(A) The intracellular distribution of She2p mutants N36S and R63K is not altered. Representative images for the intracellular distribution of She2p-myc6. Yeast strain YLM777 was transformed with pRL453 (control), pRL199 (She2p-myc6-wt), pRL324 (She2p-myc6-N36S), or pRL322 (She2p-myc6-R63K). Transformants were grown in synthetic media minus leucine; subsequently, the cells were fixed and processed for immunofluorescence using anti-myc. (B) She2p does not accumulate in the nucleus in the absence of mRNA export. Yeast strain YLM1769 was transformed with the following combinations of plasmids: pRL718 (pESC-URA-ASH1)/YCplac22 (vector), pRL718/pRL676 (YCplac22-She2p-myc6-wt), pRL718/pRL677 (YCplac22-She2p-myc6-N36S), and pRL718/pRL678 (YCplac22-She2p-myc6-R63K). Transformants were grown at the permissive and nonpermissive temperatures as described in Materials and Methods, Temperature Shift Experiment. For immunofluorescence, anti-myc antibody was used and ASH1 mRNA was detected by FISH.
