Fig. 1.

FAS is rapidly degraded under nitrogen starvation. (A) FAS1-GFP (TOS016) and FAS2-GFP (TOS017) cells of S. cerevisiae were grown to midlog phase and shifted to SD-N medium for the indicated time periods. After 16 h in SD-N, the cells were washed and shifted to YPD medium for the indicated time periods. Cell lysates were subjected to SDS/PAGE, followed by Western blot analysis using anti-GFP antibodies. (B) Quantification of Fas1-GFP and Fas2-GFP. Error bars represent the SDs of three independent experiments. n.s., not significant (Student’s t test). (C) FAS2-GFP (TOS017) and PGK1-GFP (TOS018) S. cerevisiae strains were grown to midlog phase and shifted to SD-N or KAc medium for the indicated time periods. Cell lysates were subjected to SDS/PAGE, followed by Western blot analysis using anti-GFP antibodies. Error bars represent the SDs of three independent experiments (Right); n.s., not significant; **P < 0.01 (Student’s t test). (D) FAS1-GFP (TOS016) and FAS1-GFP pep4∆ (TOS020) S. cerevisiae cells were grown to midlog phase and shifted to SD-N medium for 16 h. Fas1-GFP localization was monitored in YPD medium and after 16 h in SD-N. FM4-64 was used to detect vacuolar membranes. (Scale bar, 5 µm.) (E) FAS1-GFP (TOS016) and PGK1-GFP (TOS018) S. cerevisiae strains were grown to midlog phase, pulse-labeled with [³⁵S] cysteine/methionine for 30 min, and chased in SD-N medium for the indicated time periods. Cell lysates were subjected to immunoprecipitation using anti-GFP antibodies and the proteins were analyzed by SDS/PAGE electrophoresis followed by autoradiography.