Biomedical subjects

M Thieffry

Publications and source records attributed to M Thieffry.

At least 19 recordsLinked to original sources

PubMed2001

Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria.

Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.

Fungal Proteins↗

PubMed1998

The isolated complex of the translocase of the outer membrane of mitochondria. Characterization of the cation-selective and voltage-gated preprotein-conducting pore.

The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins. We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex. The pore is cation-selective and voltage-gated. It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages. These electrical properties distinguish it from the mitochondrial voltage-dependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel. Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer. Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner. We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane.

Biological Transport↗

PubMed1998

The preprotein translocation channel of the outer membrane of mitochondria.

The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Inactivation of the peptide-sensitive channel from the yeast mitochondrial outer membrane: properties, sensitivity to trypsin and modulation by a basic peptide.

The yeast Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane closes with slow kinetics at potentials of either polarity. The properties of this inactivation closely resemble those of the Voltage-Dependent Anion Channel (VDAC) slow kinetics closures. Addition of trypsin to one compartment suppresses the inactivation observed when this compartment is made positive, but does not affect the inactivation observed at potentials of reverse polarity. Both sides of the channel are sensitive. The reduced form of the Mast Cell Degranulating peptide (rMCD) increases the rate of inactivation, but only when the polarity of the compartment to which it is added is positive. The effect is not reversed by washing the peptide out, but is suppressed by trypsin. The peptide can bind to both sides of the membrane. The effect of rMCD on PSC closely resembles that of the "modulator" on VDAC. The similarities between PSC and VDAC suggest that the former might be a cationic porin of the mitochondrial outer membrane possessing a structure closely related to that of VDAC.

Electric Conductivity↗

PubMed1997

Relationship between the peptide-sensitive channel and the mitochondrial outer membrane protein translocation machinery.

The peptide-sensitive channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by synthetic mitochondrial presequences and by nonmitochondrial basic peptides such as dynorphin B(1-13). Both types of peptides are imported into mitochondria. However, the import of dynorphin B(1-13) had to be further characterized since its properties differed from those of the general import pathway used by mitochondrial peptides. Cross-linking experiments with iodinated dynorphin B(1-13) led to the labeling of TOM 40/ISP 42, a component of the protein import machinery of the outer membrane. Accordingly, dynorphin B(1-13) could also be used as a presequence to direct the import of a cytosolic protein into the mitochondria. Pretreatment of intact mitochondria by trypsin removed components capable of discriminating between true mitochondrial presequences and other basic peptides active on the PSC. After proteolysis, both types of peptides appeared to cross the outer membrane through the same pathway. Involvement of the PSC in the translocation complex was shown by immunoprecipitation of the PSC activity by anti-ISP 42 antibodies. Taken together, the present data reinforce the hypothesis that the PSC is the pore responsible for the translocation of protein through the outer membrane.

Animals↗

PubMed1996

Characterization and function of the mitochondrial outer membrane peptide-sensitive channel.

The PSC (peptide-sensitive Channel), a cationic channel of large conductance, has been characterized in yeast and mammalian mitochondria by three different methods, "tip-dip," patch clamp of giant liposomes, and planar bilayers. The yeast and mammalian PSC share the common property to be blocked by basic peptides such as pCyt OX IV (1-12)Y which contains the first 12 residues of the presequence of cytochrome C oxidase subunit IV. The electrophysiological data are consistent with a translocation of the peptide through the pore. Analysis of the frequency of observation of the PSC in different fractions indicates that the channel is located in the outer mitochondrial membrane. Uptake measurements of iodinated peptides by intact mitochondria from a porin-less mutant show that the peptides are translocated through the outer membrane, presumably at the level of PSC. Among the peptides active on PSC, several, such as pCyt OX IV (1-22) and the reduced form of the mast cell degranulating peptide, induce an alteration of the voltage dependence or of the inactivation rate subsisting after washing and which is eliminated only by proteolysis of the interacting peptide. These irreversible effects may account for the variability of the properties of the PSC which would interact with cytosolic or intermembrane cations, peptides, or proteins, thus modulating the channel permeability. Finally, several lines of evidence suggest the participation of the PSC in protein translocation and some interaction with the general insertion pore of the outer membrane translocation machinery.

Amino Acid Sequence↗

PubMed1995

Involvement of the peptide sensitive channel in the translocation of basic peptides into mitochondria.

The Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by several highly basic peptides. Among these peptides, the most active are pCOX IV (1-12)Y, a mitochondrial addressing peptide and dynorphin B (1-13), a peptide unrelated to mitochondrial physiology. The voltage-dependent characteristics of the block duration of the PSC induced by these peptides and the fact that these peptides are imported into mitochondria in an in vitro assay suggest the involvement of the PSC in peptide translocation into mitochondria. We have analyzed the interaction of Mast Cell Degranulating peptide (MCD), a disulfide rich basic peptide, with yeast and mammalian mitochondria. Electrophysiological experiments with native and reduced forms of this peptide (nMCD and rMCD) showed an interaction of both forms with the yeast PSC. On the other hand, only rMCD blocked the electrical activity of the bovine adrenal cortex PSC. Similarly, although both forms inhibited the import of dynorphin B (1-13) into yeast mitochondria, only rMCD inhibited this import in bovine mitochondria. The correlation between electrophysiological and biochemical data strongly suggest that dynorphin B is translocated across the outer membrane at the level of the PSC.

Adrenal Cortex↗

PubMed1994

Reversible and irreversible effects of basic peptides on the mitochondrial cationic channel.

We have previously shown that a 13-residue basic peptide, derived from the presequence of a mitochondrial precursor, blocked the cationic channel of the outer mitochondrial membrane. The properties of the blockade suggested that the peptide could go through the pore in the presence of a sufficient driving force. In an attempt to evaluate more precisely the relevance of such an interpretation, we have examined the effect on the same channel of basic peptides from 16 to 34 residues, most of which are parts of or derive from mitochondrial presequences. Two peptides were found to induce a reversible voltage-dependent blockade, the properties of which were the same as those of the blockade induced by the 13-residue peptide. The others had a similar effect, but triggered in addition a modification of the voltage gating that persisted after washing the peptide out. The modification was in turn abolished by trypsin added to the side of the channel previously exposed to the peptide. The protease acted on the bound peptide and not on the channel itself. The irreversible modification of the voltage gating, the mechanism of which remains obscure, was not specific for mitochondrial-addressing sequences.

Adrenal Cortex↗

PubMed1993

Solubilization and reconstitution of the mitochondrial peptide-sensitive channel.

In addition to the voltage-dependent anion channel (VDAC), mitochondrial outer membranes contain a cationic channel of large conductance, which is blocked by a mitochondrial addressing peptide (peptide-sensitive channel, PSC). Bovine adrenal cortex mitochondria were solubilized in 1.5% octyl beta-glucoside, and membrane vesicles were reconstituted by slow dilution with a low ionic strength buffer. The reconstituted vesicles contained a functional channel possessing the electrical characteristics of the cationic channel, including its sensitivity to the mitochondrial addressing peptide. Important features of the described protocol are the nature of the detergent, its concentration, and the addition of glycerol during the whole procedure. No solubilization could be observed in the presence of cholate.

Adrenal Cortex↗

PubMed1992

Properties of the mitochondrial peptide-sensitive cationic channel studied in planar bilayers and patches of giant liposomes.

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed by the tip-dip method from proteoliposomes derived from mitochondrial membranes. It is blocked by peptide M, a 13 residue peptide having the properties of a mitochondrial signal sequence. To verify the reliability of the experimental approach, mitochondrial membranes from bovine adrenal cortex or porin-deficient mutant yeast were either fused to planar bilayers or incorporated in giant liposomes which were studied by patch clamp. Cationic channels were found with both techniques. They had the same conductance levels and voltage-dependence as those which have been described using the tip-dip method. Moreover, they were similarly blocked by peptide M. The voltage-dependence of block duration was analyzed in planar bilayer and tip-dip records. Results strengthen the idea that peptide M might cross the channel. Other mitochondrial channels were observed in planar bilayers and patch clamp of giant liposomes. Because they were never detected in tip-dip records, they are likely to be inactivated at the surface monolayer used to form the bilayer in this type of experiment.

Adrenal Cortex↗

PubMed1991

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

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.

Animals↗

PubMed1991

A 28 kDa mitochondrial protein is radiolabelled by crosslinking with a 125I-labelled presequence.

A 13-residue peptide containing the first 12 amino acids of the N-terminal part of the signal sequence of yeast cytochrome c oxidase subunit IV is shown by chemical crosslinking to interact with a mitochondrial protein. This result is obtained with mitochondria from four different origins. Submitochondrial localization experiments suggest that the 28 kDa labelled component is present on the outer face of the inner membrane. Since such addressing peptides are imported into mitochondria through the same machinery as protein precursors, the 28 kDa protein might be a component of the translocation apparatus.

Amino Acid Sequence↗

PubMed1990

Comparison of mitochondrial cationic channels in wild-type and porin-deficient mutant yeast.

Bilayers were formed at the tip of microelectrodes from a suspension of proteoliposomes derived from wild-type and porin-deficient mutant yeast mitochondria. In both preparations, identical cationic channels of large conductance were recorded. This result rules out any relationship between this channel and the outer membrane voltage-dependent anion channel, the activity of which is carried by porin. The ionic selectivity and the voltage-dependence of the yeast cationic channel suggest that it is related to that recently described in mammalian mitochondria. This hypothesis is further supported by the fact that both channels are blocked by a mitochondrial addressing peptide.

Amino Acid Sequence↗

PubMed1989

Blockade of a mitochondrial cationic channel by an addressing peptide: an electrophysiological study.

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed at the tip of microelectrodes from proteoliposomes derived from mitochondrial membranes. This channel was blocked by a 13-residue peptide with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of cytochrome c oxidase. The blockade was reversible, voltage- and dose-dependent. The peptide did not affect the activity of a Torpedo chloride channel observed under the same conditions. From experiments with phospholipid monolayers, it is unlikely that the peptide inserts into bilayers under the experimental conditions used. The blockade was observed from both sides of the membrane, being characterized by more frequent transitions to the lower conductance states, and a maximum effect was observed around 0 mV. Channels, the gating mechanism of which had been eliminated by exposure to trypsin, were also blocked by the peptide. For trypsinized channels, the duration of the closure decreased and the blockade saturated at potentials below -30 mV. These observations are consistent with a translocation of the peptide through the channel. Dynorphin B, which has the same length and charge as the peptide, had some blocking activity. Introduction of negative charges in the peptide by succinylation suppressed the activity.

Amino Acid Sequence↗

PubMed1989

Ionic mitochondrial channels: characteristics and possible role in protein translocation.

Most of the mitochondrial proteins are synthesized in the cytoplasm as precursors which are then translocated into the organelle. These precursors have a NH2-terminal extension which functions as a mitochondrial targeting signal. The import process through mitochondrial membranes is voltage-dependent; its mechanism is still unknown. Translocation has been proposed to occur through specific channels, thus, indicating the interest of the study of mitochondrial ionic channels. Two anion channels with different electrical characteristics have been described in the outer and the inner membranes. Using the technique of "Tip-Dip", we have shown the existence of a cation channel of large conductance in mitochondria. The characteristics of this channel differ from that of the other mitochondrial anion channels. A positively charged 13-residue synthetic peptide, with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of yeast cytochrome C oxidase, induces a blockade of the cationic channel. From the characteristics of the blockade, it is likely that the channel could be permeable to the peptide. The specificity of this effect suggests that this channel might be involved in protein translocation.

Amino Acid Sequence↗

PubMed1989

[Blockage of a mitochondrial cationic channel by a mitochondrial addressing peptide].

A 13-residue peptide, including the sequence of the amino terminal end of cytochrome c oxidase subunit IV precursor, blocks a cationic channel from mitochondrial membranes. The effect is reversible and voltage-dependent. The blocking properties suggest that the peptide plugs the pore. Furthermore, when the transmembrane potential favours transfer, the peptide appears to be able to cross the channel.

Amino Acid Sequence↗

PubMed1988

Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial membranes.

Membranes from subcellular fractions of adrenal medulla were incorporated in phospholipid bilayers formed at the tip of microelectrodes. Current fluctuations recorded in the presence of a transmembrane potential revealed the existence of a voltage-dependent channel of large conductance. This channel is characterized by fast kinetics and four conductance levels separated by jumps of 100, 220 and 220 pS in 150 mM NaCl. It is permeant to Na+,K+, tetraethylammonium, Cl- and acetate and has some cation selectivity. Exposure to trypsin or pronase abolished the voltage-dependence. Upon subcellular fractionation, the activity was found to be associated with mitochondria. A similar activity was observed in mitochondrial fractions from other organs. By its kinetics, its selectivity and its potential-dependence, this channel differs from the voltage-dependent anion channel of outer mitochondrial membranes.

Adrenal Medulla↗

PubMed1987

[Demonstration of a large-conduction ion channel in subcellular fractions of the adrenal medulla].

The "tip-dip" technique (formation of a lipid bilayer at the tip of a microelectrode) allows the electrophysiological study of organelle membranes. An ionic channel of large conductance has been found by this technique in membrane preparations from adrenal medulla. This channel is voltage-sensitive and it has 4 levels of conductance. Its subcellular origin is to be determined; it is borne by a structure which sediments between 1,500 and 25,000 X g.

Adrenal Medulla↗