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PMID: 15254229 Published · ppublish English Journal Article

Functional complementation of human centromere protein A (CENP-A) by Cse4p from Saccharomyces cerevisiae.

Molecular and cellular biology ·Vol. 24 ·No. 15 ·2004-08-00 ·Pages 6620-30

Wieland G, Orthaus S, Ohndorf S, Diekmann S, Hemmerich P

Abstract

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.

MeSH Terms
Autoantigens/genetics,physiology Blotting, Western Cell Division Centromere/metabolism Centromere Protein A Chromatin/genetics,metabolism,physiology Chromosomal Proteins, Non-Histone/genetics,physiology DNA-Binding Proteins/genetics,physiology Genetic Complementation Test Genetic Techniques Humans Kinetochores/metabolism Microscopy, Fluorescence Microtubule-Associated Proteins/metabolism Mitosis Models, Biological Neoplasm Proteins Phenotype Plasmids/metabolism RNA Interference Saccharomyces cerevisiae/metabolism Saccharomyces cerevisiae Proteins/genetics,metabolism,physiology Time Factors Transfection
Chemicals
Autoantigens Bik1 protein, S cerevisiae CENPA protein, human CSE4 protein, S cerevisiae Centromere Protein A Chromatin Chromosomal Proteins, Non-Histone DNA-Binding Proteins Microtubule-Associated Proteins Neoplasm Proteins Saccharomyces cerevisiae Proteins cytoplasmic linker protein 170
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Wieland Gerhard
Department for Molecular Biology, Institute of Molecular Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Germany.
Orthaus Sandra
Ohndorf Sabine
Diekmann Stephan
Hemmerich Peter
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.24.15.6620-6630.2004
Published
2004-08-00
Pages
6620-30
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC444843
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.24.15.6620-6630.2004 PMC Full Text View journal details
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