Interactions of Ribosome-bound ppαF in the Cytosol
We first used photo-cross-linking to investigate interactions of ribosome-bound nascent ppαF with cytosolic proteins. A truncated mRNA coding for a ppαF polypeptide chain missing only the last five amino acids was translated in vitro in a reticulocyte lysate system in the presence of [35
S]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 (Plath et al. 1998
). This results in the incorporation of photoreactive lysine derivatives at positions of the polypeptide chain where lysines would normally occur. After translation, RNCs together with bound cytosolic proteins originating from the reticulocyte lysate were isolated by sedimentation, and the sample was irradiated to induce cross-links of nascent ppαF. The cross-linked products were analyzed by SDS-PAGE and autoradiography. With wt ppαF, 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 (, 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 ppαF with arginines and introduced a single lysine residue at position 5 within the signal sequence. As previously observed with cotranslational translocation substrates (Wiedmann et al. 1987
), the signal sequence could be cross-linked to SRP54 (, lane 2, asterisks). Surprisingly, two bands of SRP54 cross-links were seen, which both could be immunoprecipitated with specific antibodies (data not shown; see also ). Perhaps, ppαF 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 ppαF to Sec61p (Plath et al. 1998
). Cross-links to the NAC subunits could also be detected (, lane 2, circles; see also ), although they were weaker than those with the probes in the mature region of the chain. These results demonstrate that nascent ppαF interacts mainly with SRP and NAC. It should be noted that nascent ppαF 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 (Wiedmann et al. 1987
). Thus, the posttranslational substrate ppαF may have folding characteristics that keep the signal sequence exposed.
Figure 1 Ribosome-bound ppαF interacts mainly with SRP54 and NAC. Fragments of 160 amino acids of K5, K5Δ, wt, and M2 ppαF containing photoreactive lysine derivatives were synthesized in the reticulocyte lysate system. RNCs were isolated, (more ...)
Figure 2 SRP and NAC only interact with RNCs. A fragment of 160 amino acids of ppαF containing a single photoreactive lysine derivative at position 10 (K10 ppαF) was synthesized in vitro. RNCs were isolated, and after addition of ribosome-depleted (more ...)
Next, we tested whether the cross-linking pattern changes when ppαF carries a defective signal sequence (K5Δ and M2 ppαF mutants). M2 and wt ppαF, which contain the photoreactive probes at the same positions in the COOH-terminal domain, showed identical cross-linking patterns (, compare lane 8 with lane 6). However, the K5Δ 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 ppαF, containing an intact signal sequence (, compare lane 4 with lane 2). These data demonstrate that the interaction of ppαF with SRP, in contrast to that with NAC, requires a functional signal sequence, confirming previous results obtained with preprolactin (Wiedmann et al. 1994
Release of SRP and NAC upon Termination of Translation
Next, we studied whether the interaction of ppαF with cytosolic proteins changes upon termination of translation. To generate ribosome-released full-length ppαF, 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 ppαF mutant that carries a single photoreactive probe at position 10 of the signal sequence (K10 ppαF). Ribosome-associated, truncated K10 ppαF of 160 residues gave the same cross-linking pattern as nascent K5 ppαF studied before (, lane 3; compare with , lane 2); both SRP and NAC cross-links could be verified by IP with specific antibodies (, lanes 6 and 7). In contrast, ribosome-released full-length K10 ppαF of 165 residues could not be cross-linked to either SRP or NAC (, lane 1; IPs in lanes 9 and 10), and several new cross-linked products appeared instead (, lane 1). Similar results were obtained when the truncated nascent chain of 160 residues was released from the ribosome with EDTA (, compare lane 4 with lane 3). The resulting cross-linking pattern was similar to that of full-length K10 ppαF (, compare lane 4 with lane 2). These results demonstrate that both SRP and NAC are released from the translocation substrate ppαF upon termination of translation.
Interactions of Full-Length ppαF in the Cytosol
To analyze interactions of full-length ppαF with cytosolic proteins in more detail, we generated 37 ppαF 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 Plath et al. 1998
Each of the single lysine mutants gave cross-links to several cytosolic proteins present in reticulocyte lysate (). 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 ppαF [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 5–15) 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 ppαF could be cross-linked to several proteins suggests that there are different populations of ppαF molecules that contact either different sets of proteins or the same set with different orientations.
Figure 3 Systematic probing of the molecular environment of full-length ppαF. Full-length polypeptide chains of wt ppαF, of ppαF molecules that each contain a single lysine at the indicated positions (pos.), and of the signal sequence mutants (more ...)
With wt ppαF, 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 ppαF 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 ppαF 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 ppαF carries a defective signal sequence. Regardless of whether the photoreactive probes were located in the signal sequence (K5Δ ppαF) or in the COOH-terminal region of the polypeptide chain (M2 ppαF), the cross-linking pattern of the signal sequence mutant was indistinguishable from that of the corresponding protein with a functional signal sequence (, compare K5Δ with K5 ppαF, and M2 with wt ppαF; 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 ppαF itself (, arrow). This product is probably generated by internal cross-linking within the ppαF 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 ppαF may be in a collapsed conformation.
Full-Length ppαF Interacts with Cytosolic Chaperones
To identify the cytosolic cross-linking partners of full-length ppαF, we performed IP experiments with antibodies directed against cytosolic chaperones and their cofactors. First, cross-linked products obtained with ppαF containing the photoreactive probe at position 10 of the signal sequence were analyzed ( A). After denaturation of the irradiated sample in SDS, antibodies to Hsp70 and TCP1α, a subunit of TRiC/CCT, immunoprecipitated cross-linked products of the expected size ( 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 (Gebauer et al. 1998
), could also be identified by denaturing IP (data not shown).
Figure 4 Full-length ppαF interacts with Hsp70 and TCP1α. (A) ppαF containing a photoreactive lysine derivative at position 10 of the signal sequence (K10 ppαF) was synthesized in vitro and irradiated with UV light (UV) as indicated. (more ...)
To test whether Hsp70 and TCP1α interact with ppαF 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 TCP1α antibodies, the cross-linked product of the expected size corresponded to a major band among the total products ( A, lanes 7 [p70] and 8 [p62]; compare with lane 2), indicating that both Hsp70 and TCP1α are major interaction partners of the signal sequence of ppαF. Hsp70 antibodies also precipitated the prominent cross-linked products containing the proteins of ~50 and 20 kD ( A, lane 7). TCP1α antibodies coprecipitated cross-links to a protein slightly smaller than TCP1α itself (p60) as well as products containing the 20-kD protein ( A, lane 8). The native IPs with Hsp70 and TCP1α antibodies together account for the majority of the bands seen among the total cross-linked products ( A, lane 2). These data suggest that ppαF is part of at least two distinct complexes, explaining why different cross-linked bands could be coprecipitated with Hsp70 and TCP1α antibodies under native conditions.
Hsp70 and TCP1α were also identified as cross-linking partners when the photoreactive probes were located in the COOH-terminal region of the ppαF molecule. With the probe at position 97, the pattern of immunoprecipitation with Hsp70 and TCP1α antibodies was very similar to that of ppαF with the probe in the signal sequence, both under denaturing and native conditions ( B). p60/Hop was also identified as a cross-linking partner (data not shown). Similar results were obtained with wt ppαF containing probes in its nine lysine residues at the COOH terminus ( C). As expected from the results described above (), both Hsp70 and TCP1α were also identified as cross-linking partners with M2 ppαF containing a defective signal sequence (, compare D with C; see also , compare K5Δ with K5 ppαF, p70 and p62). The fact that Hsp70 cross-links more strongly to the signal sequence than to the mature part of ppαF may be explained by its preference for hydrophobic sequences.
Significant cross-links to TCP1α were only observed with ppαF released from the ribosomes (, compare lane 3 with lane 1, □; IP not shown), similar to results obtained with GroEL, the bacterial homologue of TRiC/CCT (Netzer and Hartl 1998
). The results are also in agreement with data obtained with a heterogeneous mixture of nascent chains (Eggers et al. 1997
) and with recent data obtained for the cotranslational translocation substrate preprolactin (McCallum et al. 2000
). For Hsp70, cross-linking of RNCs was observed, but the intensity of the cross-links became stronger upon termination of translation (, compare lane 3 with lane 1,
; 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 ppαF, 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 ( E, lanes 1 and 2). IP experiments after denaturation of cross-linked products with SDS demonstrated that proOmpA was cross-linked to Hsp70 and TCP1α ( 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 TCP1α ( 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 ppαF 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 ( A, fractions 4–6; 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 ( A, lane 7). The cross-links to TCP1α and to the slightly smaller 60-kD protein were found in a high molecular weight peak ( A, fractions 8–10), whose size is consistent with that of TRiC/CCT (970 kD). Again, the cross-linking pattern looked similar to that of native IPs with TCP1α antibodies ( 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 ppαF, although the latter was found in all fractions (quantitation not shown). These data support the existence of at least two distinct ppαF 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 ppαF were observed in all fractions of the sucrose gradient ( A, arrow), indicating that despite association with different chaperone proteins, ppαF does not attain a fully extended conformation.
Figure 5 Hsp70 and TRiC/CCT form different complexes with ppαF. (A) K10 ppαF was synthesized in vitro and irradiated with UV light (UV) as indicated (total). An aliquot of the irradiated sample was subjected to sucrose gradient centrifugation, (more ...)
Similar results were obtained with wt ppαF 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 ppαF in sucrose gradient centrifugation ( B), indicating that posttranslational translocation substrates are generally associated with different cytosolic chaperone complexes. ppαF 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 ppαF 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 non–cross-linked ppαF present in the different complexes could be translocated. Wt ppαF 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 (). 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 (). 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 A). Taken together, these results indicate that different sets of cytosolic proteins keep ppαF in a translocation-competent conformation.
Figure 6 All ppαF populations are translocation competent. Wt ppαF without photoreactive lysine derivatives was synthesized in vitro and subjected to sucrose gradient centrifugation. The top panel shows the distribution of radiolabeled ppαF (more ...)
Dissociation of ppαF from Cytosolic Proteins
We next investigated the fate of the ppαF–chaperone complexes during the next step in posttranslational protein translocation, the binding of ppαF to the Sec complex. Specifically, ppαF 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 ppαF to the cytosolic face of the Sec complex but no translocation (Plath et al. 1998
). After different incubation times at 30°C, the samples were irradiated and the cross-linked products were separated by SDS-PAGE and quantitated. For these experiments, we used ppαF with a photoreactive probe at position 11 because it gives only weak cross-links to the Sec61p subunit of the Sec complex (see 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 ( and ). Cross-links to the p60 subunit of TRiC/CCT also decreased, but more slowly ( C). The kinetics of dissociation of cytosolic proteins from ppαF are consistent with those of binding of ppαF to the Sec complex, as determined by the appearance of cross-links to Sec62p and Sec71p, two other subunits of the Sec complex ( 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).
Figure 8 ppαF bound to Sec complex is not associated with cytosolic proteins. (A) K11 ppαF was synthesized in vitro. Some samples were first irradiated with UV light and then incubated with reconstituted proteoliposomes containing Sec complex (1.X/2.B). (more ...)
Figure 7 Dissociation of cytosolic complexes of ppαF. (A) K11 ppαF was synthesized in reticulocyte lysate and incubated at 30°C either without additions (♦, control), or with a GroEL trap (), or with proteoliposomes containing (more ...)
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 (, 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 ppαF–chaperone complexes on ice resulted in all cross-links remaining constant (data not shown; see also E). These results show that, at elevated temperatures, spontaneous net dissociation of complexes between ppαF 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 ppαF molecules spontaneously released from their cytosolic partners, we used a mutant of the Escherichia coli
chaperonin GroEL as a passive trap (Fenton et al. 1994
). This mutant (D87K, in which the aspartate at position 87 is replaced by a lysine) is able to bind unfolded proteins, but cannot release them and is not expected to actively stimulate the dissociation of ppαF from cytosolic proteins (Thulasiraman et al. 1999
). In the presence of the GroEL trap, the cross-links to all cytosolic proteins disappeared with similar rapid kinetics as in the presence of the Sec complex (, A–C). In addition, cross-links to GroEL appeared with the same kinetics as those to proteins of the Sec complex ( D). We conclude that in both cases the binding partner serves as a simple trap for spontaneously released ppαF molecules. Dissociation of the complexes between ppαF 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 chaperone–substrate complexes in the absence of Sec complex leads to a reduction of translocation competence, we preincubated in vitro–synthesized ppαF at 30°C or 0°C, and then tested in parallel cross-linking of ppαF to cytosolic proteins and binding of ppαF to the Sec complex ( 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 ppαF–Sec Complex Interaction
To confirm the release of cytosolic proteins from ppαF during initiation of translocation, we analyzed interactions of the substrate bound to the Sec complex. ppαF 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 ( and , .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 ppαF. As reported previously (Plath et al. 1998
), ppαF 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; A, lane 6). ppαF with a probe at position 13 gave strong cross-links to both Sec61p and Sec62p/71p ( B, lane 6). With both positions of the probe, ppαF bound to Sec complex did not give any cross-link to cytosolic proteins ( and , 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 ppαF, 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 (, C–E). 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 ppαF bound to the Sec complex were discernible (, C–E, lane 6). Thus, during the binding of ppαF 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 ppαF to the Sec complex. To this end, samples containing ppαF 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 ppαF to the Sec complex (, A–E, 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 (, A–E, compare lanes 2 and 5). The only clear exception are the cross-links to the 55-kD protein (p55), seen with probes in wt ppαF, which were coprecipitated with the Sec complex. Thus, cross-linking of most cytosolic proteins appears to prevent the interaction of ppαF with the Sec complex, suggesting that their release is a prerequisite for initiation of translocation.