Figure 9.
Ribosome binding of Cpc2 is essential to positively regulate the translation efficiency of Pyp1, Pyp2, and Atf1 in fission yeast. (A) Increased Pmk1 phosphorylation in cells expressing a Cpc2(DE) version. Strains AN071 (pmk1-HA6H leu1:cpc2-GFP, cpc2Δ) and AN072 (pmk1-HA6H leu1:cpc2(DE)-GFP, cpc2Δ) were grown to mid-log phase in YES medium and treated with 0.6 M KCl for the indicated times. Pmk1-HA6H was purified by affinity chromatography under native conditions and the activated and total Pmk1 detected by immunoblotting with anti-phospho-p42/44 or anti-HA antibodies, respectively. (B) Decreased Pyp1 and Pyp2 protein levels in the Cpc2(DE) mutant. Strains AN091 (leu1:cpc2-GFP cpc2Δ pyp1–13myc) and AN092 (leu1:cpc2(DE)-GFP cpc2Δ pyp1–13myc) (upper panel), or AN102 (leu1:cpc2-GFP cpc2Δ pyp2–13myc) and AN103 (leu1:cpc2(DE)-GFP cpc2Δ pyp2–13myc) (lower panel) were grown in YES medium to mid-log-phase and treated with 0.6 M KCl. Aliquots were harvested at the times indicated and total extracts obtained. Pyp1–13myc or Pyp2–13myc fusions were detected by immunoblotting with anti-c-myc antibody. Anti-Cdc2 antibody was used for loading control. (C) Decreased Atf1 protein levels in the Cpc2(DE) mutant. Strains AN081 (atf1-HA6H leu1:cpc2-GFP cpc2Δ) and AN082 (atf1-HA6H leu1:cpc2(DE)-GFP cpc2Δ) were grown as above and treated with either 0.6 M KCl (top) or 1 mM H2O2 (bottom). Atf1 was purified by affinity chromatography from cell extracts of aliquots harvested at different time and detected by immunoblotting with anti-HA antibody. Anti-Cdc2 antibody was used for loading control. (D) cpc2Δ cells expressing the Cpc2(DE) version do not recover normal cell size at division. Cell morphology and size in strains AN100 (cpc2Δ), AN071 (leu1:cpc2-GFP, cpc2Δ), and AN072 (leu1:cpc2(DE)-GFP, cpc2Δ) growing in EMM2 medium and stained with calcofluor white are shown.