Fig. 6. Temperature-sensitive OSH mutants were defective for lucifer yellow fluid-phase endocytosis. Lucifer yellow uptake and delivery to the vacuole was observed at 23°C, or after a 60 minute incubation at 37°C, in wild-type (Osh⁺; SEY6210), osh OSH4 (JRY6320), osh osh4-1 (CBY886) and osh osh4-2 (CBY892) cells. For comparison, lucifer yellow uptake was examined at 23°C in wild-type LAS17 (DDY904) and las17 (DDY1438) cells. In the wild-type Osh⁺ strain, vacuoles that were brightly stained with lucifer yellow were observed in 92% of cells grown at 23°C (65 out of 71 cells counted) and an equivalent percentage was seen at 37°C (91%; 64/70). In osh OSH4 cells, 68% exhibited brightly staining vacuoles at 23°C (69/101) and an equivalent percentage was observed at 37°C (69%; 69/99). In osh osh4-1 cells at 23°C, however, 42% exhibited bright lucifer yellow staining (36/85), and at 37°C, only 13% of cells (10/75) efficiently internalized lucifer yellow. Likewise, at 23°C 44% of osh osh4-2 cells (34/78) had bright fluorescent vacuoles and vacuolar remnants, and at 37°C, only 17% of these cells (13/79) were brightly stained. This result was comparable to that observed in the endocytosis mutant las17. Only 13% of las17 cells (15/116) were as brightly stained as in the wild-type parent, where 93% of the wild-type LAS17 cells (78/84) had efficiently absorbed lucifer yellow from the medium. All photographs represent equal exposures.