Formation of nucleophosmin/B23 oligomers requires both the amino- and the carboxyl-terminal domains of the protein.
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.
Liu
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Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Chan
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003432:Cross-Linking Reagents; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008466:Mechlorethamine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000090243:Nucleophosmin; D010449:Peptide Mapping; D014176:Protein Biosynthesis; D013604:T-Phages / Q000201:enzymology; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D008466:Mechlorethamine; D012321:DNA-Directed RNA Polymerases