Specific Interaction between Human Kinetochore Protein CENP-C and a Nucleolar Transcriptional Regulator*
- From the Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
- ‡ To whom correspondence should be addressed: Dept. of Dermatology, Ross Bldg., Rm. 771, Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-550-5032; Fax: 410-955-0520; E-mail: apluta{at}welchlink.welch.jhu.edu
Abstract
CENP-C is a human kinetochore protein that was originally identified as a chromosomal autoantigen in patients with scleroderma spectrum disease. To begin to establish a comprehensive protein map of the human centromere, affinity chromatography was used to identify nuclear proteins that specifically interact with CENP-C. Whereas a number of polypeptides appeared to interact with the full-length CENP-C protein, only a pair of similarly sized proteins of ~100 kDa specifically interacted with the isolated carboxyl-terminal third of the CENP-C protein. Neither protein of the doublet bound to control affinity columns. Affinity purification and microsequence analysis of the proteins in the doublet identified them as the two highly related nucleolar transcription factors, UBF1 and UBF2 (also known as the nucleolar autoantigen NOR-90). Immunoblot analysis confirmed that both proteins also interacted with the full-length CENP-C polypeptide with similar affinities. Double indirect immunofluorescence using monospecific antibodies demonstrated that a subset of CENP-C and UBF/NOR-90 is colocalized at nucleoli of interphase HeLa cells, suggesting that the in vitro interaction detected by affinity chromatography may reflect an interaction that occurs in vivo. We discuss the implications of these findings in terms of the properties of interphase centromeres and the role of the nucleolus in scleroderma autoimmunity.
Footnotes
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↵* This work was supported by an Arthritis Investigator award (to A. F. P.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- GST
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glutathione S-transferase
- PIPES
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1,4-piperazinediethanesulfonic acid.
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↵2 I. Goldberg, unpublished data.
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↵3 A. F. Pluta, unpublished data.
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↵4 A. F. Pluta, I. Goldberg, and W. C. Earnshaw, manuscript in preparation.
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- Received November 22, 1995.
- Revision received April 23, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
