The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation

Abstract

The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the beta subunit (Sec61beta) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61beta is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61beta kinetically facilitates cotranslational translocation, but is not essential for it. Using chemical cross-linking we show that Sec61beta not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61beta and SPC25 and between Sec61beta and Sec61alpha depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.

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 extract was immunodepleted with an antibody column directed against Sec61β or was incubated with protein A–Sepharose to generate a mock-depleted extract. After reconstitution the proteoliposomes and PK-RM were analyzed by SDS-PAGE and Western blotting using a radioactively labeled secondary antibody and a PhosphorImager system for quantitation. (B) The membranes and proteoliposomes analyzed in A were tested for their competence to translocate polypeptides. Preprolactin mRNA was translated in the wheat germ system in the presence of SRP and microsomes or proteoliposomes, respectively, at 26°C (lanes 2–8). In a parallel experiment translation was started in the absence of microsomes. After addition of membranes or proteoliposomes, respectively, the targeting reaction was carried out at 0°C followed by an elongation at 26°C (lanes 10–16). Half of the sample was treated with proteinase K to assay for material that is translocated into the vesicles and is therefore protected against the added protease. DPα, α subunit of the SRP receptor; pPL, preprolactin; PL, prolactin.

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 extract was immunodepleted with an antibody column directed against Sec61β or was incubated with protein A–Sepharose to generate a mock-depleted extract. After reconstitution the proteoliposomes and PK-RM were analyzed by SDS-PAGE and Western blotting using a radioactively labeled secondary antibody and a PhosphorImager system for quantitation. (B) The membranes and proteoliposomes analyzed in A were tested for their competence to translocate polypeptides. Preprolactin mRNA was translated in the wheat germ system in the presence of SRP and microsomes or proteoliposomes, respectively, at 26°C (lanes 2–8). In a parallel experiment translation was started in the absence of microsomes. After addition of membranes or proteoliposomes, respectively, the targeting reaction was carried out at 0°C followed by an elongation at 26°C (lanes 10–16). Half of the sample was treated with proteinase K to assay for material that is translocated into the vesicles and is therefore protected against the added protease. DPα, α subunit of the SRP receptor; pPL, preprolactin; PL, prolactin.

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. To separate the bound from the unbound fraction the samples were submitted to flotation in a sucrose gradient. The number of binding sites and the apparent dissociation constants were estimated by Scatchard plot analysis. K _d, dissociation constant; eq, membrane equivalant (Walter and Blobel, 1983).

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 subunit of the Sec61 complex (lanes 1–6). To identify the cross-linked polypeptides, RM were treated with BMH as indicated. The samples were subsequently immunoprecipitated under denaturing conditions with an anti-Sec61β column. The precipitated material was analyzed by Western blotting using antibodies directed against Sec61β (lanes 7–10), Sec61α (lanes 11–14) or the 25-kD subunit of the signal peptidase complex (SPC25) (lanes 15–18), respectively. Lanes 1, 7, 11, and 15 show the results for untreated RM.

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 containing the purified Sec61 complex (Sec61p) or the purified signal peptidase complex (SPC), respectively, were treated with BMH as indicated. After SDS-PAGE the samples were analyzed by immunoblotting with antibodies against Sec61β (A) or SPC25 (B). The position of a probable cross-link between Sec61β and Sec61γ is marked by an asterisk.

Figure 5

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.

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 1–3 and 7–9) of mRNA coding for the pPl 86-mer. After addition of PK-RM (lanes 1–6) or rough microsomes (lanes 7–9), respectively, the targeting reaction was carried out at 0°C followed by an incubation at 26°C. The membranes were collected by a centrifugation through a sucrose cushion, separated into three aliquots and treated with bis-maleimidohexane (BMH) as indicated. The samples were analyzed by immunoblotting with antibodies against Sec61β using as a detection system enhanced chemiluminescence (A) or radioactively labeled secondary antibodies (B), respectively. The unknown cross-link marked with an asterisk was not observed in other experiments. (B) A PhosphorImager system was used for quantitation of the cross-linking intensities. The cross-linking intensities obtained for RM were defined as 100%.

Figure 7

Membrane topology of the signal peptidase complex and the Sec61 complex. The topologies of all subunits are schematically illustrated. The dark boxes indicate hydrophobic segments or membrane spanning segments, respectively. SPC22/23 is drawn in the glycosylated form. The position of SH-groups is marked by white circles.