Spindle checkpoint maintenance requires Ame1 and Okp1.
Kinetochore proteins are required for high fidelity chromosome segregation and as a platform for checkpoint signaling. Ame1 is an essential component of the COMA (Ctf19, Okp1, Mcm21, Ame1) sub-complex of the central kinetochore of budding yeast. In this study, we describe the isolation and characterization of an Ame1 conditional mutant, ame1-4. ame1-4 cells exhibit chromosome segregation defects and Mad2-dependent cell cycle delay similar to okp1-5 cells. However, the viability of ame1-4 cells is markedly reduced relative to wild type and okp1-5 cells after three hours at restrictive temperature. To determine if ame1-4 cells enter anaphase with mis-segregated chromosomes, we monitored the localization of Bub3:VFP as a marker for anaphase onset. ame1-4 cells containing mis-segregated sister chromatids initially accumulate Bub3:VFP at kinetochores, indicating checkpoint activation and a metaphase arrest. Subsequently, Bub3:VFP de-localizes and cells reinitiate DNA duplication and budding without cytokinesis in the presence of un-segregated chromosomes. Overexpression of OKP1 in ame1-4 cells restores ame1-4 protein localization and a stable arrest. Based on our results, we propose that Ame1 and Okp1 are required for a sustained checkpoint arrest in the presence of mis-segregated chromosomes. Our results suggest that checkpoint response might be controlled not only at the level of activation but also via signals that ensure maintenance of the response.
Pot
Isabelle
I
Department of Biochemistry & Molecular Biology and Centre for Molecular Medicine and Therapeutics, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.
Knockleby
James
J
Aneliunas
Victoria
V
Nguyen
Thao
T
Ah-Kye
Sonia
S
Liszt
Gregory
G
Snyder
Michael
M
Hieter
Philip
P
Vogel
Jackie
J
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D015825:Chromosomes, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism*
C509718:Ame1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; C509719:Okp1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins