Fig. 3.
The CPY pathway is affected in sur4Δ vps21Δ and fen1Δ vps21Δ mutants. BY4741 (wild-type), MRY90 (fen1Δ vps21Δ), MRY91 (sur4Δ vps21Δ), 1865 (vps21Δ), 5763 (fen1Δ), and 5281 (sur4Δ) cells were grown at 30°C to log phase. (A) Intracellular total lysates (I) and extracellular proteins recovered from the medium (E) were separated by SDS-PAGE, followed by immunoblotting with an anti-CPY antibody. (B) Cells were pulse-labeled with 100 μCi [35S]methionine/cysteine for 15 min and chased for 20 min. Total lysates were prepared, and CPY was immunoprecipitated with an anti-CPY antibody. Precipitates were subjected to SDS-PAGE and detected using a bioimaging analyzer BAS-2500. (C) Total lysates were prepared, separated by SDS-PAGE, and detected by immunoblotting with an anti-Pep4, anti-Pho8, or to demonstrate uniform protein loading, anti-Pgk1 antibody. m, mature form; pro, proform; s, soluble mature form.