Figure 2.

Ycf1p processing is dependent on the vacuolar proteases Pep4p and Prb1p and is End3p independent. The steady-state level of Ycf1p-GFP in wild-type (WT) and mutant strain backgrounds was examined by immunoblot (IB) analysis as described in MATERIALS AND METHODS. Unprocessed Ycf1p is indicated as full-length and the C-terminal cleavage product as C-term. The end3 mutant strain (lane 5) was shifted to 37°C for 1 h to impose the endocytosis block before preparing the cell extracts, along with its isogenic WT counterpart (lane 4). Crude yeast cell extracts (0.4 OD₆₀₀ cell equivalents per lane) were resolved by 8% SDS-PAGE and transferred to nitrocellulose. Ycf1p-GFP was detected using rabbit anti-GFP polyclonal antibodies. A molecular weight marker is indicated. The strains used are SM4605 (lane 1), SM4606 (lane 2), SM4607 (lane 3), SM4529 (lane 4), and SM4531 (lane 5).