CENP-F is a novel microtubule-binding protein that is essential for kinetochore attachments and affects the duration of the mitotic checkpoint delay.
Centromeric protein F (CENP-F) is a 367-kDa human kinetochore protein that was identified a decade ago, but its function was only recently revealed by studies that used small interfering RNA to deplete the protein from cells. All studies showed that CENP-F is important for chromosome alignment, but these studies differed as to whether CENP-F is important to the mitotic checkpoint. We report here that CENP-F is essential for cells to sustain a prolonged mitotic delay in response to unattached kinetochores. Cells depleted of CENP-F exit mitosis in the presence of defective kinetochore attachments resulting from treatment with nocodazole, or the depletion of kinetochore proteins CENP-E and hSgo1. Kinetochores depleted of CENP-F exhibited a reduction in the amounts of the mitotic checkpoint proteins Mad1, Mad2, hBUBR1, hBUB1, and hMps1. We postulate that CENP-F is not an essential component of the mitotic checkpoint but facilitates the duration of the mitotic delay. Separately, we show that CENP-F is a novel microtubule-binding protein that possesses two microtubule-binding domains at opposite ends of the molecule. The C-terminal microtubule-binding domain was found to stimulate microtubule polymerization in vitro. These activities provide a biochemical explanation for how CENP-F contributes to kinetochore attachments in vivo.
Feng
J
J
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Huang
H
H
Yen
T J
TJ
D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D002868:Chromosomal Proteins, Non-Histone; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; C094772:centromere protein F