The Spindle Pole Body Assembly Component Mps3p/Nep98p Functions in Sister Chromatid Cohesion

Lisa M Antoniacci,Margaret A Kenna,Peter Uetz,Stanley Fields,Robert V Skibbens

doi:10.1074/jbc.m404324200

Abstract

For successful chromosome segregation during mitosis, several processes must occur early in the cell cycle, including spindle pole duplication, DNA replication, and the establishment of cohesion between nascent sister chromatids. Spindle pole body duplication begins in G1 and continues during early S-phase as spindle pole bodies mature and start to separate. Key steps in spindle pole body duplication are the sequential recruitment of Cdc31p and Spc42p by the nuclear envelope transmembrane protein Msp3p/Nep98p (herein termed Mps3p). Concurrent with DNA replication, Ctf7p/Eco1p (herein termed Ctf7p) ensures that nascent sister chromatids are paired together, identifying the products of replication as sister chromatids. Here, we provide the first evidence that the nuclear envelope spindle pole body assembly component Mps3p performs a function critical to sister chromatid cohesion. Mps3p was identified as interacting with Ctf7p from a genome-wide two-hybrid screen, and the physical interaction was confirmed by both in vivo (co-immunoprecipitation) and in vitro (GST pull-down) assays. An in vivo cohesion assay on new mps3/nep98 alleles revealed that loss of Mps3p results in precocious sister chromatid separation and that Mps3p functions after G1, coincident with Ctf7p. Mps3p is not required for cohesion during mitosis, revealing that Mps3p functions in cohesion establishment and not maintenance. Mutated Mps3p that results in cohesion defects no longer binds to Ctf7p in vitro, demonstrating that the interaction between Mps3p and Ctf7p is physiologically relevant. In support of this model, mps3 ctf7 double mutant cells exhibit conditional synthetic lethality. These findings document a new role for Mps3p in sister chromatid cohesion and provide novel insights into the mechanism by which a spindle pole body component, when mutated, contributes to aneuploidy.

Highlights

Microtubule-organizing center duplication, separation, and microtubule nucleation are all essential facets of proper chromosome segregation
These findings reveal that a spindle pole body assembly factor performs a function that is critical to sister chromatid cohesion and proper chromosome segregation
Two-hybrid Assays—pJL59 containing CEN, TRP1, and GAL4 DNAbinding domain (BD) sequences was obtained by digesting pPC62 (CEN, LEU2, and GAL4(BD) and replacing the CEN-LEU2 cassette for a CEN-TRP1 cassette obtained by PvuI digestion of pRS315 [28]. pJL59 was linearized with ClaI and ligated in the presence of CTF7 obtained by BstBI digestion of pBS2 [9]

Summary

EXPERIMENTAL PROCEDURES

Two-hybrid Assays—pJL59 containing CEN, TRP1, and GAL4 DNAbinding domain (BD) sequences was obtained by digesting pPC62 (CEN, LEU2, and GAL4(BD) and replacing the CEN-LEU2 cassette for a CEN-TRP1 cassette obtained by PvuI digestion of pRS315 (a generous gift from John Lamb) [28]. pJL59 was linearized with ClaI and ligated in the presence of CTF7 obtained by BstBI digestion of pBS2 [9]. The treated beads were washed several times before bound proteins were removed using SDS-containing solubilization buffer. The lysate was centrifuged, and the resulting pellet was treated with lysis buffer supplemented to 1 M NaCl and centrifuged, and the resulting salt-extracted supernatant was harvested for immunoprecipitation. Supernatants were diluted in Buffer A (50 mM Tris (pH 7.5), 50 mM NaCl, 0.2% Triton X-100) supplemented with protease inhibitors (Roche Molecular Biochemicals), incubated with A-14 polyclonal anti-MYC (Santa Cruz Biotechnology) at 4 °C, and incubated with Protein A-Sepharose CL-4B beads (Amersham Biosciences) at 4 °C overnight. A parallel sample was treated with Protein A-Sepharose beads without anti-MYC antibody In both cases, beads were washed with Buffer A several times using low speed centrifugation, and bound proteins were eluted with SDS solubilization buffer. The remaining vector and PCR product were co-transformed into a yeast strain contain-

The abbreviations used are

RESULTS

DISCUSSION