SPECIFIC INTERACTION BETWEEN HUMAN KINETOCHORE PROTEIN CENP-C AND A NUCLEOLAR TRANSCRIPTIONAL REGULATOR

CENP-C is a human kinetochore protein that was originally identified a s a chromosomal autoantigen in patients with scleroderma spectrum dise ase. To bean to establish a comprehensive protein map of the human cen tromere, affinity chromatography was used to identify nuclear proteins that specifically interact with CENP-C. Whereas a number of polypepti des appeared to interact with the full-length CENP-C protein, only a p air of similarly sized proteins of similar to 100 kDa specifically int eracted with the isolated carboxyl-terminal third of the CENP-C protei n. Neither protein of the doublet bound to control affinity columns. A ffinity purification and microsequence analysis of the proteins in the doublet identified them as the two highly related nucleolar transcrip tion factors, UBF1 and UBF2 (also known as the nucleolar autoantigen N OR-90). Immunoblot analysis confirmed that both proteins also interact ed with the full-length CENP-C polypeptide with similar affinities. Do uble indirect immunofluorescence using monospecific antibodies demonst rated that a subset of CENP-C and UBF/NOR-90 is colocalized at nucleol i of interphase HeLa cells, suggesting that the in vitro interaction d etected by affinity chromatography may reflect an interaction that occ urs in vivo. We discuss the implications of these findings in terms of the properties of interphase centromeres and the role of the nucleolu s in scleroderma autoimmunity.