Functional complementation of human centromere protein A (CENP-A) by Cse4p from Saccharomyces cerevisiae.
We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference (RNAi) was used to block the synthesis of centromere protein A (CENP-A) and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for the ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically incorporated into kinetochore nucleosomes and was able to complement RNAi-induced cell cycle arrest in CENP-A-depleted human cells. Thus, Cse4p can structurally and functionally substitute for CENP-A, strongly suggesting that the basic features of centromeric chromatin are conserved between yeast and mammals. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized to kinetochores during mitosis, but Bik1p did not rescue CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool for studying the function and evolutionary conservation of multiprotein complexes from yeast to humans.
Wieland
Gerhard
G
Department for Molecular Biology, Institute of Molecular Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Germany.
Orthaus
Sandra
S
Ohndorf
Sabine
S
Diekmann
Stephan
S
Hemmerich
Peter
P
D001324:Autoantigens / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D002455:Cell Division; D002503:Centromere / Q000378:metabolism; D000076247:Centromere Protein A; D002843:Chromatin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D005816:Genetic Complementation Test; D005821:Genetic Techniques; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008938:Mitosis; D008954:Models, Biological; D009363:Neoplasm Proteins; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors; D014162:Transfection
D001324:Autoantigens; C488173:Bik1 protein, S cerevisiae; C000619700:CENPA protein, human; C092746:CSE4 protein, S cerevisiae; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D029701:Saccharomyces cerevisiae Proteins; C076744:cytoplasmic linker protein 170