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 approximately 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.
Highlights
The genetic material of eukaryotes is packaged into chromosomes through its interactions with both histones and nonhistone chromosomal proteins
The identification of the protein components of the human centromere initially seemed a daunting task until it was discovered that patients with scleroderma spectrum disease produce circulating autoantibodies that recognize several centromeric polypeptides [1, 2]. cDNAs encoding three centromere proteins, CENP-A, -B, and -C, have been cloned, and the proteins have been studied in detail
The yeast interaction trap4 and affinity chromatography, which forms the subject of this report, are left as the two viable methods to search for proteins that interact with CENP-C
Summary
The genetic material of eukaryotes is packaged into chromosomes through its interactions with both histones and nonhistone chromosomal proteins. Individual non-histone chromosomal proteins are considerably less abundant and have more specialized roles, including the structural and functional organization of specific chromosomal elements. One such element is the centromere, the chromosomal region responsible for the precise and accurate segregation of the genetic material during mitosis and meiosis. CENP-C is a DNA-binding protein and is located at the interface between the centromeric heterochromatin and the innermost region of the kinetochore [7]. Despite these advances, several obstacles have hindered the identification of other proteins that compose the human centromere and kinetochore. There are no good functional assays available to screen for mammalian centromere proteins, nor are there any widely applicable methods for directly purifying these chromosomal structures
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