FIG. 7.

ndc1-39 cells fail to assemble Nup49p-GFP into NPCs at the restrictive temperature. (A) Log-phase NDC1 (strain 3245; a and b) and ndc1-39 (strain 3244; c to f) cells were grown in raffinose-containing medium at 23 or 35°C for 2.5 h, and galactose was added to induce the expression of Nup49p-GFP for 1.5 h (a, c, and e). The cells were then grown in YPD medium for 2 h to repress further Nup49p-GFP expression (b, d, and f). The distribution of Nup49p-GFP (green) was visualized by fluorescence microscopy. Nup49p-GFP clusters as one or two aggregates in ndc1-39 cells (c to f, arrows). Each image shown was projected from eight consecutive images taken at 0.1-μm intervals along the z axis that have been deconvolved. Bar, 1 μm. (B) Quantification of the data in panel A. The y axis represents thepercentages of cells (n = 200) with one or more intense Nup49p-GFP signals for NDC1 cells at 35°C, ndc1-39 cells at 23°C, and ndc1-39 cells at 35°C. ndc1-39 cells at 23°C were grown in YPD medium for 4 h. NDC1 and ndc1-1 cells (strain 3299) were grown in raffinose-containing medium for 16.5 h at 15°C, and galactose was added for 11 h to induce the expression of Nup49p-GFP. Then the cells were grown in YPD medium for 6 h. A control at the permissive temperature of 30°C was performed similarly to the 23°C control, as described above. The percentages of NDC1 cells at 15°C, ndc1-1 cells at 30°C, and ndc1-1 cells at 15°C that had Nup49p-GFP assembly defects are also shown. Error bars are based on three independent trials for the experiments conducted at 23 and 35°C and two independent trials for the experiments conducted at 30 and 15°C.