Identification of novel Drosophila centromere-associated proteins.
Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins such as the centromere-specific histone H3-variant centromere protein A (CENP-A). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere-associated network (CCAN). In contrast, only one additional constitutive centromeric protein is known in Drosophila melanogaster (D.mel), the conserved CCAN member CENP-C. To gain further insights into D.mel centromere composition and biology, we analyzed affinity-purified chromatin prepared from D.mel cell lines expressing green fluorescent protein tagged histone three variants by MS. In addition to already-known centromeric proteins, we identified novel factors that were repeatedly enriched in affinity purification-MS experiments. We analyzed the cellular localization of selected candidates by immunocytochemistry and confirmed localization to the centromere and other genomic regions for ten factors. Furthermore, RNA interference mediated depletion of CG2051, CG14480, and hyperplastic discs, three of our strongest candidates, leads to elevated mitotic defects. Knockdowns of these candidates neither impair the localization of several known kinetochore proteins nor CENP-A(CID) loading, suggesting their involvement in alternative pathways that contribute to proper centromere function. In summary, we provide a comprehensive analysis of the proteomic composition of Drosophila centromeres. All MS data have been deposited in the ProteomeXchange with identifier PXD000758 (http://proteomecentral.proteomexchange.org/dataset/PXD000758).
Munich Center of Integrated Protein Science, Adolf-Butenandt Institute, Ludwig Maximilians University of Munich, Munich, Germany.
Teresa K
TK
Barth
Georg O M
GO
Schade
Andreas
A
Schmidt
Irene
I
Vetter
Marc
M
Wirth
Patrick
P
Heun
Andreas W
AW
Thomae
Axel
A
Imhof
D000818:Animals; D002460:Cell Line; D002503:Centromere / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004331:Drosophila melanogaster; D055785:Gene Knockdown Techniques; D018386:Kinetochores / Q000378:metabolism; D008856:Microscopy, Fluorescence
C581136:CENP-C protein, Drosophila; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D029721:Drosophila Proteins
Animal proteomics; CID; Centromere; Drosophila melanogaster