Figure 3.

Protein synthesis and autophagic degradation under phosphate-limited conditions. (A) Short time-course analysis. Color-converted cells were re-suspended into normal (+Pi) and Pi-free (−Pi) medium and further cultured up to 24 h. Equal amounts of proteins (5 μ g) from the cells were separated by SDS–PAGE and fluorescence in the gel was recorded. Black and white arrowheads indicate the migration position of intact and processed forms of Cyt b5-KikGR, respectively. (B) Decrease of red-converted intact-size Cyt b5-KikGR during incubation. Cells were treated as described for panel (A), and at each time point, the volume of cells in culture was measured. After the extraction of proteins from cells at the indicated time points, an equal amount (5 μ g) of protein was loaded to 9% SDS–PAGE and the intensity of the red-converted intact forms of Cyt b5-KikGR was quantified from the scanned image of the gel. Thereafter the amount of red-converted protein relative to that at the start of the experiment was calculated using the cell volume, the concentration of extracted protein, the volume of the extract, and the intensity of the protein band. The average of triplicated experiments is shown. The bar represents the SD. (C) In vitro fluorescence loss and processing with vacuole proteins of nonconverted and color-converted Cyt b5-KikGR under different pH conditions. Pellets of cell lysates from nonconverted or color-converted cells after 1000 × g centrifugation were used as the source of Cyt b5-KikGR aggregates. After incubation for the indicated times with buffers at the indicated pH, with or without vacuole-enriched fractions prepared from nontransformed cells cultured in Pi-deficient medium, the proteins were separated by SDS–PAGE and their emissions of green or red fluorescence were recorded. A merged image of the green and red fluorescence is shown. Black and white arrowheads indicate the migration position of the intact and processed forms of Cyt b5-KikGR, respectively.