The dimerization domain of Pf CENP-C is required for its functions as a centromere protein in human malaria parasite Plasmodium falciparum

Malaria Journal

Research

Namita Surolia¹  Garima Verma¹ 

[1] Molecular Parasitology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre For Advanced Scientific Research, 560064, Jakkur, Bangalore, India;

关键词: Plasmodium falciparum;    Pf;    Functional complementation;    Pf;    Pf;   

DOI  :  10.1186/1475-2875-13-475

 received in 2014-05-22, accepted in 2014-11-30,  发布年份 2014

来源: Springer

【 摘 要 】

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 Pf CENP-C, an inner kinetochore protein (the homologue of human CENP-C) and its co-localization with Pf CENH3 was recently reported. This study aims to decipher the functions of inner kinetochore protein, Pf CENP-C as a centromere protein in P. falciparum.MethodsBio-informatic tools were employed to demarcate the two conserved domains of Pf CENP-C, and the functions of Pf CENP-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 Pf CENP-C functions were determined. The chromatin immunoprecipitation was carried out to determine the in vivo binding of Pf CENP-C to the Plasmodium centromeres and the in vivo interactions of Pf CENP-C with Pf CENH3, and mitotic spindles were shown by co-immunopreciptation experiments.ResultsThe studies demonstrate that the motif and the dimerization domain of Pf CENP-C is able to functionally complement MIF2p functions. The essential role of some of the key residues: F1993, F1996 and Y2069 within the Pf CENP-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 Pf CENP-C with Pf CENH3 and mitotic spindles. The ChIP-PCR experiments confirm Pf CENP-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.ConclusionsPf CENP-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.

【 授权许可】

CC BY   
© Verma and Surolia; licensee BioMed Central. 2014