Sec61β Facilitates Cotranslational Protein Translocation
We used reconstituted proteoliposomes, immunodepleted of the β subunit of the Sec61 complex, to investigate the role of the protein in cotranslational protein translocation. Mammalian PK-RM, were solubilized in a detergent mixture that leads to the dissociation of the Sec61 complex into its subunits. The detergent extract was incubated either with antibodies directed against Sec61β that had been immobilized on protein A–Sepharose or, to generate a mock-depleted extract, with protein A–Sepharose alone. The efficiency of immunodepletion was tested by Western blotting, using a radioactively labeled secondary antibody and a PhosphorImager. In a typical experiment, the depleted proteoliposomes contained <0.5% of the original amount of Sec61β, whereas all other proteins tested remained almost unaffected (Fig. A). The only exception was Sec61α, the concentration of which was reduced to 50– 70% in the worst case. Apparently, under the conditions used, the α and the β subunits are not totally dissociated.
Figure 1 Sec61β facilitates cotranslational protein translocation. (A) To produce proteoliposomes lacking Sec61β ribosome-free membranes (PK-RM) were solubilized under conditions where the Sec61 complex dissociates into its subunits. The detergent (more ...)
We then tested the reconstituted proteoliposomes for their ability to translocate proteins synthesized in a wheat germ translation system. Microsomes (PK-RM) served as a control. Transcripts coding for full-length preprolactin were translated at 26°C in the presence of microsomes or proteoliposomes, the concentration of which was normalized for their Sec61α content (Fig. A). PK-RM and mock-depleted proteoliposomes had the same translocation activity (Fig. B, lanes 6 and 7), whereas the depleted proteoliposomes were totally inactive (lane 8). In the absence of Sec61β, processed prolactin was produced (Fig. B, lane 4) that, however, was accessible to the action of proteinase K; it therefore presumably represents material generated by signal peptidase that was incorporated into the reconstituted membrane in the inverse orientation.
Since Sec61β is not essential in yeast, we wondered if the mammalian Sec61 complex lacking this component may show in vitro translocation activity under less stringent conditions than used before. We therefore performed the translocation reaction such that more time would be allowed for the membrane binding of the ribosome– nascent chain complex. Translation of the full-length transcript coding for preprolactin was initially carried out in the presence of SRP but absence of membranes. This leads to a translational arrest when the polypeptide chain reaches a length of ~70 residues. The membranes were then added at 0°C, conditions that allow efficient membrane binding of the ribosome–nascent chain complexes but no chain elongation. The samples were then warmed up to 26°C to continue translation and concomitant translocation. With this protocol, proteoliposomes lacking Sec61β were active in translocation, although their activity was lower by a factor of three compared with the wild-type complex (Fig. B, lane 16 vs. lane 15). Thus, the depleted proteoliposomes are capable to translocate polypeptides if given enough time in the membrane targeting reaction. The results also indicate that the Sec61 complex lacking its β subunit has not been irreversibly denatured during the prolonged immunodepletion procedure. These data suggest that the ribosome–nascent chain complex was targeted to the membrane and thus brought in contact with the signal peptidase, but that a subsequent translocation step was perturbed. We also found that a fragment of preprolactin of 86 amino acids could be efficiently targeted to reconstituted proteoliposomes and reached a protease-protected state even if Sec61β was lacking (data not shown), supporting the conclusion that insertion of the nascent chain into the Sec61β-depleted translocation site can occur if no chain elongation is going on.
It should be noted that in the absence of membranes, almost no full-length preprolactin could be observed (Fig. B, lanes 1 and 9), indicating that under the conditions used, SRP produced a tight translational arrest. Both the microsomes and the two types of proteoliposomes were able to release the translational arrest (Fig. B, lanes 2–4 and 10–12), indicating that this reaction is not dependent on the presence of Sec61β.
Sec61β Is Not Required for Ribosome Binding
Our data suggested that in the absence of Sec61β, the binding of the ribosome–nascent chain complex to the ER membrane is less efficient. This could be due to either a defect in the interaction of the mutated Sec61 complex with the ribosome, or to a perturbed insertion of the nascent chain into the translocation site. We therefore analyzed whether the β subunit plays a role in ribosome binding. Depleted and mock-depleted proteoliposomes were incubated at physiological salt concentrations with radioactively labeled ribosomes and increasing amounts of unlabeled ribosomes, both lacking nascent polypeptide chains. Under these conditions, the ribosomes interact mainly with the Sec61 complex (Kalies et al., 1994
). To separate the unbound from the bound fraction, the membranes were floated in a sucrose gradient. Scatchard plot analysis was used to estimate the number of binding sites and the apparent dissociation constants (Fig. ). Both the depleted and mock-depleted proteoliposomes were found to bind ribosomes with approximately the same binding constant. Also, the number of binding sites was about the same. The measured parameters are in good agreement with published data for the binding of ribosomes to PK-RM and proteoliposomes (Kalies et al., 1994
), although the dissociation constants seem to be somewhat higher at 26° than at 0°C. Thus, steps other than the interaction between SRP and its membrane receptor or the binding of ribosomes to the Sec61 complex must be responsible for the less efficient insertion of the nascent polypeptide chains into proteoliposomes lacking Sec61β.
Figure 2 Binding of ribosomes to reconstituted proteoliposomes. Sec61β- and mock-depleted proteoliposomes were incubated with radioactively labeled ribosomes and increasing amounts of unlabeled ribosomes at physiological salt concentrations and 26°C. (more ...)
Sec61β Interacts with the 25-kD Subunit of the Signal Peptidase Complex
To further analyze the function of Sec61β, we investigated its molecular environment in the membrane by chemical cross-linking. Rough microsomes (RM) were treated with increasing amounts of BMH, a bifunctional cross-linking reagent that reacts with sulfhydryl (SH) groups. The proteins were subsequently separated by SDS-PAGE and analyzed by immunoblotting with antibodies against Sec61β (Fig. , lanes 2–6). Three cross-linked products were detected with the antibodies (Fig. , lanes 2–6 vs. lane 1). The apparent molecular weights of the cross-linked proteins were estimated to be 12, 23, and 38 kD, assuming an apparent molecular weight for Sec61β of 13 kD.
Figure 3 Analysis of the environment of Sec61β by chemical cross-linking. RM were treated with increasing amounts of bis-maleimidohexane (BMH) and were subsequently analyzed by SDS-PAGE and immunoblotting using antibodies directed against the β (more ...)
To identify the cross-linked proteins, microsomes were treated with two different concentrations of BMH and dissolved in SDS-containing buffer to dissociate noncovalent chemical bonds. The extract was subjected to immunoprecipitation with Sec61β antibodies and the precipitated material was analyzed by Western blotting using different antibodies. Fig. shows the immunoblots with antibodies directed against Sec61β (Fig. , lanes 7–10), Sec61α (Fig. , lanes 11–14) and SPC25 (lanes 15–18). The product containing the 38-kD protein could be immunoprecipitated with Sec61β antibodies (Fig. , lane 10) and was recognized by the Sec61α antibody (lane 14), indicating that it is generated by cross-linking between the α and β subunits of the Sec61 complex. The product containing a protein of ~23 kD could be immunoprecipitated with Sec61β antibodies (Fig. , lane 9) and reacted with antibodies against SPC25 (lane 17) and is thus generated by cross-linking between these two proteins. Neither Sec61α nor SPC25 were coprecipitated with Sec61β if BMH was omitted (Fig. , lanes 12 and 16), and both antibodies recognized single bands in untreated RM (lanes 11 and 15).
The product containing the protein of ~12 kD did not react with any of the antibodies tested. Considering its size, we suspected that it may represent a product generated by cross-linking of two β subunits of the Sec61 complex. To test this assumption, purified Sec61 complex was reconstituted into proteoliposomes and subjected to cross-linking with BMH. When analyzed by SDS-PAGE and immunoblotting with Sec61β antibodies, a cross-linked product containing a 12-kD protein was again observed (Fig. A, lanes 10–12), indicating that the cross-linking partner is indeed a constituent of the Sec61 complex. The smaller cross-linked product in Fig. A, lanes 11 and 12 (marked with an asterisk) is probably generated by cross-linking between Sec61β and Sec61γ, the smallest subunit of the Sec61 complex. The appearance of this cross-linked product in native microsomes was variable among different experiments.
Figure 4 Sec61β is involved in ribosome-dependent structural changes of the translocation site. Rough microsomes (RM), ribosome-free membranes (PK-RM), proteoliposomes produced from an unfractionated detergent extract of PK-RM (total), and proteoliposomes (more ...)
Ribosome-dependent Structural Changes of the Translocation Site
We were concerned that the membrane-bound ribosomes may prevent full access of the bifunctional cross-linker to Sec61β so that only a subset of its interacting partners could be detected. However, to our surprise, when PK-RM were used in cross-linking experiments, not only were no additional cross-links observed, but those between Sec61α and Sec61β and between Sec61β and SPC25 could no longer be seen (Fig. A, lanes 4–6). Treatment of RM with high salt or puromycin alone did not change these cross-links (data not shown), suggesting that their disappearance requires the dissociation of the ribosomes into subunits. In agreement with this assumption, proteoliposomes reconstituted from a crude detergent extract of microsomes or proteoliposomes containing only the purified Sec61 complex, which both lack membrane-bound ribosomes, also did not give these cross-links (Fig. A, lanes 7–12).
A similar conclusion could be drawn when the cross-linking reaction was analyzed with SPC25 antibodies (Fig. B). With RM two cross-linked products were seen, one of ~36 kD between SPC25 and Sec61β, and another of ~46 kD (Fig. B, lane 2 vs. lane 1). With PK-RM or with proteoliposomes reconstituted from a crude detergent extract, the adduct of SPC25 and Sec61β was no longer observed (Fig. B, lanes 4–6 and lanes 7–9, respectively), whereas the 46-kD cross-linked product remained unchanged. The latter was also observed with proteoliposomes containing only the purified signal peptidase complex (Fig. B, lane 11). Two SPC subunits, the nonglycosylated SPC25 and the glycoprotein SPC22/23 carry SH groups and have an appropriate molecular weight to produce this 46-kD cross-link with SPC25. As the molecular weight of the cross-linked product did not change after treatment with N-glycosidase F (data not shown), we conclude that it consists of two SPC25.
To exclude that the high salt treatment during the preparation of PK-RM was responsible for the structural alterations identified, ribosomes were detached from the membrane by an independent method. When the reaction with BMH was performed in the presence of 10 mM EDTA under low salt conditions, the cross-link between Sec61β and Sec61α and that between Sec61β and SPC25 could not be seen anymore (Fig. , lanes 5 and 4 vs. lanes 2 and 3). However, the homotypic cross-link between two Sec61β remained unchanged, indicating that the EDTA did not interfere with the reactivity of the BMH. It should be noted that the extent to which the cross-linking intensity was reduced varied in different experiments.
Structural changes of the translocation site in EDTA-treated microsomes. RM were incubated with 10 mM EDTA before BMH was added as indicated. The samples were analyzed by SDS-PAGE and immunoblotting with anti-Sec61β antibodies.
If the ribosome-dependent alteration of the cross-link pattern has a real physiological significance it should be possible to reproduce the cross-link between Sec61β and Sec61α and that between Sec61β and SPC25 by a retargeting of ribosomes carrying nascent polypeptide chains at ribosome free membranes. Ribosome–nascent chain complexes were produced by an in vitro translation of truncated mRNA coding for the first 86 amino acids of preprolactin (86-mer). Ribosome-free membranes (PK-RM) were then added to the translation mix (Fig. A, lanes 4–6). As controls PK-RM and RM were incubated with a translation mix that did not contain any preprolactin mRNA (Fig. A, lanes 1–3 and lanes 7–9). After isolation of the membranes aliquots of each sample were treated with different amounts of BMH. The samples were analyzed by Western blotting with Sec61β antibodies using enhanced chemiluminescence as a detection system (Fig. A) or by quantitative immunoblotting using radioactively labeled secondary antibodies and a PhosphorImager (Fig. B). The quantitation (Fig. B) shows that the incubation of PK-RM with ribosome–nascent chain complexes led to a clear restimulation of the cross-link intensity between Sec61β and Sec61α and between Sec61β and SPC25. Similar results were obtained if EDTA-treated membranes were analyzed (data not shown).
Figure 6 Structural changes of the translocation site are induced by the targeting of ribosomes carrying the preprolactin 86-mer (pPl 86mer). (A) Translation was carried out in the reticulocyte lysate system in the presence (lanes 4–6) or absence (lanes (more ...)
Taken together, these data provide evidence that the β subunit of the Sec61 complex is involved in ribosome- dependent conformational changes of the translocation channel and that it specifically interacts with the signal peptidase during cotranslational translocation.