The hydrophobic core of the Sec61 translocon defines the hydrophobicity threshold for membrane integration

Abstract

The Sec61 translocon mediates the translocation of proteins across the endoplasmic reticulum membrane and the lateral integration of transmembrane segments into the lipid bilayer. The structure of the idle translocon is closed by a lumenal plug domain and a hydrophobic constriction ring. To test the function of the apolar constriction, we have mutated all six ring residues of yeast Sec61p to more hydrophilic, bulky, or even charged amino acids (alanines, glycines, serines, tryptophans, lysines, or aspartates). The translocon was found to be surprisingly tolerant even to the charge mutations in the constriction ring, because growth and translocation efficiency were not drastically affected. Most interestingly, ring mutants were found to affect the integration of hydrophobic sequences into the lipid bilayer, indicating that the translocon does not simply catalyze the partitioning of potential transmembrane segments between an aqueous environment and the lipid bilayer but that it also plays an active role in setting the hydrophobicity threshold for membrane integration.

Figure 1.

Plug domain and constriction ring of Sec61p. The model of yeast Sec61 complex is shown as the polypeptide backbone (Sec61p in blue to yellow, Sbh1p in red, and Sss1p in orange) with the plug domain (residues 52-74; A) or the residues of the constriction ring (V82, I86, I181, T185, M294, and M450; B and C) in space-filling representation in gray. Views from within the membrane (A and B) or from the cytosol (C) are shown with the lateral exit gate to the front or bottom, respectively.

Figure 2.

Growth of yeast cells with wild-type or mutant Sec61p in the presence or absence of Ssh1p. SSH1 or Δssh1 cells expressing the indicated Sec61p mutants were plated at serial dilutions onto YPDA plates and incubated for 3 d at 30°C, 5 d at 37°C, or 11 d at 15°C.

Figure 3.

Translocation efficiency of wild-type and mutant Sec61p. Integration of DPAPB as a cotranslational and of CPY as a posttranslational substrate of the Sec61 translocon was analyzed in a Δssh1 background by pulse labeling for 5 min with [³⁵S]methionine, immunoprecipitation, gel electrophoresis, and autoradiography (A). The products correspond to glycosylated (g) and unglycosylated (u) forms of DPAPB and to the glycosylated first proform (p1) and the unglycosylated preproform (pp) of CPY. Results were quantified by phosphorimaging, and the fraction of untranslocated DPAPB (B) and CPY (C) was plotted (mean and SD of three determinations; single measurements for 6GΔ and 6WΔ in C). In D, C-terminally truncated CPYΔC was expressed in cells with the indicated wild-type and mutant translocons, pulse labeled for 5 min, and chased with unlabeled methionine for up to 30 min before immunoprecipitation, gel electrophoresis, and autoradiography to separate the translocated, two- and threefold glycosylated ER forms (ER) from cytosolic precursor (cyt).

Figure 4.

prl phenotype of mutant Sec61p. (A) CPYΔ3 (CPY with a signal sequence lacking 3 apolar residues) was expressed in Δssh1 cells with wild-type (wt) or the indicated mutant Sec61p, labeled, and analyzed as described in Figure 3A. (B) Translocation efficiency was quantified by phosphorimager. The average of one to three determinations is shown. The horizontal line indicates the wild-type levels.

Figure 5.

Levels of wild-type or mutant translocons in the absence (A) or presence (B) of a second wild-type copy of Sec61p. (A) Steady-state amounts of wild-type and mutant Sec61p were determined in an SSH1 background by immunoblot analysis of total cell lysate. Equal loading was approximated based on protein determination and Coomassie staining of SDS-gels. (B) Yeast cells expressing equal amounts of wild-type Sec61p and the indicated HA-tagged mutants were analyzed by immunoblot analysis using an antiserum against the C terminus of Sec61p (α61C) and an anti-HA antibody (αHA) recognizing wild-type and mutant Sec61p, respectively. Mutation of constriction residues to aspartates consistently resulted in slightly reduced electrophoretic mobility. The asterisk indicates a background band recognized by the anti-HA antibody that also serves as a loading control. Data for SSH1 cells are shown; the same result was obtained with Δssh1 cells (unpublished data).

Figure 6.

Membrane insertion of H-segments of various hydrophobicities mediated by wild-type and mutant Sec61p. (A) Schematic representation of the DPAPB-H model proteins (left) and products of the DPAPB-H substrate with four leucines expressed in cells with wild-type Sec61p after [³⁵S]methionine labeling, immunoprecipitation, incubation with (+) or without (−) endoglycosidase H (endoH), and gel electrophoresis (right). Integration of the H-segment results in a partially glycosylated double-spanning membrane protein (I), whereas its translocation yields a fully glycosylated type II protein (T). U, unglycosylated form, cyt, cytoplasmic; exo, exoplasmic. The position of molecular weight markers (in kilodaltons) is indicated. (B) SSH1 cells expressing wild-type or mutant translocons (as indicated on the left) as well as a DPAPB-H substrate (with the number of leucine or serine residues indicated above and below) were pulse labeled with[³⁵S]methionine, and the substrate products were immunoprecipitated, separated by gel electrophoresis, and visualized by autoradiography.

Figure 7.

Efficiency of H-segment integration by wild-type and mutant Sec61p. (A–C) The membrane-inserted fraction of two to five experiments like those shown in Figure 6 was quantified and plotted (with SDs) versus the number of leucines or serines in the H-segment. (A′–C′) The data for the H-segments containing zero to six leucines also were plotted as apparent free energies of membrane insertion, ΔG_app with straight lines determined by linear regression.