Figure 3.

Metabolic pulse-chase analysis of Ycf1p-GFP processing. (A) To examine the kinetics of PEP4-dependent conversion of full-length Ycf1p-GFP into the C-terminal cleavage product, pulse-chase experiments were performed in PEP4 (wild-type) (lanes 1–4) and pep4Δ (lanes 5–8) strains. Cells were pulse labeled for 10 min with Express [³⁵S] and the label was chased for the indicated times by addition of an excess of unlabeled cysteine and methionine. Ycf1p-GFP was immunoprecipitated with rabbit anti-GFP polyclonal antibodies and the samples were analyzed by 8% SDS-PAGE. The strains used are SM4523 (lanes 1–4) and SM4524 (lanes 5–8). (B) Bands corresponding to full-length Ycf1p and the C-terminal proteolytic product in lanes 1–4 were quantitated using PhosporImager and ImageQuant software. The data was graphed with KaleidaGraph to reveal a precursor–product relationship between full-length Ycf1p and the C-terminal cleavage product.