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Fig. 1. Ace2p regulates mid2 RNA and protein levels and functions downstream of Sep1p. (A) cdc25-22 ace2{Delta} (KGY3236) cells or (C) cdc25-22 mid2-myc13 ace2{Delta} (KGY4727) cells were arrested in G2 by shifting to 36°C for 4 hours. Cultures were released to 25°C and cell pellets were collected every 15 minutes for northern blot or immunoblot analysis, respectively. RNA collected from the cdc25-22 ace2{Delta} pellets was used for northern blot analysis alongside RNA from wild-type cells using the his3+ transcript as a loading control. (B) Protein lysates prepared from mid2-myc13 ace2{Delta} (KGY4518) and mid2-myc13 (KGY2432) strains were run on a 4-12% Bis-Tris gel and immunoblotted with 9E10 antibody to determine Mid2p levels and to determine the levels of Cdc2p that served as a loading control. (C) Protein lysates were prepared from the cdc25-22 mid2-myc13 ace2{Delta} (KGY4727) pellets and resolved by SDS-PAGE. Immunoblotting was performed as in (B) along with anti-Cdc13p (determined by blotting with GJG56 anti-Cdc13 rabbit serum) as a marker for cell-cycle progression. (D) The nmt1sep1-HA3 mid2-myc13 (KGY4235) strain was grown under conditions that repressed (+T) or derepressed (–T) sep1+ expression at 32°C for 20 hours. Protein lysates were prepared and resolved alongside wild-type (KGY246) lysates on a 4-12% Bis-Tris gel. Mid2p-Myc13 levels were determined by immunoblotting with the 9E10 antibody and Cdc2p (loading control) levels were detected with anti-PSTAIRE. (E) The mid2-myc13 (KGY2432) strain containing the pREP3X-ace2 plasmid (pKG3141) was grown and immunoblotted as in (D). (F) sep1{Delta} mid2-HA3 (KGY3420) and mid2-HA3 (KGY3131) cells containing either empty pREP3X vector (1) or pREP3X-ace2 vector (2) were grown in the absence of thiamine at 32°C for 20 hours to express ace2+. Protein lysates were prepared and immunoblotted as in (D). (G) A mid2-myc13 strain lacking ace2+ and with sep1 under control of the nmt1 promoter (KGY4740) was grown and immunoblotted alongside mid2-myc13 lysate as in (D).





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