Abstract
BackgroundThe conserved centromere-associated proteins, CENH3 (or CENP-A) and CENP-C are indispensable for the functional centromere-kinetochore assembly, chromosome segregation, cell cycle progression, and viability. The presence and functions of centromere proteins in Plasmodium falciparum are not well studied. Identification of PfCENP-C, an inner kinetochore protein (the homologue of human CENP-C) and its co-localization with PfCENH3 was recently reported. This study aims to decipher the functions of inner kinetochore protein, PfCENP-C as a centromere protein in P. falciparum.MethodsBio-informatic tools were employed to demarcate the two conserved domains of PfCENP-C, and the functions of PfCENP-C domains were demonstrated by functional complementation assays in the temperature sensitive (TS) mutant strains (mif2-3 and mif2-2) of Saccharomyces cerevisiae with MIF2p (the yeast homologue of CENP-C) loss-of-function. By site-directed mutagenesis, the key residues essential for PfCENP-C functions were determined. The chromatin immunoprecipitation was carried out to determine the in vivo binding of PfCENP-C to the Plasmodium centromeres and the in vivo interactions of PfCENP-C with PfCENH3, and mitotic spindles were shown by co-immunopreciptation experiments.ResultsThe studies demonstrate that the motif and the dimerization domain of PfCENP-C is able to functionally complement MIF2p functions. The essential role of some of the key residues: F1993, F1996 and Y2069 within the PfCENP-C dimerization domain in mediating its functions and maintenance of mitotic spindle integrity is evident from this study. The pull-down assays show the association of PfCENP-C with PfCENH3 and mitotic spindles. The ChIP-PCR experiments confirm PfCENP-C-enriched Plasmodium centromeres. These studies thus provide an insight into the roles of this inner kinetochore protein and establish that the centromere proteins are evolutionary conserved in the parasite.ConclusionsPfCENP-C is a true CENP-C homologue in P. falciparum which binds to the centromeric DNA and its dimerization domain is essential for its in vivo functions as a centromere protein. The identification and functional characterization of the P. falciparum centromeric proteins will provide mechanistic insights into some of the mitotic events that occur during the chromosome segregation in human malaria parasite, P. falciparum.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-475) contains supplementary material, which is available to authorized users.
Highlights
The conserved centromere-associated proteins, CENH3 and CENP-C are indispensable for the functional centromere-kinetochore assembly, chromosome segregation, cell cycle progression, and viability
The BLASTp between PfCENP-C, MIF2p and CENP-C shows the maximum sequence homology in the conserved regions of the CENP-C motif and the C-terminal dimerization domain (Figure 1A,i) and these conserved domains were mapped in PfCENP-C (Figure 1A,ii)
The PfCENP-C dimerization domain shares 50 and 46% amino acid sequence similarities with S. cerevisiae and human dimerization domains, respectively (Figure 1C)
Summary
The conserved centromere-associated proteins, CENH3 (or CENP-A) and CENP-C are indispensable for the functional centromere-kinetochore assembly, chromosome segregation, cell cycle progression, and viability. CENP-C is an integral part of the inner kinetochore plate and serves as an interface between the centromeric chromatin and the outer kinetochore plate to which spindle microtubules attach [1,2,3,4,5]. Loss of CENP-C function results in abolishing the centromere-kinetochore assembly, chromosome mis-segregation, metaphase/anaphase block and cell death, suggesting its indispensable role in proper chromosome segregation and viability in the eukaryotes [2,4,13,18,21,22,23,24,25,26]
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