for the majority of the bands seen among the total cross-linked products (Fig 4 A, lane 2). These data suggest that pp
F is part of at least two distinct complexes, explaining why different cross-linked bands could be coprecipitated with Hsp70 and TCP1
antibodies under native conditions.
Hsp70 and TCP1 were also identified as cross-linking partners when the photoreactive probes were located in the COOH-terminal region of the pp
F molecule. With the probe at position 97, the pattern of immunoprecipitation with Hsp70 and TCP1
antibodies was very similar to that of pp
F with the probe in the signal sequence, both under denaturing and native conditions (Fig 4 B). p60/Hop was also identified as a cross-linking partner (data not shown). Similar results were obtained with wt pp
F containing probes in its nine lysine residues at the COOH terminus (Fig 4 C). As expected from the results described above (Fig 3), both Hsp70 and TCP1
were also identified as cross-linking partners with M2 pp
F containing a defective signal sequence (Fig 4, compare D with C; see also Fig 3, compare K5
with K5 pp
F, p70 and p62). The fact that Hsp70 cross-links more strongly to the signal sequence than to the mature part of pp
F may be explained by its preference for hydrophobic sequences.
Significant cross-links to TCP1 were only observed with pp
F released from the ribosomes (Fig 2, compare lane 3 with lane 1,
; IP not shown), similar to results obtained with GroEL, the bacterial homologue of TRiC/CCT (
; IP not shown).
We also tested a second posttranslational translocation substrate, proOmpA. Wt proOmpA contains lysine residues both in the signal sequence and in the mature part (a total of 19 lysines). Like ppF, it could be cross-linked to several cytosolic proteins in the reticulocyte lysate, although a high background was seen in the absence of irradiation, probably caused by ubiquitination (Fig 4 E, lanes 1 and 2). IP experiments after denaturation of cross-linked products with SDS demonstrated that proOmpA was cross-linked to Hsp70 and TCP1
(Fig 4 E, lanes 4 [p70] and 5 [p62]). Native IP suggested again that the translocation substrate is contained in at least two distinct complexes, one with Hsp70 and the other with TCP1
(Fig 4 E, lanes 7 and 8).
Distinct Translocation-competent Complexes
To confirm that posttranslational translocation substrates are present in at least two distinct complexes with cytosolic proteins, we performed sucrose gradient centrifugation. Specifically, a ppF mutant containing a single photoreactive probe at position 10 was synthesized in vitro, irradiated, and separated in a sucrose gradient. The cross-links to Hsp70 and the 50-kD protein were found in fractions of relatively low molecular weight (Fig 5 A, fractions 46; IPs for Hsp70 not shown). In fact, the pattern of cross-links in these fractions was remarkably similar to that seen in native IPs with Hsp70 antibodies (Fig 4 A, lane 7). The cross-links to TCP1
and to the slightly smaller 60-kD protein were found in a high molecular weight peak (Fig 5 A, fractions 810), whose size is consistent with that of TRiC/CCT (970 kD). Again, the cross-linking pattern looked similar to that of native IPs with TCP1
antibodies (Fig 4 A, lane 8). p60 is thus likely a subunit of TRiC/CCT that contains several polypeptides of almost the same size. The Hsp70 and TRiC/CCT peaks also contained the maximum amounts of non-cross-linked pp
F, although the latter was found in all fractions (quantitation not shown). These data support the existence of at least two distinct pp
F populations, one in a complex with Hsp70 and the other with TRiC/CCT. The other cross-linked products were also found in distinct fractions of the sucrose gradient, and only the cross-links to the 20-kD protein were distributed throughout the gradient. It should be noted that internal cross-links of pp
F were observed in all fractions of the sucrose gradient (Fig 5 A, arrow), indicating that despite association with different chaperone proteins, pp
F does not attain a fully extended conformation.
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Similar results were obtained with wt ppF and ppaF carrying a photoreactive probe at position 97 of the mature region, with a signal sequence mutant, or if the order of sucrose gradient centrifugation and cross-linking was changed (data not shown). Moreover, proOmpA behaved similarly to pp
F in sucrose gradient centrifugation (Fig 5 B), indicating that posttranslational translocation substrates are generally associated with different cytosolic chaperone complexes. pp
F synthesized in yeast lysate was also found to be contained in different complexes, since it showed a broad distribution in sucrose gradients (data not shown). Unfortunately, cross-linking experiments with pp
F synthesized in yeast lysates did not give distinct cross-linked bands, most likely because of the fast hydrolysis of the modified lysyl tRNA (data not shown).
Next, we tested whether noncross-linked ppF present in the different complexes could be translocated. Wt pp
F without photoreactive probes was separated in a sucrose gradient, and the various fractions of the gradient were incubated with proteoliposomes containing Sec complex in the membrane and Kar2p and ATP in the lumen (Fig 6). Translocation was assessed after treatment with protease. Regardless of whether equal volumes or equal amounts of substrate of the individual fractions were tested, all were clearly active in translocation (Fig 6). The fact that translocation competence was found for fractions between the TRiC and Hsp70 peaks may be explained either by significant overlap of the peaks or by the presence of a chaperone in all fractions (e.g., p20; see Fig 5 A). Taken together, these results indicate that different sets of cytosolic proteins keep pp
F in a translocation-competent conformation.
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Dissociation of ppF from Cytosolic Proteins
We next investigated the fate of the ppFchaperone complexes during the next step in posttranslational protein translocation, the binding of pp
F to the Sec complex. Specifically, pp
F containing a photoreactive probe at a single position was synthesized in vitro in a reticulocyte lysate system, the ribosomes were removed by centrifugation, and proteoliposomes containing the purified yeast Sec complex were added. The vesicles lack Kar2p and thus allow binding of pp
F to the cytosolic face of the Sec complex but no translocation (
F with a photoreactive probe at position 11 because it gives only weak cross-links to the Sec61p subunit of the Sec complex (see Fig 8 A, lane 6), which could obscure the presence of some cross-links to cytosolic proteins in SDS gels. When the Sec complex was present, the cross-links to the 50-kD protein and to Hsp70 both diminished very rapidly with the same kinetics (Fig 7A and Fig B). Cross-links to the p60 subunit of TRiC/CCT also decreased, but more slowly (Fig 7 C). The kinetics of dissociation of cytosolic proteins from pp
F are consistent with those of binding of pp
F to the Sec complex, as determined by the appearance of cross-links to Sec62p and Sec71p, two other subunits of the Sec complex (Fig 7 D). The cross-links to the proteins of
20 kD also disappeared rapidly (those to the 180- and 200-kD proteins could not be well quantitated; data not shown).
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When the proteoliposomes were omitted, cross-links to Hsp70, p60, and the 50-kD protein also diminished with time but remained significantly stronger than in the presence of Sec complex throughout the experiment (Fig 7AC, control; cross-links to the 20-kD proteins behaved similarly [data not shown]). Liposomes lacking Sec complex gave the same result (data not shown). Incubation of ppFchaperone complexes on ice resulted in all cross-links remaining constant (data not shown; see also Fig 7 E). These results show that, at elevated temperatures, spontaneous net dissociation of complexes between pp
F and cytosolic proteins occurs; net dissociation is significantly faster in the presence of Sec complex.
To test whether the Sec complex stimulates dissociation in an active manner or simply captures free ppF molecules spontaneously released from their cytosolic partners, we used a mutant of the Escherichia coli chaperonin GroEL as a passive trap (
F from cytosolic proteins (
F molecules. Dissociation of the complexes between pp
F and cytosolic proteins seems to be the rate-limiting step in the transfer of the substrate to the respective binding partner.
To test whether the spontaneous dissociation of chaperonesubstrate complexes in the absence of Sec complex leads to a reduction of translocation competence, we preincubated in vitrosynthesized ppF at 30°C or 0°C, and then tested in parallel cross-linking of pp
F to cytosolic proteins and binding of pp
F to the Sec complex (Fig 7 E). While after preincubation on ice both the cross-linking to cytosolic chaperones and the binding to the Sec complex remained unchanged compared with a sample without preincubation, at 30°C both were reduced to the same extent (
50%). These data suggest that substrate molecules released from cytosolic chaperones aggregate and become incompetent for translocation if they cannot immediately interact with the Sec complex.
Dissociation of Cytosolic Complexes Required for ppFSec Complex Interaction
To confirm the release of cytosolic proteins from ppF during initiation of translocation, we analyzed interactions of the substrate bound to the Sec complex. pp
F molecules with probes in the signal sequence at positions 11 or 13 were first incubated with proteoliposomes containing the purified Sec complex and then irradiated (Fig 8A and Fig B, Fig 1.B/2.X). The samples were either analyzed directly by SDS-PAGE (total) or solubilized in digitonin, and were then subjected to IP with antibodies to Sec62p to isolate the Sec complex and the associated cross-linked and non-cross-linked pp
F. As reported previously (
F with a probe at position 11 cross-linked weakly to Sec61p and strongly to Sec62p and Sec71p (the latter are not separated in the gel; Fig 8 A, lane 6). pp
F with a probe at position 13 gave strong cross-links to both Sec61p and Sec62p/71p (Fig 8 B, lane 6). With both positions of the probe, pp
F bound to Sec complex did not give any cross-link to cytosolic proteins (Fig 8A and Fig B, lane 6), indicating that the cytosolic binding partners were released from the signal sequence upon its binding to the Sec complex. To test whether cytosolic proteins are also released from the mature portion of pp
F, we repeated the experiments with mutants containing probes at positions 117, 138, and with the wild-type protein containing the probes at nine COOH-terminal positions (Fig 8, CE). With all three proteins, strong cross-links to Sec62/71p and Sec72p were seen, while the single-lysine mutants showed additional very weak cross-links to Sec63p, Sec61p, and Sbh1p (Sec72p, Sec63p, and Sbh1p are also subunits of the Sec complex). Significantly, in all cases no cytosolic cross-links of pp
F bound to the Sec complex were discernible (Fig 8, CE, lane 6). Thus, during the binding of pp
F to the Sec complex, all cytosolic proteins must have been released, even from COOH-terminal parts of the polypeptide chain which are not inserted into the translocation channel.
Finally, we tested whether the release of cytosolic proteins is required for the binding of ppF to the Sec complex. To this end, samples containing pp
F with photoreactive probes at different positions were first irradiated on ice, conditions that maximize the extent of cross-linking to cytosolic proteins, and then proteoliposomes containing the Sec complex were added to allow binding of pp
F to the Sec complex (Fig 8, AE, 1.X/2.B). Again, both the total products and those associated with the Sec complex were analyzed. Although a large number of cross-links to cytosolic proteins were visible among the total products, most of them either were not bound or were only inefficiently bound to the Sec complex (Fig 8, AE, compare lanes 2 and 5). The only clear exception are the cross-links to the 55-kD protein (p55), seen with probes in wt pp
F, which were coprecipitated with the Sec complex. Thus, cross-linking of most cytosolic proteins appears to prevent the interaction of pp
F with the Sec complex, suggesting that their release is a prerequisite for initiation of translocation.
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Discussion |
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We have used photo-cross-linking to follow the route of a posttranslational translocation substrate from its synthesis on a free ribosome to its binding to the Sec complex, the component in the yeast ER membrane that forms the translocation channel. Our data suggest the following sequence of events (Fig 9). First, while still bound to the ribosome, the nascent polypeptide chain interacts with SRP and NAC. Second, when the chain is completed and released from the ribosome, it no longer interacts with these proteins and instead associates with several different cytosolic proteins. At least two distinct complexes could be identified, one containing Hsp70 and the other TRiC/CCT. Third, the cytosolic complexes dissociate spontaneously, without active participation of the Sec complex. Finally, substrate bound to the Sec complex is no longer associated with any cytosolic proteins and can now begin translocation through the membrane channel without interference by cytosolic proteins.
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For technical reasons, we have used in our experiments a heterologous system comprised of cytosol from rabbit reticulocytes and Sec complex from S. cerevisiae. However, we believe that the results are also relevant to the homologous system in yeast, and even to in vivo conditions, for the following reasons. First, ppF and proOmpA synthesized in a reticulocyte lysate system are posttranslationally transported into yeast ER vesicles with the same high efficiency as substrates synthesized in yeast lysate (data not shown). Although posttranslational translocation appears to be more prominent with yeast ER membranes than with mammalian ones, the difference is not due to cytosolic factors. Second, the cytosolic chaperone proteins are highly conserved among species, making it unlikely that entirely different pathways are employed in different eukaryotes. Furthermore, cytosolic chaperone proteins are equally abundant both in reticulocyte and yeast lysates (
F synthesized in yeast lysate runs as a heterogeneous population, similarly to pp
F made in reticulocyte lysate. Fourth, in yeast, Hsp70 is involved in the translocation of pp
F both in vivo and in vitro (
Our results show that a posttranslational translocation substrate differs from a polypeptide lacking a signal sequence, as long as it is associated with the ribosome, by being associated with SRP. However, once released from the ribosome, both classes of proteins seem to interact with the same cytosolic proteins. These include Hsp70 and TRiC/CCT, but also p60/Hop. Several other, unidentified binding partners of posttranslational translocation substrates may also be identical to those known to interact with cytosolic polypeptides. The 50-kD protein might be Hsp40, a mammalian cytosolic J protein, since it comigrated with Hsp70 in sucrose gradients and could be coimmunoprecipitated with it. The cross-linked proteins of 20 kD might be subunits of prefoldin, a cofactor of TRiC/CCT that has been shown to interact with some newly synthesized polypeptides (
Our data suggest that the cytosolic proteins continuously associate with and dissociate from a completed polypeptide chain. The addition of an unspecific GroEL trap accelerated the net dissociation of all chaperones, indicating that in its absence a portion of the substrate is able to reassociate with them. Reassociation of chaperones is apparently not very efficient in our in vitro system. In vivo, the chaperones may associate and dissociate as long as the translocation substrate remains in the cytosol (see also
Given that completed polypeptides with and without a signal sequence apparently interact with the same cytosolic proteins, and that there is continuous binding and dissociation, it may not be very surprising that we found that the Sec complex plays no active role in releasing these proteins from the translocation substrate. This conclusion is based on the result that the Sec complex and the unspecific GroEL trap accelerate net dissociation to the same extent. The binding of the substrate to these components appears to prevent the reassociation with cytosolic proteins. Surprisingly, Sec complex and the GroEL trap even stimulated the disappearance of all cross-links after depletion of ATP by addition of hexokinase and glucose (data not shown), suggesting that dissociation of the chaperones can occur in their ADP form. A similar observation has been made for the release of cytosolic Hsp70 from mitochondrial precursor proteins (
Eventually, the polypeptide chain bound to the Sec complex is entirely stripped of all cytosolic proteins. It is possible that the polypeptide chain needs to be fully stripped before its binding to the Sec complex because, with the exception of a 55-kD protein, all cytosolic proteins need to be released from the substrate to allow its stable interaction with the Sec complex. Alternatively, only the signal sequence may have to be stripped initially, allowing substrate binding to the Sec complex. The chaperones would then dissociate from the mature part of the chain but reassociation would be prevented, possibly by the large cytosolic domains of the Sec62p and Sec63p subunits of the Sec complex.
Remarkably, once bound to the Sec complex, the substrate is free of cytosolic proteins even in regions that are protruding into the cytosol or are only loosely associated with the cytosolic aspect of the Sec complex. As the bound polypeptide chain is fully stripped, it can move freely through the channel, uninhibited by any interaction with cytosolic proteins. In fact, thermal motion has been demonstrated to be sufficient for the movement of a chain through the channel (
Posttranslational translocation appears to be quite different from the tightly regulated process of cotranslational translocation in which the signal sequence is recognized first by SRP in the cytosol and then by the Sec61p complex in the membrane. In the posttranslational pathway, the only essential recognition step seems to be the binding of the signal sequence to the Sec complex. Although we have found that the signal sequence of a ribosome-bound posttranslational substrate can bind to SRP, the interaction may be of low affinity in living yeast cells, explaining why the substrate escapes cotranslational membrane targeting. The recognition of the signal sequence by the Sec complex must be sufficient to distinguish proteins to be exported from those staying in the cytosol, despite the fact that both can associate with the same chaperones. The situation may be similar for at least some proteins imported into mitochondria. Although some targeting sequences may be recognized by specific cytosolic receptors, others associate with Hsp70 and are only recognized by receptors on the outer mitochondrial membrane (
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Footnotes |
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Dr. Plath's present address is Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143.
1 Abbreviations used in this paper: CCT, chaperonin containing TCP1; IP, immunoprecipitation; NAC, nascent polypeptideassociated complex; p60/Hop, Hsp70/Hsp90 organizing protein; ppF, prepro-
factor; RNC, ribosomenascent chain complex; SRP, signal recognition particle; TCP1, tailless complex polypeptide; TRiC, TCP1ring complex; wt, wild-type.
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Acknowledgements |
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We thank N. Cowan for providing antibodies to a prefoldin subunit, M. Wiedmann for providing antibodies to NAC, F.U. Hartl for providing the purified GroEL trap, and A. Horwich for providing a plasmid coding for the GroEL trap. We are grateful to J. Brunner for the generous supply of cross-linking reagent. We thank L. Dreier and P. Stein for stimulating discussions, and C. Shamu, M. Wiedmann, K. Matlack, S. Heinrich, B. Tsai, and P. Stein for critical reading of the manuscript.
T.A. Rapoport is a Howard Hughes Medical Institute Investigator. The work was further supported by a grant from the National Institutes of Health (GM54238) to T.A. Rapoport.
Submitted: 14 June 2000
Revised: 11 August 2000
Accepted: 21 August 2000
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