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. 2021 Jul 26;10:e67390. doi: 10.7554/eLife.67390

Figure 1. Phosphorylation analysis of the essential constitutive centromere-associated network (CCAN) subunit Ame1.

(A) Organization of the essential CCAN component Ame1CENP-U and localization of phosphorylation sites. Ame1 shows a Cdk1 phosphorylation cluster (T31, S41, S45, S52, S53, S59, S101) at the N-terminus. The first 15 amino acids are essential for Mtw1c binding, the coiled-coil region (aa 199–254) is required for heterodimerization with Okp1CENP-Q. Schematic overview on the right shows the four-protein complex COMA, consisting of Ame1CENP-U, Okp1CENP-Q, Ctf19CENP-P, and Mcm21CENP-O. The COMA complex binds to the outer kinetochore component Mtw1 complex and to the centromeric nucleosome. (B) In vitro kinase assay with recombinant Ame1-Okp1c with either Cdk1-Clb2 or Cdk1-Clb5. The migration pattern of Ame1 is shifted to a slowly migrating form when incubated with Cdk1-Clb2. Asterisk denotes a contaminating protein. (C) List of all mapped Ame1 phosphorylation sites either in vivo or in vitro. T31, S41, S45, S53, and S101 show the minimal motif for Cdk1 (S/TP). (D) Stably integrated Ame1 variants display distinct migration patterns in SDS-PAGE. Ame1-WT shows multiple slowly migrating forms that are eliminated in Ame1-7A and Ame1-7E. (E) Serial dilution assay of Ame1 variants using the FRB anchor-away system. Ame1-WT and both mutants can rescue the growth defect when endogenous Ame1 is anchored away from the nucleus. (F) Serial dilution assay of internal Ame1 truncation mutants in the anchor-away system.

Figure 1—source data 1. Mass spectrometry analysis of native constitutive centromere-associated network (CCAN) complexes.
Figure 1—source data 2. Mass spectrometry analysis of in vitro phosphorylated COMA.

Figure 1.

Figure 1—figure supplement 1. Quantitative phosphorylation analysis of recombinant Ame1-Okp1 by S-Cdk1 and M-Cdk1 complexes.

Figure 1—figure supplement 1.

(A) In vitro phosphorylation of recombinant AO with Cdc28-Clb5 and Cdc28-Clb2 complexes or their hydrophobic patch mutants (hpm) that eliminate docking-dependent phosphorylation was analyzed. ΔN: N-terminal truncation mutant of Okp1. Ratio between phosphorylation of the different variants is given below. Quantification shows mean values of Ame1 or Okp1 phosphorylation (a.u.) and standard error of the mean is indicated, n = 3. (B) Multiple-sequence alignment of Ame1 N-terminus (aa 1–63) from different yeast species. Conserved residues are colored according to the ClustalW scheme. The identified phosphorylation sites are conserved in the most closely related Saccharomyces species.