Figure 6.

Reduction of Iss10 levels leads to disappearance of Red1 during meiosis. (A) Localization of Iss10 during meiosis. Wild-type (JT971) cells expressing CFP-Mmi1, Iss10-GFP and Mei2-mCherry from the respective endogenous promoters were examined by fluorescence microscopy under mitotically growing and meiotic conditions. Merged images: blue, CFP-Mmi1; green, Iss10-GFP; red, Mei2-mCherry. The dotted lines indicate the shape of cells. Bar, 5 µm. (B) Iss10 expression levels during meiosis. Native cell extracts prepared from exponentially growing (+N) and meiotic (−N) wild-type (JT973) cells expressing Iss10-13myc from the endogenous promoter were subjected to western blot analysis by using an anti-myc antibody. α-Tubulin was used as a loading control. (C) Expression of iss10 mRNAs during meiosis. Transcripts of iss10 were analyzed by northern blot analysis in exponentially growing (+N) and meiotic (−N) wild-type (JY362) cells. rRNAs stained with ethidium bromide are shown in the bottom panel as loading controls. (D) Localization of Red1 in cells overexpressing Iss10. Meiotic wild-type (JT972) cells expressing Iss10-GFP from the expression plasmid and CFP-Mmi1 and Red1-mCherry from endogenous promoters were examined. Merged images: blue, CFP-Mmi1; green, Iss10-GFP; red, Red1-mCherry. The dotted lines indicate the shape of cells. Bar, 5 µm. (E) Inhibition of sporulation by Iss10 overexpression in temperature-sensitive mei2 mutants. JY450 (wt) and JV393 (mei2-ts) were transformed with a multicopy plasmid expressing Iss10 (iss10) or an empty plasmid (-). Transformants were incubated on SSA medium at the indicated temperatures for 4 days, and sporulation frequency was measured. Error bars indicate standard deviations from three measurements (total n > 500). **P < 0.01 (Student’s t-test).