Degradation of cyclin Cln3 by exceeding CENs: Mad3 physical and functional interactions with SCF.

Abstract

(A) Analysis of Cln3 stability by promoter shut-off experiments in the presence (orange circles) or absence (gray circles) of two YCp–CENGALp vectors in wild-type cells grown under permissive conditions. After tetracycline addition, cells were collected at the indicated times, and obtained Cln3–6FLAG levels are plotted relative to an unspecific cross-reacting band (asterisk) used as loading control. (B) Analysis of Cln3 stability in Mad3-deficient cells as in (A). (C) Analysis of mCitrine–Cln3–11A stability by time-lapse microscopy in the presence (orange circles) or absence (gray circles) of three YCp vectors. Nuclear levels of mCitrine–Cln3–11A in cells were determined at the indicated times after cycloheximide addition, and mean values (N = 100) are plotted. (D) Analysis of mCitrine–Cln3–11A accumulation in the nucleus in the presence (orange circles) or absence (gray circles) of three YCp vectors. Nuclear levels of mCitrine–Cln3–11A were determined in G1 daughter cells with 50–60 μm3 of volume. Individual data (N = 90) and median values are plotted. (E) Cell extracts (input) and GST PDs of cdc4ts grr1 cells expressing Cln3–13myc and GST fusions to Cdc4ΔFbox, Mad3, or Mad3ΔGLEBS were analyzed by immunoblotting with either αmyc (top panels) or αGST (bottom panel) antibodies. (F) Cell extracts (input) and GST PDs of cells expressing Mad3–3HA or Mad3 ΔGLEBS–3HA and either GST or GST–Cdc4 were analyzed by immunoblotting with either αHA (top panels) or αGST (bottom panel) antibodies. (G) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in Fig 1B to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, N = 50) and a regression line are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in S1 Data. GST, glutathione S-transferase; PD, pulldown; YCp, yeast centromeric plasmid.</p