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Chemical cross-linking of the Spindly-RZZ complex followed by mass spectrometry

ABSTRACT: Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules during mitosis. The metazoan-specific ≈800 kilodalton ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores prior to microtubule attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. Here, we combine biochemical reconstitutions, single particle electron cryo microscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Victor Solis

LAB HEAD: Andrea Musacchio

PROVIDER: PXD005287 | Pride | 2022-02-28

REPOSITORIES: Pride

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			Action	DRS
	AG_Herzog_herzog_E1303_296.raw	Raw
	AG_Herzog_herzog_E1303_297.raw	Raw
	AG_Herzog_herzog_E1303_299.raw	Raw
	AG_Herzog_herzog_E1303_300.raw	Raw
	AG_Herzog_herzog_E1303_302.raw	Raw

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

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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