Journal
BIOCHEMICAL JOURNAL
Volume 427, Issue -, Pages 445-454Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20091690
Keywords
apoptosis; cross-linking; cysteine residue; mitochondrion; thiol-modifying agent; voltage-dependent anion channel (VDAC)
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Funding
- Israel Science Foundation [1013/05]
- Israel Cancer Association
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The VDAC (voltage-dependent anion channel) is proposed to control metabolic cross-talk between mitochondria and the cytosol, as well as apoptotic cell death. It has been suggested that apoptosis is modulated by the oxidation state of VDAC. Since cysteine residues are the major target for oxidation/reduction, we verified whether one or both VDAC1 cysteine residues are involved in VDAC I-mediated transport or apoptosis activities. To assess the function of VDAC I cysteine residues in channel activity and to probe cysteine topology with respect to facing the pore or the bilayer, we used thiol-modifying agents, namely membrane-permeable NEM (N-ethylmaleimide), bulky charged 5-FM (fluorescein-5-maleimide) and the cross-linking reagent BMOE [bis(maleimido)ethane]. Bilayer-reconstituted VDAC conductance was decreased by 5-FM, but not by NEM, whereas 5-FM had no effect on NEM-labelled VDAC conductance. BMOE caused the formation of dimeric VDAC1, suggesting that one of the two VDAC1 cysteine residues is exposed and available for cross-linking. The results thus suggest that one of the VDAC1 cysteine residues faces the VDAC pore, whereas the second is oriented towards the lipid bilayer. Mutated rat VDAC1 in which the two cysteine residues, Cys(127) and Cys(232), were replaced by alanine residues showed channel activity like native VDAC1 and, when expressed in cells, was localized to mitochondria. Human VDAC1-shRNA (small hairpin RNA)or -siRNA (small interfering RNA)-treated cells, expressing low levels of endogenous human VDAC I together with native or cysteine-less rat VDAC I, undergo apoptosis as induced by overexpression of these VDAC1 or upon treatment with reactive oxygen species-producing agents, H2O2, As2O3 or selenite, suggesting that the two cysteine residues are not required for apoptosis or VDAC1 oligomerization.
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