Specific interaction between human kinetochore protein CENP-C and a nucleolar transcriptional regulator.
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.
Pluta
A F
AF
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Earnshaw
W C
WC
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000378:metabolism; D001483:Base Sequence; D002466:Cell Nucleolus / Q000378:metabolism*; D002846:Chromatography, Affinity; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D018386:Kinetochores / Q000378:metabolism*; D008969:Molecular Sequence Data; D035463:Pol1 Transcription Initiation Complex Proteins; D011485:Protein Binding; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001324:Autoantigens; D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D004268:DNA-Binding Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; D014157:Transcription Factors; C060905:centromere protein C; C063844:transcription factor UBF