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.