Formation of nucleophosmin/B23 oligomers requires both the amino-and the carboxyl-terminal domains of the protein

Authors


Correspondence to P. K. Chan, Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA

Abstract

Nucleophosmin/B23 is a nucleolar phosphoprotein which forms oligomers. To determine the domain essential for oligomer formation, various deletion and point mutation clones of nucleophosmin/B23 were constructed. Nucleophosmin/B23 and the mutant proteins were produced by (a) coupled in vitro transcription and translation and (b) expression in Escherichia coli with T7 RNA polymerase expression vector (pET-8c). Nucleophosmin/B23 synthesized in vitro has the same peptide map as that synthesized in HeLa cells. Similarly, it formed oligomers which could be detected in SDS/PAGE and were cross-linked with nitrogen mustard in vivo. Substitution of Met5, Met7, and Met9 with Leu or deletion of five amino acids at the C-terminus abolished the oligomerization. Deletion of portions of amino acids in the middle of the molecule (amino acid residues 83–152, 117–186 and 185–240) had little effect on the oligomerization. Co-expression of the N- and C-terminal mutant clones in vitro did not produce oligomers. These results indicate that intra-molecular interactions with both the N- and C-terminal domains are essential for oligomer formation.

Abbreviation
PCR

polymerase chain reaction

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