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CENP-C is a blueprint for constitutive centromere-associated network assembly within human kinetochores.

Klare K, Weir JR, Basilico F, Zimniak T, Massimiliano L, Ludwigs N, Herzog F, Musacchio A - J. Cell Biol. (2015)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.

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Characterization of the CENP-C–CENP-HIKM interaction. (A) GST pull-down assay of GST–CENP-C2–545 with CENP-M or CENP-I57–281. CENP-C does not bind to CENP-M or CENP-I57–281 in this assay. (B) GST pull-down assay of GST–CENP-C2–545 with CENP-HK. (C) GST pull-down assay of GST–CENP-C189–400 with CENP-HK. In B and C, CENP-C2–545 and CENP-C189–400, respectively, bind to CENP-HK. (D) Summary of cross-links within the CENP-C2–545–CENP-HK complex. Intermolecular cross-links are shown in blue. Intramolecular cross-links are not shown but are listed in Table S1. CENP-HK binds to CENP-C within its PEST-rich domain. (E) Within the boxed regions are two conserved hydrophobic motifs in CENP-C189–400. In the 3A mutant, L265, F266A, and L267A were mutated to alanine. The 4A mutant combines 3A with W317A. Two Lys residues found to cross-link with CENP-HK are shown with blue circles. Budding yeast Mif2 is shown for comparison. Curiously, the regions conserved in Mif2 seem to be inverted. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, X. laevis; Sc, S. cerevisiae. Red and pink boxes indicate positions in the CENP-C sequence that are either fully conserved (per residue class, e.g., hydrophobic and aromatic) or conserved in at least four in five sequences, respectively. (F) GST–CENP-C189–290-3A was unable to bind HIKM in a GST pull-down assay, in contrast to wild-type CENP-C189–290. (G) GST–CENP-C290–400-W317A was unable to bind HIKM in a GST pull-down assay, in contrast to wild-type GST–CENP-C290–400. (H) GST pull-down assay of GST–CENP-C2–545 shows binding to HIKM, whereas GST–CENP-C2–545-4A is effectively impaired in HIKM binding. White lines indicate that intervening lanes have been spliced out. MWM, molecular weight marker.
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fig2: Characterization of the CENP-C–CENP-HIKM interaction. (A) GST pull-down assay of GST–CENP-C2–545 with CENP-M or CENP-I57–281. CENP-C does not bind to CENP-M or CENP-I57–281 in this assay. (B) GST pull-down assay of GST–CENP-C2–545 with CENP-HK. (C) GST pull-down assay of GST–CENP-C189–400 with CENP-HK. In B and C, CENP-C2–545 and CENP-C189–400, respectively, bind to CENP-HK. (D) Summary of cross-links within the CENP-C2–545–CENP-HK complex. Intermolecular cross-links are shown in blue. Intramolecular cross-links are not shown but are listed in Table S1. CENP-HK binds to CENP-C within its PEST-rich domain. (E) Within the boxed regions are two conserved hydrophobic motifs in CENP-C189–400. In the 3A mutant, L265, F266A, and L267A were mutated to alanine. The 4A mutant combines 3A with W317A. Two Lys residues found to cross-link with CENP-HK are shown with blue circles. Budding yeast Mif2 is shown for comparison. Curiously, the regions conserved in Mif2 seem to be inverted. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, X. laevis; Sc, S. cerevisiae. Red and pink boxes indicate positions in the CENP-C sequence that are either fully conserved (per residue class, e.g., hydrophobic and aromatic) or conserved in at least four in five sequences, respectively. (F) GST–CENP-C189–290-3A was unable to bind HIKM in a GST pull-down assay, in contrast to wild-type CENP-C189–290. (G) GST–CENP-C290–400-W317A was unable to bind HIKM in a GST pull-down assay, in contrast to wild-type GST–CENP-C290–400. (H) GST pull-down assay of GST–CENP-C2–545 shows binding to HIKM, whereas GST–CENP-C2–545-4A is effectively impaired in HIKM binding. White lines indicate that intervening lanes have been spliced out. MWM, molecular weight marker.

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.


CENP-C is a blueprint for constitutive centromere-associated network assembly within human kinetochores.

Klare K, Weir JR, Basilico F, Zimniak T, Massimiliano L, Ludwigs N, Herzog F, Musacchio A - J. Cell Biol. (2015)

Characterization of the CENP-C–CENP-HIKM interaction. (A) GST pull-down assay of GST–CENP-C2–545 with CENP-M or CENP-I57–281. CENP-C does not bind to CENP-M or CENP-I57–281 in this assay. (B) GST pull-down assay of GST–CENP-C2–545 with CENP-HK. (C) GST pull-down assay of GST–CENP-C189–400 with CENP-HK. In B and C, CENP-C2–545 and CENP-C189–400, respectively, bind to CENP-HK. (D) Summary of cross-links within the CENP-C2–545–CENP-HK complex. Intermolecular cross-links are shown in blue. Intramolecular cross-links are not shown but are listed in Table S1. CENP-HK binds to CENP-C within its PEST-rich domain. (E) Within the boxed regions are two conserved hydrophobic motifs in CENP-C189–400. In the 3A mutant, L265, F266A, and L267A were mutated to alanine. The 4A mutant combines 3A with W317A. Two Lys residues found to cross-link with CENP-HK are shown with blue circles. Budding yeast Mif2 is shown for comparison. Curiously, the regions conserved in Mif2 seem to be inverted. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, X. laevis; Sc, S. cerevisiae. Red and pink boxes indicate positions in the CENP-C sequence that are either fully conserved (per residue class, e.g., hydrophobic and aromatic) or conserved in at least four in five sequences, respectively. (F) GST–CENP-C189–290-3A was unable to bind HIKM in a GST pull-down assay, in contrast to wild-type CENP-C189–290. (G) GST–CENP-C290–400-W317A was unable to bind HIKM in a GST pull-down assay, in contrast to wild-type GST–CENP-C290–400. (H) GST pull-down assay of GST–CENP-C2–545 shows binding to HIKM, whereas GST–CENP-C2–545-4A is effectively impaired in HIKM binding. White lines indicate that intervening lanes have been spliced out. MWM, molecular weight marker.
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fig2: Characterization of the CENP-C–CENP-HIKM interaction. (A) GST pull-down assay of GST–CENP-C2–545 with CENP-M or CENP-I57–281. CENP-C does not bind to CENP-M or CENP-I57–281 in this assay. (B) GST pull-down assay of GST–CENP-C2–545 with CENP-HK. (C) GST pull-down assay of GST–CENP-C189–400 with CENP-HK. In B and C, CENP-C2–545 and CENP-C189–400, respectively, bind to CENP-HK. (D) Summary of cross-links within the CENP-C2–545–CENP-HK complex. Intermolecular cross-links are shown in blue. Intramolecular cross-links are not shown but are listed in Table S1. CENP-HK binds to CENP-C within its PEST-rich domain. (E) Within the boxed regions are two conserved hydrophobic motifs in CENP-C189–400. In the 3A mutant, L265, F266A, and L267A were mutated to alanine. The 4A mutant combines 3A with W317A. Two Lys residues found to cross-link with CENP-HK are shown with blue circles. Budding yeast Mif2 is shown for comparison. Curiously, the regions conserved in Mif2 seem to be inverted. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, X. laevis; Sc, S. cerevisiae. Red and pink boxes indicate positions in the CENP-C sequence that are either fully conserved (per residue class, e.g., hydrophobic and aromatic) or conserved in at least four in five sequences, respectively. (F) GST–CENP-C189–290-3A was unable to bind HIKM in a GST pull-down assay, in contrast to wild-type CENP-C189–290. (G) GST–CENP-C290–400-W317A was unable to bind HIKM in a GST pull-down assay, in contrast to wild-type GST–CENP-C290–400. (H) GST pull-down assay of GST–CENP-C2–545 shows binding to HIKM, whereas GST–CENP-C2–545-4A is effectively impaired in HIKM binding. White lines indicate that intervening lanes have been spliced out. MWM, molecular weight marker.
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.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany.

Show MeSH
Related in: MedlinePlus