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In vitro phosphorylation of Dam1 complex and Bim1 by Mps1

ABSTRACT: The Dam1 complex (Dam1c) of Saccharomyces cerevisiae is a major microtubule binding component of the yeast kinetochore. Phosphorylation of various kinetochore proteins by mitotic kinases such as Mps1 are known to modulate the function of these proteins and regulate protein-protein interaction. The Dam1 subunit of Dam1c was previously demonstrated to be phosphorylate by Mps1 (residues S13, S49, S217, S218, S221, S232. In this project we mapped additional phosphor sites on components of the Dam1 complex by mass spectrometry after in vitro phosphorylation as a first step to understand the physiological function of these phosphorylation events.

INSTRUMENT(S): Orbitrap Fusion Lumos

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

SUBMITTER: Farnusch Kaschani

LAB HEAD: Farnusch Kaschani

PROVIDER: PXD047005 | Pride | 2024-05-22

REPOSITORIES: Pride

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			Action	DRS
	ACE_0655-02_AD01.raw	Raw
	ACE_0655-02_AD02.raw	Raw
	ACE_0655-02_AD03.raw	Raw
	ACE_0655-02_AD04.raw	Raw
	ACE_0655_SOI_v01.fasta	Fasta

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A common molecular mechanism underlies the role of Mps1 in chromosome biorientation and the spindle assembly checkpoint

Project description:The conserved Mps1 kinase corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical targets in this process remain elusive. Mps1 is also involved in the spindle assembly checkpoint (SAC), the surveillance mechanism halting chromosome segregation until biorientation is attained. Its role in SAC activation is antagonized by the PP1 phosphatase and involves phosphorylation of Knl1/Spc105, which recruits Bub1 to kinetochores to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single device or separable pathways. Here we characterise a novel yeast mutant, mps1-3, defective in chromosome biorientation and SAC activation. Through an unbiased screen for suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1-3 cells. Our data strongly argue that Mps1-dependent phosphorylation of the Knl1/Spc105 kinetochore scaffold is critical for Mps1 function in both chromosome biorientation and SAC activation, thus supporting the idea that a common sensory apparatus simultaneously elicits error correction and SAC signalling.

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Mps1 Phosphorylation of the Molecular Chaperone Hsp90 Regulates Mitotic Checkpoint

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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.

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