Dominant-negative VDAC1 mutants reveal oligomeric VDAC1 to be the active unit in mitochondria-mediated apoptosis.
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.
Mader
Asaf
A
Department of Life Sciences, National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Abu-Hamad
Salah
S
Arbel
Nir
N
Gutiérrez-Aguilar
Manuel
M
Gutierr-Asuilar
Manuel
M
Shoshan-Barmatz
Varda
V
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002460:Cell Line; D005799:Genes, Dominant / Q000502:physiology; D006801:Humans; D008928:Mitochondria / Q000235:genetics*; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D050995:Voltage-Dependent Anion Channel 1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; C494764:Vdac1 protein, rat; D050995:Voltage-Dependent Anion Channel 1