Dominant-negative VDAC1 mutants reveal oligomeric VDAC1 to be the active unit in mitochondria-mediated apoptosis

Asaf Mader, Salah Abu-Hamad, Nir Arbel, Manuel Gutierr-Suilar, Varda Shoshan-Barmatz

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Mitochondria play a central role in the intrinsic pathway of apoptosis. Oligomerization of the mitochondrial protein VDAC1 (voltage-dependent anion channel 1) has been proposed to play a role in apoptosis in various studies. In the present study, we have generated dimeric fusion proteins consisting of tandem-linked wild-type and RuR (Ruthenium Red)-insensitive mutant VDAC1 monomers and studied the capacity of RuR to protect against apoptosis, as induced by various means. Fusion proteins composed of wild-type and/or E72Q-VDAC1 were successfully expressed in T-REx-293 cells. Bilayer-reconstituted dimeric rVDAC1 (rat VDAC1) functions as a channel-forming protein, showing typical voltage-dependence conductance, but with a unitary conductance higher than that of monomeric VDAC. As with wild-type VDAC1, overexpression of either the wild-type or mutated VDAC1 dimeric fusion protein induced apoptotic cell death. In addition, as shown previously, the anti-apoptotic effect of RuR was not observed in cells expressing E72Q-VDAC1, despite endogenous VDAC1 being present in these cells. Similar RuR insensitivity governed the VDAC1 fusion proteins comprising the E72Q mutation in either the first, second or both VDAC1 monomers of the same dimer. RuR-mediated protection against apoptosis in T-REx-293 cells, as induced by staurosporine, was observed in cells expressing VDAC1 or dimeric wild-type VDAC1. However, RuR offered no protection against staurosporine-induced apoptosis in cells expressing E72Q-VDAC1 or E72Q-containing dimeric VDAC1. These results suggest that E72Q-VDAC1 has a dominant-negative effect and implies that VDAC1 homo-oligomerization, involving intermolecular interactions, might be involved in the apoptotic process.

Original languageEnglish
Pages (from-to)147-155
Number of pages9
JournalBiochemical Journal
Volume429
Issue number1
DOIs
StatePublished - 1 Jul 2010

Keywords

  • Apoptosis
  • Dominant-negative effect
  • Mitochondrion
  • Ruthenium Red (RUR)
  • Voltage-dependent anion channel 1 (VDAC1)
  • Voltage-dependent anion channel 1 (VDAC1) oligomerization

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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