Synthetic partial extension peptides of P-450(SCC) and adrenodoxin precursors: effects on the import of mitochondrial enzyme precursors

Various portions of the extension peptides of P-450(SCC) precursor were chemically synthesized. The effects of these peptides on the import of enzyme precursors into mitochondria were examined. Peptides SEP1-15 and SEP1-20, corresponding to the amino terminal portion of the extension peptides, strongly inhibited the import of P-450(SCC) precursor into mitochondria. These peptides were effective at concentrations above 30 microM, and complete inhibition was observed at 100 microM. SEP1-11, which is shorter than SEP1-15 and SEP1-20, showed very weak inhibition. SEP25-39, which corresponds to the carboxy terminal portion of the extension peptide, did not affect the import of the precursor. The import of P-450(11 beta) and adrenodoxin precursors were also inhibited by SEP1-15. Another peptide, AEP1-14, which corresponds to the amino terminal portion of the extension peptide of adrenodoxin precursor, was also synthesized. The peptide inhibited the import of both adrenodoxin and P-450(SCC) precursors into mitochondria. The import of malate dehydrogenase was also inhibited by SEP1-15 and AEP1-14. The rate of the internalization of the precursor into mitochondria was decreased by the peptides. The amount of the precursor bound to the surface of mitochondria and the processing of adrenodoxin precursor were not affected. The respiratory activities of isolated mitochondria were not influenced by SEP1-15 even at 100 microM. We conclude that the inhibitory activities of the synthetic partial extension peptides on the import of enzyme precursors into mitochondria require the presence of about fifteen amino acid residues in the amino terminal portion of the extension peptides, and the inhibition of the import by the peptides was dependent on the blockage of the internalization of the precursors into mitochondria.     

Critical region in the extension peptide for the import of cytochrome P-450(SCC) precursor into mitochondria

In order to establish the role of the extension peptide of the precursor of P-450(SCC), a mitochondrial inner membrane protein, in the import into the organella, three deletion mutants of the precursor, in which the deletions were in the mature portion, were constructed. These mutant precursors were imported into mitochondria in vitro as efficiently as the original precursor, indicating that the extension peptide contains sufficient information for the import of the precursor into mitochondria. To investigate which portion of the extension peptide contains the mitochondrial targeting signal, various lengths of the amino-terminal portion of the extension peptide of P-450(SCC) precursor were fused to the mature portion of adrenodoxin. The fusion proteins consisting of 44 and 19 amino-terminal amino acids and mature adrenodoxin were imported into mitochondria, whereas those containing 14, 7, and 2 amino-terminal amino acid residues were not. The importance of the amino-terminal portion of the extension peptide was confirmed by the deletion from the amino-terminal end of a fusion protein consisting of the amino-terminal 44 amino acid residues of P-450(SCC) precursor and mature adrenodoxin, SCC44RAd. The amino-terminal deletions abolished the import of the fusion proteins into mitochondria. Substitution of all of the three basic amino acids, Arg(4), Arg(9), and Lys(14) in the extension peptide of SCC44RAd to Ser or Thr inhibited the binding of the fusion protein to mitochondria as well as its import.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of basic amino acid residues in the extension peptide of P-450(SCC) precursor: effects on the import of the precursor into mitochondria

The precursor of cytochrome P-450(SCC) (preP-450(SCC], an inner membrane protein of adrenal cortex mitochondria, has an extension peptide consisting of 39 amino acids which is thought to play an essential role in the import of the precursor into mitochondria. The amino terminal portion of the extension peptide contains three positively charged amino acid residues, Arg(4), Arg(9), and Lys(14). To investigate their role in the import of preP-450(SCC) into mitochondria, they were replaced by other amino acids, Ser or Thr, by site-directed mutagenesis. The import of mutated preP-450(SCC)s with single amino acid substitution was much less efficient than with the original precursor. The mutated preP-450(SCC)s with two or three substitutions were not imported. These results suggest that the positively charged amino acid residues in the amino terminal portion of the extension peptide are essential for the import of preP-450(SCC) into mitochondria.     

Specific binding of mitochondrial protein precursors to liposomes containing cardiolipin

In vitro synthesized precursors of several mitochondrial proteins, including P-450(SCC), adrenodoxin, and malate dehydrogenase, bound to liposomes prepared from mitochondrial phospholipids, but not to those from microsomal phospholipids. When liposomes were prepared from various pure phospholipids, adrenodoxin precursor was bound only to the liposomes that contained cardiolipin. The liposomes containing other phospholipids did not show the binding affinity for the precursor. The binding was observed only with the precursor peptides of adrenodoxin and malate dehydrogenase, and their mature forms were not bound to the liposomes. The binding of the precursors was dependent on the concentration of cardiolipin in the liposomes. Liposomes containing various cardiolipin derivatives with modified polar head groups showed very different binding affinity for adrenodoxin precursor, suggesting the importance of the structure of the polar head of the cardiolipin molecule. Two or three positively charged amino acid residues in the extension peptide of P-450(SCC) precursor were replaced by neutral amino acid residues by site-directed mutagenesis. The mutated P-450(SCC) precursors did not bind to the liposomes containing cardiolipin. The results indicated that mitochondrial protein precursors have specific affinity for cardiolipin, and the affinity was due to the interaction between the extension peptides of the precursors and the polar head of the cardiolipin molecule.     

The role of arginine residues in the rat mitochondrial malate dehydrogenase transit peptide

Arginine residues in the transit peptides of mitochondrial precursors are proposed to be important for uptake into mitochondria. To study this further, we have used cassette mutagenesis to create site-specific amino acid replacements within the transit peptide of rat mitochondrial malate dehydrogenase. Plasmids containing mutant sequences were expressed in vitro and tested in a mitochondrial uptake system utilizing isolated rat liver mitochondria. Substitution for arginine at position 14 with asparagine, glutamine, or alanine decreased the relative import level by 20-30% compared to the wild-type sequence when assayed in 1-h uptake experiments. Although lysine substitution did not alter import, substitution with glutamic acid decreased import by 40%. Alanine substitution for arginines at both positions 14 and 15 also dramatically decreased import. Uptake was partially restored in this mutant when positive charge was inserted at a new location within the transit peptide. Time course experiments showed that the initial rates of import were decreased in these mutants, as were the relative amounts of incorporated protein. These results were best explained by the loss of positive charge following amino acid substitutions for the arginine residues and suggest that the role of the charge is to enhance the efficiency of membrane translocation.     

Synthetic transit peptides inhibit import and processing of mitochondrial precursor proteins

We have demonstrated that a synthetic peptide corresponding to the rat mitochondrial malate dehydrogenase (mMDH) transit peptide (TP-28) inhibits the binding of pre-mMDH to isolated mitochondria. Synthetic peptides derived from chloroplast transit peptide sequences, which have a similar net charge, did not inhibit import. In addition, this peptide (TP-28) inhibits import of ornithine transcarbamylase, another mitochondrial matrix protein, thus suggesting that common import pathways exist for both mMDH and ornithine transcarbamylase. A smaller synthetic peptide corresponding to residues 1-20 of the mMDH transit peptide (TP-20) also inhibits binding. However, several substitutions for leucine-13 in the smaller peptide relieve import inhibition, thus providing evidence that this neutral residue plays a crucial role in transit peptide binding to the mitochondrial surface. Proteolytic processing of pre-mMDH by a mitochondrial matrix fraction to both the mature and intermediate forms of mMDH was also inhibited by TP-28. The ability of synthetic peptides to inhibit distinct steps in the import of mitochondrial precursor proteins corresponds precisely to their ability to interact with the same components used by transit peptides on intact precursors. Furthermore, inhibition at multiple points along the import pathway reflects the functions of several independent structures contained within transit peptides.     

Characterization of a mitochondrial matrix protease catalyzing the processing of adrenodoxin precursor

Adrenodoxin (Ad) is synthesized as a larger precursor (preAd) by cytoplasmic polysomes and then transported into mitochondria concomitant with its proteolytic processing to the mature form. The protease in bovine adrenal cortex mitochondria, which converts preAd to the mature form, is a metalloprotease in the matrix (Sagara, Y., Ito, A. & Omura, T. (1984) J. Biochem. 96, 1743-1752). In this study, the protease was purified about 100-fold from the matrix fraction of bovine adrenal cortex mitochondria. The partially purified protease converted not only preAd, but also the precursors of malate dehydrogenase (MDH) and 27 kDa protein (P-27) to the corresponding mature forms. However, it was inactive toward the precursors of P-450(SCC) and of P-450(11 beta). Since isolated rat liver mitochondria can import and process preAd as efficiently as bovine adrenal cortex mitochondria, we partially purified a preAd-processing protease from rat liver mitochondria and compared its properties with those of the bovine adrenal cortex enzyme. The properties of the rat liver protease were indistinguishable from those of the bovine adrenal cortex enzyme in molecular weight determined from Sephadex G-150 gel filtration, metal requirement and ability to process preMDH and preP-27. The rat liver enzyme was also inactive toward the precursors of P-450(SCC) and P-450(11 beta). These results indicate the presence in both adrenal cortex and liver mitochondria of the same type of processing protease, which processes preAd and also the precursors of some other mitochondrial proteins.     

Signal peptide analogs derived from two chloroplast precursors interact with the signal recognition system of the chloroplast envelope

We have used synthetic peptides representing segments of the signal sequences of preferredoxin (pFd) and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pS) to study interactions with the signal sequence recognition system at the chloroplast surface. Peptides representing the COOH-terminal 30 amino acids of the pFd and pS signal peptides were able to completely and reversibly inhibit the import of their homologous precursors into isolated chloroplasts at a 2.5 microM concentration. Import was blocked at the level of precursor binding to the chloroplast. This inhibition of precursor binding and import was not due to disruption of chloroplast integrity as incubation of isolated chloroplasts with the peptides did not cause measurable perturbation of the envelope membranes. The peptides also were able to block the import of the heterologous precursor protein, suggesting that pS and pFd share a common signal sequence recognition system. Visualization of the bound peptides at the chloroplast surface by indirect immunofluorescence microscopy using antipeptide antibodies gave a marked punctate staining pattern. This pattern is consistent with the localization of chloroplast import receptor(s) at contact zones between the inner and outer envelope membranes.     

Binding of basic peptides to acidic lipids in membranes: effects of inserting alanine(s) between the basic residues.

We studied the binding of peptides containing five basic residues to membranes containing acidic lipids. The peptides have five arginine or lysine residues and zero, one, or two alanines between the basic groups. The vesicles were formed from mixtures of a zwitterionic lipid, phosphatidylcholine, and an acidic lipid, either phosphatidylserine or phosphatidylglycerol. Measuring the binding using equilibrium dialysis, ultrafiltration, and electrophoretic mobility techniques, we found that all peptides bind to the membranes with a sigmoidal dependence on the mole fraction of acidic lipid. The sigmoidal dependence (Hill coefficient greater than 1 or apparent cooperativity) is due to both electrostatics and reduction of dimensionality and can be described by a simple model that combines Gouy-Chapman-Stern theory with mass action formalism. The adjustable parameter in this model is the microscopic association constant k between a basic residue and an acidic lipid (1 less than k less than 10 M-1). The addition of alanine residues decreases the affinity of the peptides for the membranes; two alanines inserted between the basic residues reduces k 2-fold. Equivalently, the affinity of the peptide for the membrane decreases 10-fold, probably due to a combination of local electrostatic effects and the increased loss of entropy that may occur when the more massive alanine-containing peptides bind to the membrane. The arginine peptides bind more strongly than the lysine peptides: k for an arginine residue is 2-fold higher than for a lysine residue. Our results imply that a cluster of arginine and lysine residues with interspersed electrically neutral amino acids can bind a significant fraction of a cytoplasmic protein to the plasma membrane if the cluster contains more than five basic residues.     

Processing-independent in vitro translocation of cytochrome P-450(SCC) precursor across mitochondrial membranes

In the presence of a membrane-permeable metal chelator, bovine adrenal cortex mitochondria imported P-450(SCC) precursor without processing of the amino-terminal extension peptide. The imported precursor was bound to the matrix side surface of the inner membrane. When the inhibition due to the metal chelator was removed, the imported precursor was processed to the mature form. Unprocessed precursor was also detected in mitochondria when the import reaction was carried out at relatively low temperature. These results suggest that the translocation of P-450(SCC) precursor across mitochondrial membranes is independent of its processing to the mature form. Both membrane-bound and solubilized P-450(SCC) could be cleaved by trypsin into two fragments with molecular weights of 29 kDa and 26 kDa, respectively, suggesting a two-domain structure of the molecule. The in vitro-imported and processed P-450(SCC) was also cleaved by trypsin in the same way. This finding indicated that the in vitro-imported and processed P-450(SCC) has the same conformation as the native form.     

Mitochondrial import and processing of mutant human ornithine transcarbamylase precursors in cultured cells.

We have investigated mitochondrial import and processing of the precursor for human ornithine transcarbamylase (OTC; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) in HeLa cells stably transformed with cDNA sequences encoding OTC precursors carrying mutations in their leader peptides. The mutant precursors studied included two with amino acid substitutions in the 32-amino-acid leader peptide (glycine for arginine at position 23, designated gly23; glycines for arginines at positions 15, 23, and 26, designated gly15,23,26) and two with deletions (deletion of residues 8 to 22, designated d8-22; deletion of residues 17 to 32, designated N16). Specific immunoprecipitation with anti-OTC antiserum of extracts of L-[35S]methionine-labeled cells expressing these mutations yielded only precursor species; neither mature nor intermediate-size OTC subunits were observed. Fractionation of radiolabeled cells, however, revealed important differences among the various mutants: the gly23 precursor was associated with mitochondria and was not detected in the cytosol; the d8-22 and N16 precursors were found with both the mitochondrial fraction and the cytosol; only the gly15,23,26 precursor was detected exclusively in the cytosol. A large fraction of each of the mitochondrially associated OTC species was in a trypsin-protected compartment. In particular, the gly23 precursor behaved in trypsin protection and mitochondrial fractionation studies in a manner consistent with its translocation into the mitochondrial matrix. On the other hand, the lack of binding of the gly23 protein to a delta-N-phosphonoacetyl-L-ornithine affinity column, which specifically recognizes active OTC enzyme, indicated that, despite its intramitochondrial location, the mutant protein did not assemble into the normal, active trimer. Further, the gly23 mutant precursor was unstable within the mitochondria and was degraded with a t1/2 of less further than 4 h. Thus, we have shown that, in intact HeLa cells, cleavage of the OTC leader peptide is not required for translocation into mitochondria, but is required for assembly into active enzyme.     

Interaction of peptides corresponding to mitochondrial presequences with membranes.

The transport of the F1-ATPase beta-subunit precursor into mitochondria is dependent upon a presequence at its amino terminus. Within the mitochondrial membrane translocation site the potential amphiphilic character of the presequence region may be necessary to stabilize binding to the mitochondrial inner membrane. To better understand its role in protein import, the interaction of the F1 beta-presequence with lipid membranes was measured using circular dichroism and surface tensiometry. These studies reveal that a 20-residue peptide containing the F1 beta-presequence binds to phospholipid vesicles (Kd = 4.5-6.0 x 10(-8)M and adopts a predominantly alpha-helical structure. Although the presequence peptide binds avidly to lipids, it does not appear to penetrate deeply into the bilayer to perturb a reporter probe in the membrane interior. Compared with the effect of the peptides with demonstrated membrane insertion and lytic properties, the F1-beta-presequence appears to displace phospholipid head groups but not insert deeply into the bilayer. High concentrations (greater than 50 microM) of presequence peptides are required to noticibly perturb import of the full length F1 alpha- or F1 beta-subunit precursors. Thus, the F1 beta-presequence alone is not sufficient to efficiently compete for import but may require a protein context or a minimal length to assist insertion into the transport site. These observations are discussed in light of the different requirements for import of various presequence containing precursors into mitochondria.     

Nucleotide sequence of the cDNA encoding the precursor for mitochondrial serine:pyruvate aminotransferase of rat liver

The nucleotide sequence of the mRNA coding for the precursor of mitochondrial serine:pyruvate aminotransferase of rat liver was determined from those of cDNA clones. The mRNA comprises at least 1533 nucleotides, except the poly(A) tail, and encodes a polypeptide consisting of 414 amino acid residues with a molecular mass of 45,834 Da. Comparison of the N-terminal amino acid sequence of mitochondrial serine:pyruvate aminotransferase with the nucleotide sequence of the mRNA showed that the mature form of the mitochondrial enzyme consisted of 390 amino acid residues of 43,210 Da. The amino acid composition of mitochondrial serine:pyruvate aminotransferase deduced from the nucleotide sequence of the cDNA showed good agreement with the composition determined on acid hydrolysis of the purified protein. The extra 24 amino acid residues correspond to the N-terminal extension peptide (pre-sequence) that is indispensable for the specific import of the precursor protein into mitochondria. In the extension peptide there are four basic amino acids distributed among hydrophobic amino acids and, as revealed on helical wheel analysis, the putative alpha-helical structure of the peptide was amphiphilic in nature. The secondary structures of the mature serine:pyruvate aminotransferase and three other aminotransferases of rat liver were predicted from their amino acid sequences. Their secondary structures exhibited a common feature and so we propose the specific lysine residue which binds pyridoxal phosphate as the active site of serine:pyruvate aminotransferase.     

Partitioning of malate dehydrogenase isoenzymes into glyoxysomes, mitochondria, and chloroplasts

Malate dehydrogenase isoenzymes catalyzing the oxidation of malate to oxaloacetate are highly active enzymes in mitochondria, in peroxisomes, in chloroplasts, and in the cytosol. Determination of the primary structure of the isoenzymes has disclosed that they are encoded in different nuclear genes. All three organelle-targeted malate dehydrogenases are synthesized with an amino terminal extension that is cleaved off in connection with the import of the enzyme precursor into the organelle. The sequence of the 27 amino acids of the mitochondrial transit peptide is unrelated to the 37-residue glyoxysomal transit peptide, which in turn is entirely different in sequence from the 57-residue chloroplastic transit peptide. With the exception of malate dehydrogenase and 3-ketoacyl thiolase, peroxisomal enzymes are synthesized without transit peptides and are frequently translocated into the organelle with a peroxisomal targeting signal consisting of a conserved tripeptide at the carboxy terminus of the protein. Based on the observation that this tripeptide (Ala-His-Leu) occurs in the transit peptides of glyoxysomal malate dehydrogenase and peroxisomal 3-ketoacyl thiolase, the possible significance of amino terminal transit peptides for peroxisome import is discussed.     

Import and processing of P-450SCC and P-450(11) beta precursors by corpus luteal mitochondria: a processing pathway recognizing homologous and heterologous precursors

Maturation of the precursor forms of bovine cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) and 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta) was investigated using mitochondria from bovine corpus luteum. The results show that both precursors, whose synthesis was directed by bovine adrenocortical RNA, can be imported and proteolytically processed to their corresponding mature forms by bovine corpus luteal mitochondria, even though P-450(11)beta is not expressed in this tissue. Furthermore, the efficiency of processing of pre-P-450(11)beta by corpus luteal mitochondria is similar to that of pre-P-450SCC, an endogenous enzyme of these mitochondria. However, the P-450(11)beta precursor is not processed by mitochondria from a nonsteroidogenic tissue (heart), a result observed previously for the P-450SCC precursor (M. F. Matocha and M. R. Waterman (1984) J. Biol. Chem. 259, 8672-8678). This discriminatory processing of pre-P-450(11)beta by heterologous mitochondria suggests that the precursor forms of P-450SCC and P-450(11)beta are processed via a common pathway in steroidogenic mitochondria and that this pathway is absent in nonsteroidogenic mitochondria.     

Early events in the transport of proteins into mitochondria. Import competition by a mitochondrial presequence.

Studies with a synthetic presequence peptide, F1 beta 1-20, corresponding to the NH2-terminal 20 amino acids of the F1-ATPase beta-subunit precursor (pF1 beta) show that although this peptide binds avidly to phospholipid bi-layers it does not efficiently compete for import of full-length precursor into mitochondria, Ki approximately 100 microM (Hoyt, D.W., Cyr, D.M., Gierasch, L.M., and Douglas, M.G. (1991) J. Biol. Chem. 266, 21693-21699). Herein we report that longer F1 beta presequence peptides F1 beta 1-32 + 2, F1 beta 1-32SQ + 2, and F1 beta 21-51 + 3 compete for mitochondrial import at 1000-, 250-, and 25-fold lower concentrations, respectively, than F1 beta 1-20. A longer peptide, F1 beta 1-51 + 3, was no more effective as an import competitor than F1 beta 1-32 + 2. Both minimal length and amphiphilic character appear required in order for F1 beta peptides to block mitochondrial import. Import competition by longer F1 beta peptides seems to occur at a step common to all precursors since they blocked import of precursors to F1-ATPase alpha- and beta-subunits and the ADP/ATP carrier protein. Dissipation of membrane potential (delta psi) across the inner mitochondrial membrane is observed in the presence of F1 beta-peptides, but this mechanism alone does not account for the observed import inhibition. F1 beta 1-32 + 2 and 21-51 + 3 block import of pF1 beta 100% at peptide concentrations which dissipate delta psi less than 25%. In contrast, experiments with valinomycin demonstrate that when mitochondrial delta psi is reduced 25% import of pF1 beta is inhibited only 25%. Therefore, at least 75% of maximal import inhibition observed in the presence of F1 beta 1-32 + 2 and F1 beta 21-51 + 3 does not result from dissipation of delta psi. Import inhibition by F1 beta-peptides is reversible and can be overcome by increasing the amount of full-length precursor in import reactions. F1 beta presequence peptides and full-length precursor are therefore likely to compete for a common import step. Presequence dependent binding of pF1 beta to trypsin-sensitive elements on the outer mitochondrial membrane is insensitive to inhibitory concentrations of F1 beta presequence peptide. We conclude that import inhibition by F1 beta presequence peptides is competitive and occurs at a site beyond initial interaction of precursor proteins with mitochondria.     

The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.     

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

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.