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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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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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