(A) List of histone and kinetochore proteins identified by MS after purification of 3xFLAG-Cse4^16R (SBY5442). (B) Psh1 has a consensus RING domain (residues 29–71, black) and a pair of C4-type zinc finger motifs (residues 150–171, gray). RING domain cysteine and histidine residues predicted to bind zinc are in large typeface and the mutants are indicated in bold. “B” is a bulky residue in the RING domain. (C) Psh1 autoubiquitinates in vitro. Recombinant GST-Psh1 was added to reactions in the presence (+) or absence (−) of ubiquitin-activating enzyme (Uba1), ubiquitin-conjugating enzyme (UbcH5a), ubiquitin (Ub), and ATP (lanes 1–6). A recombinant GST-Psh1 RING mutant with C45S and C50S mutations was used in a complete reaction instead of WT protein in lane 7. Lane 8 contains GST-Psh1 alone. The reactions were run on two gels and immunoblotted with either anti-ubiquitin antibody (left) or anti-GST antibody (right). (D) Recombinant Cse4 octamers were added to reactions containing ubiquitin-activating enzyme (Uba1), ubiquitin-conjugating enzyme (UbcH5a), ubiquitin (Ub), and ATP in the absence (−) or presence (+) of GST-Psh1 (lanes 2 and 3). GST-Psh1 (lane 1) or Cse4 octamers (lane 4) alone were also included in the immunoblot as controls. The blot was probed with anti-Cse4 antibodies.

(A) Wild-type (SBY8281) or ndc10-1 mutant (SBY8282) cells containing Psh1-FLAG were shifted to 37 °C for 3 hrs and Psh1-FLAG was immunoprecipitated. The corresponding immunoblot was probed with anti-FLAG and anti-Cse4 antibodies. (B) WT (SBY8281) or ndc10-1 mutant (SBY8282) cells were grown as in (A). Psh1-FLAG was immunoprecipitated from the soluble (S) and chromatin (P) fractions and the resulting immunoblots were probed with anti-FLAG and anti-Cse4 antibodies. Pgk1 and K4-Me₂ H3 are shown as markers for S and P fractions, respectively. Note that there is loss of material during the procedure so the levels between soluble and chromatin fractions cannot be compared.

(A) Wild-type (SBY3570) and psh1Δ (SBY8355) cells expressing pGAL-Myc-CSE4 were grown in galactose and Cse4 was immunoprecipitated using anti-Myc antibodies. The immunoblot was probed with anti-Myc antibodies. Wild-type cells expressing pGAL-Myc-CSE4^16R (SBY3571) were used as a control. (B) Wild-type (SBY3) and psh1Δ (SBY8336) cells were arrested in G1-, S- or M-phase with α-factor (G1), hydroxyurea (S) or nocodazole (M), respectively. Protein synthesis was inhibited by cycloheximide addition at time zero and lysates were analyzed for endogenous Cse4 levels using anti-Cse4 antibodies at the indicated time points. Pgk1 is shown as a loading control. See also Figure S1.

Extracts (WCE) from wild-type (SBY3570) and psh1Δ (SBY8355) cells expressing pGAL-Myc-CSE4 were fractionated into soluble (S) and chromatin (P) fractions. Cse4 levels were assayed in each fraction using anti-Myc antibodies. Tubulin and K4-Me₂ H3 are markers for S and P fractions, respectively. Note that there is loss of material during the procedure so the levels between soluble and chromatin fractions cannot be compared. (B) FLAG-Cse4 was transiently overexpressed for 1 hour in wild-type (SBY8918) and psh1Δ (SBY8917) cells and its chromatin localization was assayed by immunofluorescence on chromosome spreads. DAPI, Alexafluor-GFP and anti-Cse4 staining recognize the DNA, Mtw1, and Cse4, respectively. Scale bar = 5 μm.

(A) 5- fold dilutions of the indicated strains (SBY674, SBY8904, SBY4471, SBY9007, SBY8903 and SBY8857) were plated on glucose or galactose media at 23 °C. (B) WT (SBY3), WT+pGAL-FLAG-CSE4 (SBY8904) and psh1Δ+pGAL-FLAG-CSE4 (SBY8903) were released from G1 into galactose and plated at the indicated timepoints on glucose media to determine relative viability. (C) Wild-type (SBY8976), psh1Δ (SBY 8982) and psh1Δ mad2Δ (SBY8975) cells containing pGAL-FLAG-CSE4 were released from G1 into galactose media. Lysates were prepared at the indicated time points and analyzed for Pds1 levels by immunoblotting. See also Figure S2.

(A) Anti-Myc-conjugated beads were used to immunoprecipitate Psh1-Myc (SBY5364 and SBY8027). The samples were subsequently analyzed by immunoblotting with anti-Myc, anti-Cse4 or anti-T7 antibodies. (B) Psh1-FLAG was immunoprecipitated from strains expressing Myc-tagged fusions to full length Cse4 (SBY7268), the N-terminal domain (NTD, SBY7269), or the Histone Fold Domain (HFD, SBY7270). The immunoprecipitates were subsequently probed with anti-FLAG or anti-Myc antibodies. (C) Schematic of the chimeric constructs Cse4^−CATD and H3^CATD. Cse4 and H3 are included as a reference. (D) Psh1-FLAG was immunoprecipitated from cells carrying pGAL-Myc-CSE4^−CATD (SBY9126) and subsequently probed with anti-Myc antibodies to detect the Cse4 chimeric protein and anti-Cse4 antibodies to detect endogenous Cse4 protein as a control. (E) Extracts from cells containing Psh1-FLAG and either pGAL-Myc-H3^CATD (SBY 8932) or pGAL-Myc-H3 (SBY9134) were fractionated into soluble (S) and chromatin (P) fractions. Psh1-FLAG was imunoprecipitated and subsequently probed for the Myc tagged proteins. Cells lacking Psh1-FLAG were used as controls (SBY8994 and SBY9133). The samples were immunoblotted with anti-FLAG or anti-Myc antibodies.

(A) The stability of Myc-Cse4^−CATD was assayed in WT (SBY8700) and psh1Δ (SBY8760) cells after repressing transcription and translation. The lysates were immublotted with anti-Myc antibodies. Pgk1 is a loading control. The (−) timepoint indicates samples taken before induction of the protein. (B) The stability of Myc-H3^CATD (SBY8932) and Myc-H3 (SBY 9134) in WT cells as in (A). The lysates were immunoblotted with anti-Myc antibodies. Tubulin is a loading control. The (−) timepoint indicates samples taken before induction of the proteins. (C) The stability of Myc-H3^CATD was assayed in wild-type (SBY8932) and psh1Δ (SBY8959) cells as in (A). (D) Model: Cse4 recognition by its chaperone occurs via the CATD, which targets it to the centromere where it is protected from degradation by kinetochore assembly. Psh1 also recognizes Cse4 via the CATD. Psh1 prevents mislocalization of Cse4 in at least two possible ways, by (a) recognizing mis-incorporated Cse4 on the chromatin and targeting it for degradation, or by (b) by regulating the level of soluble Cse4, thereby limiting its misincorporation rate into the euchromatin. The exclusive localization of Cse4 to the centromere is therefore maintained by the CATD through a combination of targeting Cse4 to the centromere and degrading mislocalized Cse4. See also Figure S3.