The plug domain of yeast Sec61p is important for efficient protein translocation, but is not essential for cell viability

Abstract

The Sec61/SecY translocon mediates translocation of proteins across the membrane and integration of membrane proteins into the lipid bilayer. The structure of the translocon revealed a plug domain blocking the pore on the lumenal side. It was proposed to be important for gating the protein conducting channel and for maintaining the permeability barrier in its unoccupied state. Here, we analyzed in yeast the effect of introducing destabilizing point mutations in the plug domain or of its partial or complete deletion. Unexpectedly, even when the entire plug domain was deleted, cells were viable without growth phenotype. They showed an effect on signal sequence orientation of diagnostic signal-anchor proteins, a minor defect in cotranslational and a significant deficiency in posttranslational translocation. Steady-state levels of the mutant protein were reduced, and when coexpressed with wild-type Sec61p, the mutant lacking the plug competed poorly for complex partners. The results suggest that the plug is unlikely to be important for sealing the translocation pore in yeast but that it plays a role in stabilizing Sec61p during translocon formation.

Figure 1.

Plug domain of the yeast Sec61 complex. A stereo representation of the structure of the yeast Sec61 complex as modeled on the experimental structure of the M. jannaschii SecYEβ translocon (van den Berg et al., 2004) is shown with the cytosolic side facing up. The model is shown as a backbone in stereo with the plug domain (residues 52–74), which was replaced by a glycine in the Δplug mutant, displayed as a space-filling contour. Sbh1p is shown in red, Sss1p in orange, and Sec61p is colored from N to C terminus in blue to yellow. Segments P200-E212 and D227-N240 of Sec61p are not present in the model.

Figure 2.

Mutations in the plug domain have no growth defect. (A) Growth curves were measured for liquid cultures of Δssh1 cells expressing wild-type Sec61p or the indicated plug mutations. (B) Δssh1 cells expressing wild-type Sec61p, or the mutants lacking the entire plug domain (Δplug) or TM2 (ΔTM2), were plated at serial dilutions onto YPDA plates and incubated for 3 d at 15 or 39°C, or onto YPDA plates containing 0.3 μg/ml tunicamycin and incubated for 3 d at 30°C.

Figure 3.

All plug mutations have the same effect on the topology of model proteins. (A) Diagnostic protein substrates to analyze topology changes are shown schematically with the signal-anchor sequence in dark gray (Leu₁₆) or black (of the asialoglycoprotein receptor H1), flanking charges as + and −, and glycosylation sites as black dots. Their names indicate the length of the N-terminal hydrophilic sequence preceding the signal, the hydrophobic signal core (H1 or Leu16) in brackets, and the charge difference Δ(C–N) according to Hartmann et al. (1989) in parentheses. (B) The three substrates were expressed in Δssh1 cells with wild-type, Δplug, or ΔTM2 Sec61p, pulse labeled for 5 min with [³⁵S]methionine, and analyzed by immunoprecipitation, SDS-gel electrophoresis, and autoradiography. The forms corresponding to the polypeptides with zero, one, two, or three glycans are indicated with arrowheads. The dash designates the position of the 37-kDa molecular weight standard. (C) Effect of indicated Sec61p mutants on the orientation of the model proteins was determined by PhosphorImager quantitation of labeling experiments like those shown in B. Two- and threefold glycosylated species together represent polypeptides with a translocated C terminus. Their fraction of the total (in case of 60[H1](+1) excluding the unglycosylated, not integrated products) are represented as the deviation from the value for the wild-type in percentage points). The results of single determinations (no error bars) or of two to six measurements (average with SD) are shown. The absolute fraction of C-terminally translocated products with wild-type Sec61p was 29.1% for [Leu16](−3), 46.3% for 40[Leu16](+5), and 60.2% for 60[H1](+1).

Figure 4.

Co- and posttranslational integration defects of Sec61p mutations. Integration of DPAPB as a cotranslational substrate 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 different products correspond to glycosylated (g) and unglycosylated (u) forms of DPAPB and to the glycosylated first proform (p1) and the unglycosylated pre-pro-form (pp) of CPY. As a control, labeled protein from cells expressing wild-type Sec61p (wt) were analyzed after deglycosylation with endoglycosidase H (EndoH). The unglycosylated forms correspond to nonintegrated polypeptides and were quantified as a fraction of the total in B. In C, the steady-state levels of mature CPY (m) was determined by immunoblot analysis. Equal amounts of cell lysates were analyzed in lanes 2–4.

Figure 5.

Plug deletion impairs translocon levels. (A) Steady-state amounts of wild-type and mutant Sec61p were determined by immunoblot analysis of equal amounts of protein. (B) The half-lives of wild-type Sec61p and the Δplug and ΔTM2 mutants were estimated by immunoblot analysis of cells incubated for 0, 1, 2, 4, or 8 h in the presence of the translation inhibitor cycloheximide. Similar starting signals of Δplug and ΔTM2 versus wild type are shown. (C) Sec61p levels were determined by immunoblot analysis for cells simultaneously expressing wild-type Sec61p from a chromosomal SEC61 gene, and the Δplug or ΔTM2 mutants from a CEN plasmid (wt/Δplug and wt/ΔTM2, respectively). The cells contained no additional plasmid (−), or a 2μ plasmid with SBH1 (+β), SSS1 (+γ), or both (+β+γ) with their natural promoters, or a 2μ plasmid with SBH1 or SSS1 with a GPD promoter (+β* and +γ*, respectively). Alternatively, the cells were incubated for 3 h with 0.3 μg/ml tunicamycin (+Tun) to induce an unfolded protein response. As controls, cells expressing only wild-type Sec61p (lanes 1, 9, 11, 13, and 15) or the Δplug or ΔTM2 mutants (lanes 2 and 16, respectively) were analyzed. Lanes 9–14 are from the identical immunoblot.