Fig. 6. Cells depleted of Osh protein accumulate PMN structures under non-starving conditions. (A) Localization of Nvj1p-EYFP as a function of Osh protein depletion. An Osh depletion strain (osh1-osh7 P_MET3-OSH2) and its parental wild-type background harboring P_CUP1-NVJ1-EYFP were grown to low log phase in the presence or absence of methionine. To deplete all Osh protein, the Osh depletion strain was shifted into methionine-containing medium for 8 hours. Nvj1p-EYFP was induced for 1.5 hours with CuSO₄, and its localization was monitored with respect to vacuolar membranes (red) and nuclear chromatin (blue). PMN structures increase in frequency upon the depletion of Osh protein (v,vi, arrows). (B) Kinetic analysis of Nvj1p-EYFP degradation in cells depleted of Osh protein. Starvation-induced degradation of a limited pool of Nvj1p-EYFP (arrows) was analysed by immunoblot in wild-type, pep4 and osh cells (depleted of Osh protein). Bar graph represents the average percentage loss of Nvj1p-EYFP from two independent experiments. Extract of cells lacking Nvj1p-EYFP is denoted C; a non-specific band cross-reacts above Nvj1p-EYFP on the blots. (C) Cells depleted of Osh protein (osh) show normal sorting and processing of aminopeptidase I precursor (prApe1p) into its mature form (mApe1p) by autophagy. Extracts of pep4 cells, which accumulate unprocessed prApe1p in the vacuole, were used as controls.