Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437.

An increasing number of experimental chemotherapeutic agents induce apoptosis by directly triggering mitochondrial membrane permeabilization (MMP). Here we examined MMP induced by lonidamine, arsenite, and the retinoid derivative CD437. Cells overexpressing the cytomegalovirus-encoded protein vMIA, a protein which interacts with the adenine nucleotide translocator, were strongly protected against the MMP-inducing and apoptogenic effects of lonidamine, arsenite, and CD437. In a cell-free system, lonidamine, arsenite, and CD437 induced the permeabilization of ANT proteoliposomes, yet had no effect on protein-free liposomes. The ANT-dependent membrane permeabilization was inhibited by the two ANT ligands ATP and ADP, as well as by recombinant Bcl-2 protein. Lonidamine, arsenite, and CD437, added to synthetic planar lipid bilayers containing ANT, elicited ANT channel activities with clearly distinct conductance levels of 20+/-7, 100+/-30, and 47+/-7 pS, respectively. Altering the ATP/ADP gradient built up on the inner mitochondrial membrane by inhibition of glycolysis and/or oxidative phosphorylation differentially modulated the cytocidal potential of lonidamine, arsenite, and CD437. Inhibition of F(0)F(1)ATPase without glycolysis inhibition sensitized to lonidamine-induced cell death. In contrast, only the combined inhibition of glycolysis plus F(0)F(1)ATPase sensitized to arsenite-induced cell death. No sensitization to cell death induction by CD437 was achieved by glucose depletion and/or oligomycin addition. These results indicate that ANT is a target of lonidamine, arsenite, and CD437 and unravel an unexpected heterogeneity in the mode of action of these three compounds.

Isolation and characterisation of the major outer membrane protein of Erwinia carotovora.

The purified major outer membrane protein (37275 Da) from the psychrotrophic phytopathogen Erwinia carotovora MFCL0 was structurally characterised by MALDI-TOF mass spectrometry, N-terminal microsequencing and DNA sequence determinations, and secondary structure prediction analyses. The deduced amino acid sequence showed 76% and 72% of similarities with the Serratia marcescens and Escherichia coli OmpA proteins respectively. Dendrogram analysis allowed to point out that E. carotovora is close to the genus Serratia. After reconstitution into planar lipid bilayers, this major protein induced ion channels with a major conductance level of 630 pS in 1 M NaCl and a weak cationic selectivity. These functional and structural features allowed to identify this major outer membrane component of E. carotovora as an OmpA-like protein, i.e., a channel-forming protein which could be involved in the infection process of this phytopathogen agent.

The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal.

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2.

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties.

The major outer-membrane protein, OprF, from the psychrotrophic bacterium Pseudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28 degrees C to 8 degrees C. The involvement of changes in tertiary or quaternary structure in this behaviour, was implied by enzymatic digestion experiments in which OprFs purified from 8 degrees C and 28 degrees C cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtained from both OprF preparations, were identified as the N-terminal half of the native protein. These 19 kDa fragments induced ion channels in planar lipid bilayers with similar conductance values of 65-75 pS in 1 M NaCl, in contrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-forming properties. LPS was not detected on the proteolytic fragments while it was found in similar amounts on the native OprFs. These results suggest the LPS/porin association occurs through the C-terminal part of the porin. Radiolabelling experiments showed different phosphorylation levels of LPS for 8 degrees C and 28 degrees C cultures. Thus, in response to growth temperature, the structural modification of the LPS could be associated to the modulation of OprF pore size.

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli.

The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight beta-strand OmpA N-terminal domain (OmpA171) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA171, provides strong evidence of the existence of an eight-stranded beta-barrel in the N-terminal domain of OprFs.

Evidence for association of lipopolysaccharide with Pseudomonas fluorescens strain MF0 porin OprF.

Lipopolysaccharide (LPS) was found to be associated with the major outer membrane protein OprF of the psychrotrophic bacterium Pseudomonas fluorescens MF0, using two OprF purification procedures. OprF, purified under mild conditions, presented two types of association with LPS: tight (tLPS) and slight (sLPS), both of type R. LPS protected OprF from heat modification and trypsin degradation and facilitated the reincorporation of purified OprF into an artificial lipid bilayer without affecting its pore-forming activity. The size of the OprF channel depended on cell growth temperature, as did the extent of LPS phosphorylation: we suggest that LPS may be involved in modifications of OprF pore formation.

Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator.

Mitochondrial membrane permeabilization can be a rate limiting step of apoptotic as well as necrotic cell death. Permeabilization of the outer mitochondrial membrane (OM) and/or inner membrane (IM) is, at least in part, mediated by the permeability transition pore complex (PTPC). The PTPC is formed in the IM/OM contact site and contains the two most abundant IM and OM proteins, adenine nucleotide translocator (ANT, in the IM) and voltage-dependent anion channel (VDAC, in the OM), the matrix protein cyclophilin D, which can interact with ANT, as well as apoptosis-regulatory proteins from the Bax/Bcl-2 family. Here we discuss that ANT has two opposite functions. On the one hand, ANT is a vital, specific antiporter which accounts for the exchange of ATP and ADP on IM. On the other hand, ANT can form a non-specific pore, as this has been shown by electrophysiological characterization of purified ANT reconstituted into synthetic lipid bilayers or by measuring the permeabilization of proteoliposomes containing ANT. Pore formation by ANT is induced by a variety of different agents (e.g. Ca(2+), atractyloside, thiol oxidation, the pro-apoptotic HIV-1 protein Vpr, etc.) and is enhanced by Bax and inhibited by Bcl-2, as well as by ADP. In isolated mitochondria, pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, and OM permeabilization, presumably due to physical rupture of OM. Although alternative mechanisms of mitochondrial membrane permeabilization may exist, ANT emerges as a major player in the regulation of cell death. Cell Death and Differentiation (2000) 7, 1146 - 1154

Bid acts on the permeability transition pore complex to induce apoptosis.

Similar to most if not all pro-apoptotic members of the Bcl-2 family, Bid (and its truncated product t-Bid) triggers cell death via mitochondrial membrane permeabilization (MMP). This effect can be monitored in intact cells, upon microinjection of recombinant Bid protein into the cytoplasm, as well as in purified mitochondria, upon addition of Bid protein. Here we show that Bid-induced MMP can be inhibited, both in cells and in the cell-free system, by three pharmacological inhibitors of the permeability transition pore complex (PTPC), namely cyclosporin A, N-methyl-4-Val-cyclosporin A, and bongkrekic acid (a ligand of the adenine nucleotide translocase, ANT, one of the PTPC components). Bid effects on synthetic membranes were studied either in proteoliposomes or in synthetic bilayers subjected to electrophysiological measurements. Full length Bid preferentially permeabilizes membranes and induces the formation of large conductance channels at neutral pH, when added to liposomes or bilayers containing both purified ANT and Bax, yet has no or little effect combined with ANT or Bax alone. t-Bid acts on membranes containing ANT alone with the same efficiency as on those containing both ANT and Bax. These results suggest that the proapoptotic effects of Bid are mediated, at least in part, by its functional interaction with ANT, one of the major components of PTPC.

Channel formation by FhaC, the outer membrane protein involved in the secretion of the Bordetella pertussis filamentous hemagglutinin.

Many virulence factors of pathogenic microorganisms are presented at the cell surface. However, protein secretion across the outer membrane of Gram-negative bacteria remains poorly understood. Here we used the extremely efficient secretion of the Bordetella pertussis filamentous hemagglutinin (FHA) to decipher this process. FHA secretion requires a single specific accessory protein, FhaC, the prototype of a family of proteins necessary for the extracellular localization of various virulence proteins in Gram-negative bacteria. We show that FhaC is heat-modifiable and localized in the outer membrane. Circular dichroism spectra indicated that FhaC is rich in beta-strands, in agreement with structural predictions for this protein. We further demonstrated that FhaC forms pores in artificial membranes, as evidenced by single-channel conductance measurements through planar lipid bilayers, as well as by liposome swelling assays and patch-clamp experiments using proteoliposomes. Single-channel conductance appeared to fluctuate very fast, suggesting that the FhaC channels frequently assume a closed conformation. We thus propose that FhaC forms a specific beta-barrel channel in the outer membrane for the outward translocation of FHA.