Whole-proteome genetic analysis of dependencies in assembly of a vertebrate kinetochore

• Published in: The Journal of cell biology Vol. 211; no. 6; pp. 1141 - 1156
• Main Authors: Samejima, Itaru, Spanos, Christos, Alves, Flavia de Lima, Hori, Tetsuya, Perpelescu, Marinela, Zou, Juan, Rappsilber, Juri, Fukagawa, Tatsuo, Earnshaw, William C
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 21-12-2015; The Rockefeller University Press
• Subjects: Animals; Cell Line, Tumor; Cellular biology; Chickens; Chromosomes; Chromosomes - genetics; Genetics; Kinetochores - chemistry; Kinetochores - metabolism; Mass Spectrometry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Proteins; Proteome - genetics; Proteome - metabolism; Proteomics; Vertebrates
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.201508072

Kinetochores orchestrate mitotic chromosome segregation. Here, we use quantitative mass spectrometry of mitotic chromosomes isolated from a comprehensive set of chicken DT40 mutants to examine the dependencies of 93 confirmed and putative kinetochore proteins for stable association with chromosomes. Clustering and network analysis reveal both known and unexpected aspects of coordinated behavior for members of kinetochore protein complexes. Surprisingly, CENP-T depends on CENP-N for chromosome localization. The Ndc80 complex exhibits robust correlations with all other complexes in a "core" kinetochore network. Ndc80 associated with CENP-T interacts with a cohort of Rod, zw10, and zwilch (RZZ)-interacting proteins that includes Spindly, Mad1, and CENP-E. This complex may coordinate microtubule binding with checkpoint signaling. Ndc80 associated with CENP-C forms the KMN (Knl1, Mis12, Ndc80) network and may be the microtubule-binding "workhorse" of the kinetochore. Our data also suggest that CENP-O and CENP-R may regulate the size of the inner kinetochore without influencing the assembly of the outer kinetochore.