Figure 3.

Pmt2p is required for Δgpαf mannosylation but not for its degradation. (A) Microsomes were prepared from PMT wild-type SEY6210 (lane 1) and SEY6211 (lane 2) and the indicated mutant strains. Equal amounts of in vitro translated, [³⁵S]methionine-labeled pΔgpαf were translocated into wild-type yeast microsomes at 24°C in the presence of ATP and an ATP-regenerating system for 50 min, followed by membrane lysis and ConA precipitation. Translocation efficiencies were similar for all microsome preparations tested. Lectin-bound material was analyzed by SDS-PAGE and autoradiography. (B) PMT2 wild-type and Δpmt2 cells expressing pΔgpαf were pulse-labeled for 5 min with [³⁵S]methionine/cysteine and chased for the indicated periods of time. At each time point, cells were lysed and alpha-factor precursor immunoprecipitated and quantified by SDS-PAGE and phosphorimager analysis. The experiment was repeated twice.