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 journalArticle

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

Fingerprint

Proteolysis
Eukaryotic Initiation Factor-3
Arabidopsis
Proteins
Transition Temperature
Prokaryotic Initiation Factor-3
Peptide Hydrolases
Hot Temperature
Eukaryotic Initiation Factors
Amino Acids
Circular Dichroism
Solubility
Dichroism
Mass Spectrometry
Escherichia coli
Mass spectrometry
Thermodynamic stability

Keywords

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

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Biophysics
  • Biochemistry

Cite this

Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor. / Murai, Marcelo J.; Carneiro, Flávia R G; Gozzo, Fabio C.; Ierardi, Daniela F.; Pertinhez, Thelma A.; Zanchin, Nilson I T.

In: Cell Biochemistry and Biophysics, Vol. 44, No. 3, 2006, p. 522-529.

Research output: Contribution to journalArticle

Murai, Marcelo J. ; Carneiro, Flávia R G ; Gozzo, Fabio C. ; Ierardi, Daniela F. ; Pertinhez, Thelma A. ; Zanchin, Nilson I T. / Identification and characterization of a proteolysis-resistant fragment containing the PCI domain in the Arabidopsis thaliana INT6/eIF3e translation factor. In: Cell Biochemistry and Biophysics. 2006 ; Vol. 44, No. 3. pp. 522-529.
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