Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor

Marcelo J. Murai, Flávia R G Carneiro, Fabio C. Gozzo, Daniela F. Ierardi, Thelma A. Pertinhez, Nilson I T Zanchin

Research output: Contribution to journalArticlepeer-review

Abstract

The PCI domain comprises approx 200 amino acids and is found in subunits of the eukaryotic translation initiation factor 3 (eIF3), the 26S proteasome and the COP9/signalosome complexes. The PCI domain is involved in protein-protein interaction, and mouse INT6 truncated proteins lacking the PCI domain show cell malignant-transforming activity. In this work, the Arabidopsis thaliana INT6/eIF3e (AtINT6) protein was dissected using limited proteolysis, and a protease-resistant fragment containing the PCI domain was identified. Based on mass spectrometry analyses of the protease-resistant fragments and on secondary structure prediction, AtINT6-truncated proteins were cloned and expressed in Escherichia coli. Stability studies using thermal unfolding followed by circular dichroism revealed a midpoint transition temperature of 44°C for the full-length AtINT6 protein, whereas the truncated proteins comprising residues 125-415 (AtINT6TR2) and 172-415 (AtINT6TR3) showed transition temperatures of 49 and 58°C, respectively. AtINT6TR3 contains the PCI domain with additional amino acids at the N and C termini. It shows high solubility, and together with the high thermal stability, should facilitate further characterization of the PCI domain structure, which is important to understand its function in protein-protein interaction.

Original languageEnglish
Pages (from-to)522-529
Number of pages8
JournalCell Biochemistry and Biophysics
Volume44
Issue number3
DOIs
Publication statusPublished - 2006

Keywords

  • Circular dichroism
  • elF3
  • INT6
  • Limited proteolysis
  • PCI domain

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Biophysics
  • Biochemistry

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