The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore

Josef Fischböck-Halwachs,Sylvia Singh,Medini Ghodgaonkar-Steger,Florian Weissmann,Stephan Woike,Götz Hagemann,Franz Herzog,Alwin Köhler,Victor Solis-Mezarino,Mia Potocnjak,Julie Rojas,Jessica Andreani,Laura D Gallego

doi:10.7554/elife.42879

Abstract

Kinetochores are macromolecular protein complexes at centromeres that ensure accurate chromosome segregation by attaching chromosomes to spindle microtubules and integrating safeguard mechanisms. The inner kinetochore is assembled on CENP-A nucleosomes and has been implicated in establishing a kinetochore-associated pool of Aurora B kinase, a chromosomal passenger complex (CPC) subunit, which is essential for chromosome biorientation. By performing crosslink-guided in vitro reconstitution of budding yeast kinetochore complexes we showed that the Ame1/Okp1CENP-U/Q heterodimer, which forms the COMA complex with Ctf19/Mcm21CENP-P/O, selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus. The Sli15/Ipl1INCENP/Aurora-B core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affected chromosome segregation fidelity in Sli15 wild-type cells. Tethering Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19 depletion in a Sli15 centromere-targeting deficient mutant. This study shows molecular characteristics of the point-centromere kinetochore architecture and suggests a role for the Ctf19 C-terminus in mediating CPC-binding and accurate chromosome segregation.

Highlights

The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore Josef Fischbock-Halwachs et al
Eluted proteins were visualized by SDS-PAGE and Coomassie staining. (D) Size exclusion chromatography (SEC) analysis of Ame1/Okp1 (AO) preincubated with Cse4D34–46- or Cse4D48–61-NCPs
The Cse4 wildtype and indicated mutant proteins were ectopically expressed in a Cse4 anchor-away strain (Cse4-FRB) and cell growth was monitored by plating 1:10 serial dilutions on YPD medium at 30 ̊C in the absence or presence of 1 mg/ml rapamycin

Summary

Introduction

The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore Josef Fischbock-Halwachs et al Fischbock-Halwachs et al eLife 2019;8:e42879. The Cse4 wildtype and indicated mutant proteins were ectopically expressed in a Cse4 anchor-away strain (Cse4-FRB) and cell growth was monitored by plating 1:10 serial dilutions on YPD medium at 30 ̊C in the absence or presence of 1 mg/ml rapamycin.

Results

Conclusion