A peptide‐sensitive channel of large conductance is localized on mitochondrial outer membrane

Abstract

Applying the technique of 'tip-dip' to mitochondria, we have shown the existence in this organelle of a cationic channel of large conductance, which is blocked by a 13-residue peptide possessing the sequence of the N-terminal extremity of the cytochrome c oxidase subunit IV precursor. To study the submitochondrial localization of the channel, the effect of trypsin on isolated channels and on entire mitochondria were compared. One side of isolated channels is sensitive to trypsin, which eliminates the voltage dependence. Channels isolated from trypsinized mitochondria were devoid of voltage dependence and were blocked by the peptide. This suggests a localization of the channel on the outer membrane. Consistent with this hypothesis, the channel was observed with the highest frequency in outer membrane fractions purified by different procedures, either from bovine adrenal cortex or from rat liver mitochondria. Such a localization is also consistent with digitonin solubilization experiments. The channel was solubilized before the inner membrane marker, cytochrome c oxidase. The orientation of the channel was inferred from its trypsin sensitivity and its potential dependence: a transmembrane potential (inside negative) will close the channel.Mitochondria are classically viewed as possessing a very permeable outer membrane, the permeability of which is attributed to a voltage-dependent anion channel (VDAC) [l], and an impermeable inner membrane equipped only with transporters. This view was based on the different functions of the two membranes and on the differences between channels and transporters, since mitochondrial ionic gradients seem to exclude the presence through the inner membrane of the large ionic flux generally associated with ionic channels. However, methodological progress has been made in the investigation of the electrical properties of organelle membranes and new mitochondrial channels have been described [2 -71. These new channels are ascribed to outer and inner membranes and also to contact sites between these membranes.We have described a cationic channel of large conductance and with rapid kinetics [2]. The most striking property of this channel is that it is blocked by a 13-residue peptide possessing the sequence of the N-terminal extremity of cytochrome c oxidase subunit IV precursor: MLSLRQSIRFFKY (peptide M) [8]. This peptide-sensitive channel (PSC) was characterized by the technique of 'tip-dip', which allows the study of membranes irrespective of their size [9 -111. However, because total mitochondria membrane fragments were used, it was necessary to determine the sub-mitochondrial origin of PSC. In the present communication, this question was investigated by comparing the distribution of the channel, as determined from the frequency of observation of the characteristic electrical activity in membrane fragments, to the distribution of various mitochondrial markers. EXPERIMENTAL PROCEDURES M a terialsTrypsin (treated with N-tosyl-L-phenylalanylchloromethane) and digitonin were purchased from...

Relationship between the Peptide-sensitive Channel and the Mitochondrial Outer Membrane Protein Translocation Machinery

“…The channel content of a given fraction was quantified by the frequency of the characteristic electrical activity (7) and defined as the total number of channels found divided by the total number of seals (i.e. seals exhibiting PSC activity plus seals with no electrical activity).…”

“…Trypsin-sensitive Components of the Outer Membrane Discriminate between Dynorphin B (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) and the Synthetic Mitochondrial Presequence pCytOX IV (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)Y-A possible explanation for the difference observed between the import of natural presequences and that of dynorphin B(1-13) is the presence of receptors on the outer membrane surface (11). These receptors would discriminate between the two types of peptides that would then be transported along the same pathway.…”

“…The solubilized fraction, which contained most of the radioactivity, was immunoprecipitated overnight at 4°C in the presence of either anti-ISP 42 antiserum or a nonimmune serum, immobilized on protein A-Sepharose 4B. The beads were washed with increasing concentrations of salt (up to 300 mM NaCl) and/or 1% Nonidet P-40 in order to prevent immu- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) and ISP 42, a component of the outer membrane import machinery, it was interesting to test the ability of dynorphin B(1-13) to act as a mitochondrial presequence. We thus constructed a chimeric protein (see Table I) containing the sequence of dynorphin B(1-13) fused to the N terminus of cytosolic mouse DHFR using a PCR-directed gene fusion technique (13).…”

“…The PSC is also blocked by basic peptides unrelated to mitochondrial physiology, such as dynorphin B (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). However, dynorphin B(1-13) failed to inhibit the import of a chimeric protein, cytochrome b 2 -dihydrofolate reductase (DHFR), into the matrix of yeast mitochondria (11).…”

“…Following this recognition step, protein translocation appears to occur through an unique pore, the general insertion pore (GIP) 1 (5). The mitochondrial outer membrane contains a voltage-dependent cationic channel of large conductance, the peptide-sensitive channel (PSC) (6,7). The PSC is blocked by small basic peptides derived from mitochondrial presequences (e.g.…”