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

TY - JOUR

T1 - A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

AU - Hornung, Peter

AU - Troc, Paulina

AU - Malvezzi, Francesca

AU - Maier, Michael

AU - Demianova, Zuzana

AU - Zimniak, Tomasz

AU - Litos, Gabriele

AU - Lampert, Fabienne

AU - Schleiffer, Alexander

AU - Brunner, Matthias

AU - Mechtler, Karl

AU - Herzog, Franz

AU - Marlovits, Thomas C

AU - Westermann, Stefan

N1 - © 2014 Hornung et al.

PY - 2014/8/18

Y1 - 2014/8/18

N2 - 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.

AB - 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.

KW - Autoantigens

KW - Cell Cycle Proteins

KW - Centromere

KW - Chromatin

KW - Chromosomal Proteins, Non-Histone

KW - DNA-Binding Proteins

KW - Kinetochores

KW - Microtubule-Associated Proteins

KW - Multiprotein Complexes

KW - Saccharomyces cerevisiae Proteins

KW - Saccharomycetales

KW - Sequence Alignment

U2 - 10.1083/jcb.201403081

DO - 10.1083/jcb.201403081

M3 - SCORING: Journal article

C2 - 25135934

VL - 206

SP - 509

EP - 524

JO - J CELL BIOL

JF - J CELL BIOL

SN - 0021-9525

IS - 4

ER -