Mutational analysis of the central centromere targeting domain of human centromere protein C, (CENP-C).
Human centromere protein C (CENP-C) is an essential component of the inner kinetochore plate. A central region of CENP-C can bind DNA in vitro and is sufficient for targeting the protein to centromeres in vivo, raising the possibility that this domain mediates centromere localization via direct DNA binding. We performed a detailed molecular dissection of this domain to understand the mechanism by which CENP-C assembles at centromeres. By a combination of PCR mutagenesis and transient expression of GFP-tagged proteins in HeLa cells, we identified mutations that disrupt centromere localization of CENP-C in vivo. These cluster in a 12 amino acid region adjacent to the core domain required for in vitro DNA binding. This region is conserved between human and mouse, but is divergent or absent in invertebrate and plant CENP-C homologues. We suggest that these 12 amino acids are essential to confer specificity to DNA binding by CENP-C in vivo, or to mediate interaction with another as yet unidentified centromere component. A differential yeast two-hybrid screen failed to identify interactions specific to this sequence, but nonetheless identified 14 candidate proteins that interact with the central region of CENP-C. This collection of mutations and interacting proteins comprise a useful resource for further elucidating centromere assembly.
Song
Kang
K
Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan 48202, USA.
Gronemeyer
Bobbi
B
Lu
Wei
W
Eugster
Emily
E
Tomkiel
John E
JE
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002503:Centromere / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016384:Consensus Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity
D001324:Autoantigens; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C060905:centromere protein C