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

Eur J Biochem. 1991 Sep 15;200(3):715-21. doi: 10.1111/j.1432-1033.1991.tb16236.x.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cross-Linking Reagents
  • DNA-Directed RNA Polymerases
  • Escherichia coli / genetics*
  • Gene Expression
  • HeLa Cells
  • Humans
  • Macromolecular Substances
  • Mechlorethamine
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / biosynthesis*
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Peptide Mapping
  • Protein Biosynthesis
  • T-Phages / enzymology
  • Transcription, Genetic

Substances

  • Cross-Linking Reagents
  • Macromolecular Substances
  • Nuclear Proteins
  • nucleophosmin
  • Mechlorethamine
  • DNA-Directed RNA Polymerases