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Fig. 1. Electron micrographs of Osh protein-depleted cells and OSH temperature-sensitive mutants. (A) Wild-type, (B) osh P^MET3-OSH2, and (C and D) osh osh4(ts) cells, were examined by electron microscopy. V, vacuole; N, nucleus; LD, lipid droplets. Thick arrows indicate the large dark irregular structures that correspond to fragmented and collapsed vacuoles, thin arrows indicate vesicles, asterisks indicate cell wall abnormalities. (A) Wild-type cells (SEY6210) contained an average of 2.5 lipid droplets (s.d.=2.1; 54 random sections counted); only 2% of wild-type cells (1/54) accumulated more than twenty-five 50 nm vesicles; 2% of wild-type cells (1/54) had pronounced cell wall defects; 2% exhibited vacuolar fragmentation (1/54); and in only 10% of sections with vacuoles (5/50) were <100 nm vacuolar droplets observed. (B) In the absence of methionine (-Met), OSH2 was expressed in osh P^MET3-OSH2 cells (JRY6326), which were viable, but compared with wild-type cells defects were nonetheless observed. In random sections, an average of 2.6 lipid droplets were counted (s.d.=2.5; 70 sections counted); 6% of growing osh P^MET3-OSH2 cells (4/63) exhibited cell wall defects; 5% had an accumulation of more than 25 50 nm vesicles (4/75); 39% exhibited vacuolar fragmentation (25/64); and in 61% of vacuole-containing sections (37/61), small <100 nm vacuolar lipid droplets were observed. After methionine addition (+Met) and the gradual depletion of Osh proteins in osh P^MET3-OSH2 cells, the average number of lipid droplets was 8.1 (s.d.=6.7; 30 sections counted); 47% of Osh-depleted cells (14/30) had pronounced cell wall defects; in 30% of random sections (9/30) more than twenty-five 50 nm vesicles accumulated; 80% of random sections (24/30) contained aberrant, dark-staining vacuolar remnants; and in 81% of sections with vacuoles (26/32), <100 nm vacuolar lipid droplets were observed. (C) osh osh4-1 cells, and (D) osh osh4-2 cells were cultured either at 23°C or at 23°C and then for 1 hour at 37°C. For osh osh4-2 cells at 23°C, an average of 3.5 lipid droplets were observed (s.d.=3.4; 41 sections counted); 37% exhibited vacuolar fragmentation (18/49); 9% accumulated more than twenty-five 50 nm vesicles (4/46); and 10% had pronounced cell wall defects (6/62). At 37°C, osh osh4-2 cells contained an average of 4.4 lipid droplets (s.d.=4.8; 52 sections counted); 62% exhibited vacuolar fragmentation (29/47); 10% accumulated more than twenty-five 50 nm vesicles (6/61); and 15% had pronounced cell wall defects (10/65). Bars, 1 µm.

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