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

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 G 1 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 G 1 , 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.Microtubule-organizing center duplication, separation, and microtubule nucleation are all essential facets of proper chromosome segregation. In budding yeast, microtubule-organizing centers, called spindle pole bodies, function on both sides of the nuclear envelope to nucleate microtubules in both the cytoplasmic and nuclear volumes. Spindle pole body duplication is a multistep process that includes formation of a satellite, expansion into a cytoplasmic duplication plaque, half-bridge growth, and subsequent insertion into the nuclear envelope (1, 2). Evidence from genetic and electron microscopy studies indicates that spindle pole body duplication starts in G 1 and continues into early S-phase. Mps3p/Nep98p (herein termed Mps3p) is an essential nuclear envelope protein that is concentrated at the nuclear envelope half-bridge and required for spindle pole body assembly. Early in spindle pole body assembly, Mps3p functions to recruit and anchor Cdc31p to the half-bridge. Later, Mps3p is required for integration of Spc42p into the spindle pole duplication plaque (3, 4).Coincident with the latter portion of spindle pole body maturation and separation are the processes ...

The Nuclear Envelope and Spindle Pole Body-Associated Mps3 Protein Bind Telomere Regulators and Function in Telomere Clustering

“…However, ctf7 est1 double mutant strains were completely inviable at 30˚C and exhibited a significant growth defect even at 23˚. In fact, each pair-wise combination of mps3, est1 and ctf7 alleles exhibit conditional synthetic lethality, 10 providing indirect evidence for a tripartite complex.…”

“…Given the pair-wise interactions between Mps3p and Est1p and between Mps3p and Ctf7p, 10 we also tested for an interaction between Est1p and Ctf7p. GST-Ctf7p was bacterially expressed and coupled to glutathione-sepharose beads prior to coincubation with lysates from yeast cells expressing Est1-12MYCp.…”

“…In support of this model, Mps3p binds Ctf7p in vivo and mps3 cells exhibit precocious sister chromatid separation. [10][11] In the second model, Mps3p directly links telomeres to the nuclear envelope. This model is in part supported by evidence that Mps3p is critical for efficient movement of telomeres into discrete clusters (this study).…”

“…For instance, Mps3p is critical for proper sister chromatid cohesion and interacts both physically and genetically with the soluble cohesion establishment factor Ctf7p-an essential and highly conserved protein required for sister chromatid pairing during S-phase. [10][11][12][13][14] Based on these findings and previous reports that Ctf7p associates with DNA replication factors, 12,13,15 one model is that Mps3p recruits Ctf7p to the nuclear envelope-potentially activating Ctf7p before its release to the DNA replication fork to establish sister chromatid pairing. 10 Note that other spindle pole body components such as Kar3p and Bim1p were previously identified as functioning in sister chromatid cohesion.…”

“…[10][11][12][13][14] Based on these findings and previous reports that Ctf7p associates with DNA replication factors, 12,13,15 one model is that Mps3p recruits Ctf7p to the nuclear envelope-potentially activating Ctf7p before its release to the DNA replication fork to establish sister chromatid pairing. 10 Note that other spindle pole body components such as Kar3p and Bim1p were previously identified as functioning in sister chromatid cohesion. 16,17 The finding that Mps3p plays a critical role in sister chromatid cohesion raises the possibility that the nuclear envelope may serve similar regulatory functions in other DNA metabolism pathways.…”