Figure 3.

Specific sec61 mutants are defective in glycopeptide export from the ER. Microsomes were prepared from SEC61 wild-type and mutant strains grown at their respective permissive temperatures (see Methods). (Upper) Glycopeptide export. Each strain was assayed in duplicate for glycopeptide export for 30 min at 24°C in the presence or individual absence of ATP and cytosol, as described in Methods. Release at 30 min is shown. Nonspecific release in the absence of ATP was less than 5% and subtracted. Mutants with significantly different effects on glycopeptide and misfolded protein export are marked with asterisks. (Lower) Misfolded protein export. Mutant α-factor precursor (pΔgpαf) was translocated into wild type or sec61 mutant microsomes and Δgpαf export and degradation initiated by the addition of ATP and cytosol, as described in Methods. After 30 min at 24°C, proteins were precipitated with TCA, resolved by gel electrophoresis, and Δgpαf quantified by using a Bio-Rad phosphorimager. Samples were done in duplicate and the experiment repeated twice.