FIG 4.

Phosphoproteomic analysis of bub mutants after HO-induced DSB at the MAT locus. (A) Strategy to identify phosphoprotein substrates that are dependent (case 1) or independent (cases 2 and 3) of Bub1/Bub2 in response to DSB induced by HO endonuclease. (B) The distribution of protein copy number per cell gathered from Ghaemmaghami et al. (60) is shown for phosphoproteins identified in this study versus the total yeast cellular proteome. (C) The number and percentage of Bub-dependent or -independent kinase and nonkinase phosphorylation sites identified by MS at high confidence (left), and the relative ion abundances (right) are shown for two representative Bub-dependent kinase-phosphorylated substrates. (D) Comparative analysis on the frequency of amino acids at the +1 position of phosphorylated serine or threonine for Bub kinase-dependent phosphorylation (denoted as a hydrophobic or hydrophilic residue) against all identified phosphopeptides. (E) Motif analysis showing the distribution of amino acid residues surrounding the phospho-SQ sites on the Bub-dependent substrates after HO induction. (F) Network showing the connectivity between Bub and phosphorylated kinase substrates from various processes in response to DSB induction. (G) Immunoblot analyses of HO-induced DSB with cells expressing a hemagglutinin (HA)-tagged Akl1 and Ste20 Bub-dependent kinase substrates in the anti-HA immunoprecipitates using anti-phospho-S/T-Q antibody. Anti-HA antibody was used as the loading control (LC).