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Molecular and Cellular Biology, August 2004, p. 6620-6630, Vol. 24, No. 15
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.15.6620-6630.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Functional Complementation of Human Centromere Protein A (CENP-A) by Cse4p from Saccharomyces cerevisiae

Gerhard Wieland,{dagger} Sandra Orthaus, Sabine Ohndorf, Stephan Diekmann, and Peter Hemmerich*

Department for Molecular Biology, Institute of Molecular Biotechnology, Jena, Germany

Received 20 January 2004/ Returned for modification 8 March 2004/ Accepted 26 April 2004

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.

* Corresponding author. Mailing address: Department for Molecular Biology, Institute of Molecular Biotechnology, Beutenbergstr. 11, D-07745 Jena, Germany. Phone: 49 3641 656262. Fax: 49 3641 656225. E-mail: phemmer{at}imb-jena.de.

{dagger} Present address: Hans-Knöll-Institut für Naturstofforschung, D-07745 Jena, Germany.

Molecular and Cellular Biology, August 2004, p. 6620-6630, Vol. 24, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.15.6620-6630.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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