FIG. 6.

Rpf1 is required for the export of large ribosomal subunits. (A) The rpf1-1 mutant is temperature sensitive for growth at 37°C. RPF1 wild-type cells, rpf1-1 mutants, and diploid cells generated by crossing the wild type with rpf1-1 cells were spotted onto plates containing rich yeast-peptone-dextrose media and incubated for 2 days at 25°C, 30°C, or 37°C. (B) The Rpf1-1 protein is stable at 37°C. Logarithmically grown RPF1 cells and rpf1-1 cells were cultured in liquid yeast-peptone-dextrose media at 25°C or cultured at 25°C and then shifted to 37°C for 2 h. The cells were analyzed by SDS-PAGE and Western blotting with affinity-purified Rpf1-specific antibodies. Molecular mass markers are in kDa. (C) rpf1-1 mutants are defective in export of large ribosomal subunits. RPF1 cells and rpf1-1 mutants containing a chromosomal integration of L11-GFP were grown in liquid media at 25°C and analyzed by immunofluorescence microscopy. The samples were probed with GFP-specific antibodies and with FITC-conjugated secondary antibodies and stained with the DNA dye DAPI.