CENP-C is a blueprint for constitutive centromere-associated network assembly within human kinetochores.
Bottom Line: A 16-subunit complex named the constitutive centromere-associated network (CCAN) creates the centromere-kinetochore interface.We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex.When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.
Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.Show MeSH
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Mentions: Next, we tried to identify which subunits of the CENP-HIKM complex might be involved in the interaction with CENP-C. To this end, we immobilized a GST–CENP-C2–545 fusion protein to glutathione beads and used it as bait in pull-down assays with CENP-M, the CENP-HK complex, and CENP-I57–281 (full-length CENP-I could not be produced because it is not soluble in the absence of CENP-M and CENP-HK; Basilico et al., 2014). Neither CENP-M nor CENP-I57–281 bound the GST–CENP-C2–545 bait (Fig. 2 A). In contrast, the CENP-HK complex bound the GST–CENP-C2–545 bait (Fig. 2 B) and also the GST–CENP-C189–400 bait (Fig. 2 C), indicating that the CENP-HK subcomplex is sufficient for a tight interaction with CENP-C. We conclude that the PEST-rich domain of CENP-C and the CENP-HK subunits of the CENP-HIKM complex are the main determinants of the interaction of CENP-C with the CENP-HIKM complex.
Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.