Figure 6.

Sec61p, but not a mutant of Sec62p, are close to the nascent chain of a translocated protein. (A) These assays employed S. cerevisiae expressing Mfα₃₇-C_ub-Dha (construct 8, Figure 2) and one of the following N_ub fusions (Figure 2): N_ub-Sec62p, either integrated (lane a) or plasmid borne (lane b); N_ub-Sec62(ΔC60)Dha (lane c); and N_ub-Sec61p (lane d). Whole-cell extracts from these strains were subjected to immunoblot analysis with anti-ha antibody. (B) Same as panel A but the same N_ub fusions were coexpressed with Tpi1-C_ub-Dha (construct 14; Figure 2). Numbers 2 and 14 indicate the positions of the corresponding (uncleaved) fusions. (C) Lane a: S. cerevisiae expressing Suc2₂₃-C_ub-Dha (construct 10; Figure 2) together with either N_ub-Sec61p, N_ua-Sec61p, N_ug-Sec61p, N_ub-Sec62p, N_ua-Sec62p, or N_ug-Sec62p; lane b: same as lane a but cells expressed Mfα₃₇-C_ub-Dha (construct 8; Figure 2) instead of Suc2₂₃-C_ub-Dha. (D) S. cerevisiae cells expressing Mfα₃₇-C_ub-Dha together with N_ub-Sec61p (a and b) or N_ub-Sec62p (e and f), and cells expressing Suc2₂₃-C_ub-Dha together with N_ub-Sec61p (c and d) or N_ub-Sec62p (g and h) were labeled for 5 min with ³⁵S-methionine. Whole-cell extracts were immunoprecipitated with anti-ha antibody, followed by SDS-PAGE, and quantitation of the cleaved and uncleaved C_ub fusions using PhosphorImager. Shown are the relative amounts of the cleaved (white bars: a, c, e, and g) and uncleaved (black bars: b, d, f, and h) C_ub fusions. The sum of a cleaved and uncleaved fusion was set at 100 in each of the three independent experiments. SDs are also indicated.