Cell Biochemistry and Biophysics

, Volume 44, Issue 3, pp 522–529 | Cite as

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
Original Article
  • 78 Downloads

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.

Index Entries

PCI domain eIF3 INT6 limited proteolysis circular dichroism 

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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Marcelo J. Murai
    • 1
  • Flávia R. G. Carneiro
    • 1
  • Fabio C. Gozzo
    • 1
  • Daniela F. Ierardi
    • 1
  • Thelma A. Pertinhez
    • 1
  • Nilson I. T. Zanchin
    • 1
  1. 1.Center for Structural Molecular BiologyBrazilian Synchrotron Light Laboratory-LNLSCampinas-SPBrazil

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