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An Mtw1 C terminal motif is cruical for cooperative outer kinetochore assembly in budding yeast

ABSTRACT: We employed chemical crosslinking and mass spectrometry on in vitro reconstituted outer kinetochore KMN network.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Mia Potocnjak

LAB HEAD: Franz Herzog

PROVIDER: PXD011775 | Pride | 2020-09-30

REPOSITORIES: Pride

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			Action	DRS
	161118_in_vitro.xlsx	Xlsx
	AG_Herzog_busslehner_E1609_096.raw	Raw
	AG_Herzog_busslehner_E1609_097.raw	Raw
	AG_Herzog_busslehner_E1609_100.raw	Raw
	AG_Herzog_busslehner_E1609_102.raw	Raw

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An Mtw1 C-terminal motif is crucial for cooperative outer kinetochore assembly in budding yeast

Project description:We employed chemical crosslinking and mass spectrometry on KMN complexes isolated from S. cerevisiae.

2020-09-30 | PXD011774 | Pride

CX-MS of Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Project description:Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the S. cerevisiae outer kinetochore, assembling into 3 MDa-sized microtubule-embracing rings, but how ring assembly is specifically initiated at kinetochores remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi-orientation. We show that Dam1c and the general microtubule plus-end-associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c-Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c-Bim1 binding by relieving an intramolecular inhibition of the Dam1 C-terminus. In addition, Bim1 recruits Bik1/CLIP-170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end-on attachments are formed during the process of attachment error correction.

2021-06-28 | PXD026935 | Pride

AOCM-CPC - Kinetochores are macromolecular protein complexes that ensure accurate chromosome segregation by linking chromosomes to spindle microtubules and integrating safeguard mechanisms

Project description:Kinetochores are macromolecular protein complexes that ensure accurate chromosome segregation by linking chromosomes to spindle microtubules and integrating safeguard mechanisms. In yeast, the inner kinetochore, also known as Constitutive Centromere Associated Network (CCAN), is specifically established at point centromeres and has been implicated in contributing to Aurora-BIpl1 function. In an attempt to gain a more detailed picture of the budding yeast kinetochore architecture, crosslink-guided in vitro reconstitution revealed novel direct interactions of the inner kinetochore assembled on Cse4CENP-A nucleosomes. The Ame1/Okp1CENP-U/Q heterodimer selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus, providing an explanation for the essential role of the COMA complex in budding yeast. Moreover, the Sli15/Ipl1 core chromosomal passenger complex was found to directly interact with COMA in vitro, suggesting a hitherto unknown role of the COMA complex in establishing biorientation. In line with this finding, in vivo artificial tethering of Sli15 to inner kinetochore proteins rescued synthetically lethal subunit deletion phenotypes in a Sli15 centromere targeting deficient mutant. This study reveals characteristics of the inner kinetochore architecture assembled at point centromeres and its implications on chromosomal passenger complex function.

2019-04-12 | PXD011235 | Pride

The COMA complex plays a role in positioning Ipl1 activity proximal to Cse4 nucleosomes

2019-04-12 | PXD011236 | Pride

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A.

Project description:Kinetochores are megadalton-sized protein complexes that mediate chromosome-microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1(CENP-U) and Okp1(CENP-Q) form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1-Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.

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Pcs1 and Mde4 are inner centromere proteins required to prevent merotelic kinetochore orientation

Project description:Chromosome segregation depends on proper attachment of sister kinetochores to microtubules. Merotelic kinetochore orientation is an error which occurs when a single kinetochore is attached to microtubules emanating form opposite poles. Mechanisms preventing or correcting the merotelic attachment must operate to avoid chromosome missegregation. Pcs1 has been implicated in preventing merotelic attachment in mitosis and meiosis II. We describe here the identification of Mde4 protein which forms a complex with the Pcs1. Both Mde4 and Pcs1 localize to the central core of the centromere. Similarly to the pcs1 mutant, in the absence of mde4 lagging chromosomes are frequently observed during mitosis and meiosis II . We provide the first evidence that the lagging chromosomes in pcs1 and mde4 mutants are due to merotelic kinetochore orientation. Keywords: ChIP-chip analysis ChIP-chip analysis: In all cases, hybridization data for ChIP fraction was compared with that of SUP (supernatant) fraction. Pombe whole chromosome array was used.

2010-06-10 | E-GEOD-5257 | biostudies-arrayexpress

Genome-wide maps of heterochromatin state (H3K9me2) in kinetochore and other centromere related mutants.

Project description:We report the high-throughput profiling of histone modification (H3K9me2) in fission yeast Schizosaccharomyces pombe. By obtaining 1-10 ng immunoprecipitated DNA, we generated genome-wide H3K9me2 maps of fission yeast mutants with either deletions of non-essential kinetochore genes or conditional inactivation (temperature sensitive, ts) mutations in essential ones. We find that the impairment of the kinetochore componnets cause various levels (from no to prominent) of heterochroamatin spreading into centromeric core regions. Hence, we conclude that the integrity of the inner kinetochore is required to maintain normal centromeric chromatin organization as well as distinct centromere identity.

2019-09-28 | GSE117948 | GEO

Spo11-oligo mapping in S. cerevisiae Ctf19/CCAN kinetochore sub-complex mutant mcm21

Project description:During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. Two samples total: two biological replicate Spo11-oligo maps of S. cerevisiae SK1 mcm21 null mutant

2015-12-15 | E-GEOD-72683 | biostudies-arrayexpress

Cross-linking mass spectrometry analyses of three different kinetochore protein complexes (KMN, NDC80C, MIS12C) using an MS-cleavable cross-linker, BuUrBu (DSBU)

Project description:In order to develop a simplified cross-linking mass spectrometry protocol, we applied one-step size-exclusion chromatography (SEC) for peptide purification after proteolysis with Lys-C and trypsin. Three benchmark protein complexes (KMN, NDC80C, MIS12C) from human kinetochore were cross-linked with an MS-cleavable cross-linker (BuUrBu). After proteolysis, digested peptides were purified with SEC omitting the step of C18 column to avoid loss of cross-linked peptides. Our protocol showed improvement of existing protocols, and we could successfully identify over 700 cross-links per experiment of each of the three protein complexes. We also observed that about half of the non-redundant cross-links were not lysine-lysine cross-links but cross-links between lysine and side chains with alcohols (serine, threonine, tyrosine).

2019-04-01 | PXD010070 | Pride

Pcs1 and Mde4 are inner centromere proteins required to prevent merotelic kinetochore orientation

2007-06-18 | GSE5257 | GEO