Figure 2.
Identification of Ipl1p-dependent phosphorylation sites in Mad3p. (A) Recombinant Mad3p was incubated with either Ipl1p or Ipl1p-D227A (kinase dead) in the presence or absence of Sli15p, together with Mg2+-[γ-32P]ATP. Phosphorylation of Mad3p was visualized following polyacrylamide gel electrophoresis and autoradiography, locating the position of the Mad3p band by Coomassie staining. (B) Following phosphorylation with Ipl1p–Sli15p complex, 32P-labeled Mad3p was digested with trypsin and separated on a C18 column, monitoring the 32P elution profile. Peaks containing 32P-labeled peptides are labeled 1–5. Peak 2 contained 57% of the applied 32P radioactivity. (C) Peak 2 was analyzed by MALDI-TOF-TOF mass spectrometry and Edman degradation: The deduced amino acid sequence from the mass spectrometric analysis is plotted against the radioactivity released in each cycle of Edman degradation. Maximal release of 32P occurred in the cycle corresponding to Ser 337. (D) Escherichia coli-expressed Mad3p and Mad3p-S337A were incubated with Ipl1p–Sli15p complex in the presence of Mg2+-[γ-32P]ATP. Phosphorylation of Mad3p and Mad3p-S337A was determined following electrophoresis on a polyacrylamide gel and autoradiography, locating the position of the Mad3p band by Coomassie staining. The relevant section of the stained gel is shown to confirm equal loadings. (E) The Ipl1p consensus phosphorylation site (Cheeseman et al. 2002) is shown together with the four Ipl1p phosphorylation sites identified by LC-MS with precursor ion scanning (one of which had been previously identified in C), highlighting the phosphorylated serine, the basic residue at −2, and the hydrophobic residue at +1 present at two of the sites. A phosphopeptide containing a fifth site that could not be unambiguously placed within the sequence is also shown (possible sites of phosphorylation are indicated in gray). The location of each defined phosphoserine within the Mad3p sequence is also indicated in relation to two conserved domains (Hardwick et al. 2000) involved in binding Cdc20p/Mad2p (I) and Bub3p (II).