High-affinity binding sites involved in the import of porin into mitochondria.

The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane.     

The carboxyl-terminal two-thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria

The precursor of the mitochondrial inner membrane protein ADP/ATP carrier is cytoplasmically synthesized without an amino-terminal peptide extension. We constructed a truncated precursor lacking the 103 amino acids from the amino terminus (about a third of the protein). Import of the truncated precursor into mitochondria showed the import characteristics of the authentic precursor, including nucleoside triphosphate dependence, requirement for a protease-sensitive component on the mitochondrial surface, two-step specific binding to the outer membrane, and membrane potential-dependent translocation into the inner membrane. We conclude that, in contrast to all other mitochondrial precursor proteins studied so far, domains of the ADP/ATP carrier distant from the amino terminus can carry specific targeting information for transport into mitochondria.     

Different transport pathways of individual precursor proteins in mitochondria

Transport of mitochondrial precursor proteins into mitochondria of Neurospora crassa was studied in a cell-free reconstituted system. Precursors were synthesized in a reticulocyte lysate programmed with Neurospora mRNA and transported into isolated mitochondria in the absence of protein synthesis. Uptake of the following precursors was investigated: apocytochrome c, ADP/ATP carrier and subunit 9 of the oligomycin-sensitive ATPase. Addition of high concentrations of unlabelled chemically prepared apocytochrome c (1-10 microM) inhibited the appearance in the mitochondrial of labelled cytochrome c synthesized in vitro because the unlabelled protein dilutes the labelled one and because the translocation system has a limited capacity [apparent V is 1-3 pmol X min-1 X (mg mitochondrial protein)-1]. Concentrations of added apocytochrome c exceeding the concentrations of precursor proteins synthesized in vitro by a factor of about 10(4) did not inhibit the transfer of ADP/ATP carrier or ATPase subunit 9 into mitochondria. Carbonylcyanide m-chlorophenylhydrazone, an uncoupler of oxidate phosphorylation, inhibited transfer in vitro of ADP/ATP carrier and of ATPase subunit 9, but not of cytochrome c. These findings suggest that cytochrome c and the other two proteins have different import pathways into mitochondria. It can be inferred from the data presented that different 'receptors' on the mitochondria. It can be inferred from the data presented that different 'receptors' on the mitochondrial surface mediate the specific recognition of precursor proteins by mitochondria by mitochondria as a first step in the transport process.     

Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocation.     

Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier.

The transfer of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane is dependent on energization of the membrane. To investigate the role of this energy requirement, a buffer system was developed in which efficient import of ADP/ATP carrier into mitochondria from the receptor-bound state occurred. This import was rapid and was dependent on divalent cations, whereas the binding of precursor proteins to the mitochondrial surface was slow and was independent of added divalent cations. Using this buffer system, the import of ADP/ATP carrier could be driven by a valinomycin-induced potassium diffusion potential. The protonophore carbonylcyanide m-chlorophenyl-hydrazone was not able to abolish this import. Imposition of a delta pH did not stimulate the import. We conclude that the membrane potential delta psi itself and not the total protonmotive force delta p is the required energy source.     

Cell-free synthesis of the mitochondrial ADP/ATP carrier protein of Neurospora crassa.

ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence.     

Distinct steps in the import of ADP/ATP carrier into mitochondria

Transport of the precursor to the ADP/ATP carrier from the cytosol into the mitochondrial inner membrane was resolved into several consecutive steps. The precursor protein was trapped at distinct stages of the import pathway and subsequently chased to the mature form. In a first reaction, the precursor interacts with a protease-sensitive component on the mitochondrial surface. It then reaches intermediate sites in the outer membrane which are saturable and where it is protected against proteases. This translocation intermediate can be extracted at alkaline pH. We suggest that it is anchored to the membrane by a so far unknown proteinaceous component. The membrane potential delta psi-dependent entrance of the ADP/ATP carrier into the inner membrane takes place at contact sites between outer and inner membranes. Completion of translocation into the inner membrane can occur in the absence of delta psi. A cytosolic component which is present in reticulocyte lysate and which interacts with isolated mitochondria is required for the specific binding of the precursor to mitochondria.     

70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria

We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide- sensitive protein(s). Both factors act synergistically.     

Transfer of proteins into mitochondria. Precursor to the ADP/ATP carrier binds to receptor sites on isolated mitochondria

The precursor form of Neurospora crassa mitochondrial ADP/ATP carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. If the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. Upon reestablishment of the membrane potential, the bound precursor is imported. This occurs without dissociation of the bound precursor from the mitochondrial surface. We conclude that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway.     

Coupling between proteolytic processing and translocation of the precursor of the F1-ATPase beta-subunit during its import into mitochondria of intact cells

The intracellular transport of newly synthesized beta-subunits of the F1-ATPase (beta F1) and of newly synthesized ADP/ATP carrier was followed in isolated rat hepatoma cells. As tested by rapid fractionation of [35S]methionine pulse- and pulse-chase-labeled cells and by sensitivity of labeled polypeptides to externally added protease, the import of beta F1 into mitochondria was strongly inhibited by the additional low concentrations of rhodamine 6G (R6G). In contrast, the import of the ADP/ATP carrier into mitochondria was not affected by the inhibitor. The results imply that the proteolytic processing of the precursor of beta F1 is coupled to its translocation across the mitochondrial membrane.     

Actin in mung bean mitochondria and implications for its function

Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin-green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography-tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed.     

Reinvestigation of the requirement of cytosolic ATP for mitochondrial protein import

Protein import into mitochondria requires the energy of ATP hydrolysis inside and/or outside mitochondria. Although the role of ATP in the mitochondrial matrix in mitochondrial protein import has been extensively studied, the role of ATP outside mitochondria (external ATP) remains only poorly characterized. Here we developed a protocol for depletion of external ATP without significantly reducing the import competence of precursor proteins synthesized in vitro with reticulocyte lysate. We tested the effects of external ATP on the import of various precursor proteins into isolated yeast mitochondria. We found that external ATP is required for maintenance of the import competence of mitochondrial precursor proteins but that, once they bind to mitochondria, the subsequent translocation of presequence-containing proteins, but not the ADP/ATP carrier, proceeds independently of external ATP. Because depletion of cytosolic Hsp70 led to a decrease in the import competence of mitochondrial precursor proteins, external ATP is likely utilized by cytosolic Hsp70. In contrast, the ADP/ATP carrier requires external ATP for efficient import into mitochondria even after binding to mitochondria, a situation that is only partly attributed to cytosolic Hsp70.     

The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria

The ADP/ATP carrier (AAC) is a major representative of mitochondrial preproteins lacking an N-terminal presequence. AAC contains targeting information in each of its three modules, which has led to a search for the dominant targeting region. An alternative, not yet tested model would be that several distinct targeting signals function simultaneously in import of the preprotein. We report that the three AAC modules cooperate in binding to the receptor Tom70 such that three Tom70 dimers are recruited to one preprotein. The modules are transferred to the import pore in a stepwise manner and cooperate again in the accumulation of AAC in the general import pore complex. AAC can cross the outer membrane with an internal segment first, i.e. in a loop formation. Each module of AAC is required for dimerization in the inner membrane. We propose a new concept for import of the hydrophobic carrier proteins into mitochondria where multiple signals cooperate in receptor recruitment, outer membrane translocation via loop formation and assembly in the inner membrane.     

Transport of cytoplasmically synthesized proteins into the mitochondria in a cell free system from Neurospora crassa.

Synthesis and transport of mitochondrial proteins were followed in a cell-free homogenate of Neurospora crassa in which mitochondrial translation was inhibited. Proteins synthesized on cytoplasmic ribosomes are transferred into the mitochondrial fraction. The relative amounts of proteins which are transferred in vitro are comparable to those transferred in whole cells. Cycloheximide and puromycin inhibit the synthesis of mitochondrial proteins but not their transfer into mitochondria. The transfer of immunoprecipitable mitochondrial proteins was demonstrated for matrix proteins, carboxyatractyloside-binding protein and cytochrome c. Import of proteins into mitochondria exhibits a degree of specificity. The transport mechanism differentiates between newly synthesized proteins and preexistent mitochondrial proteins, at least in the case of matrix proteins. In the cell-free homogenate membrane-bound ribosomes are more active in the synthesis of mitochondrial proteins than are free ribosomes. The finished translation products appear to be released from the membrane-bound ribosomes into the cytosol rather than into the membrane vesicles. The results suggest that the transport of cytoplasmically synthesized mitochondrial proteins is essentially independent of cytoplasmic translation; that cytoplasmically synthesized mitochondrial proteins exist in an extramitochondrial pool prior to import; that the site of this pool is the cytosol for at least some of the mitochondrial proteins; and that the precursors in the extramitochondrial pool differ in structure or conformation from the functional proteins in the mitochondria.     

The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria.

Human monoamine oxidase A that had been synthesized in a reticulocyte lysate translation system was capable of binding to and inserting into either rat liver mitochondria or isolated mitochondrial outer membranes. The inserted form was as resistant to proteinase K as endogenous mitochondrial monoamine oxidase A. The insertion, but not the binding, of monoamine oxidase A was prevented by depleting the reaction mixture of either ATP (with apyrase) or ubiquitin (with purified antibodies against this polypeptide). Addition of ATP or ubiquitin, respectively, to these depleted mixtures restored the insertion of the enzyme. In the absence of mitochondria, in vitro synthesized monoamine oxidase A did not catalyze its own alkylation by the mechanism-based inhibitor, [3H]clorgyline. However, both monoamine oxidase A that had been membrane-inserted in vitro and monoamine oxidase A that had been bound to the mitochondria under conditions of ATP depletion catalyzed adduct formation. Furthermore, reaction of either clorgyline or another mechanism-based inhibitor, pargyline, with the membrane-bound enzyme during ATP depletion inhibited the insertion of monoamine oxidase A when ATP was restored. These observations indicate that monoamine oxidase A acquired a catalytically active conformation on interaction with the mitochondrial outer membranes prior to its ATP and ubiquitin-dependent insertion into the membrane.     

Uncoupling proteins in mitochondria of plants and some microorganisms.

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.     

Insertion into the mitochondrial inner membrane of a polytopic protein, the nuclear-encoded Oxa1p.

Oxa1p, a nuclear-encoded protein of the mitochondrial inner membrane with five predicted transmembrane (TM) segments is synthesized as a precursor (pOxa1p) with an N-terminal presequence. It becomes imported in a process requiring the membrane potential, matrix ATP, mt-Hsp70 and the mitochondrial processing peptidase (MPP). After processing, the negatively charged N-terminus of Oxa1p (approximately 90 amino acid residues) is translocated back across the inner membrane into the intermembrane space and thereby attains its native N(out)-C(in) orientation. This export event is dependent on the membrane potential. Chimeric preproteins containing N-terminal stretches of increasing lengths of Oxa1p fused on mouse dehydrofolate reductase (DHFR) were imported into isolated mitochondria. In each case, their DHFR moieties crossed the inner membrane into the matrix. Thus Oxa1p apparently does not contain a stop transfer signal. Instead the TM segments are inserted into the membrane from the matrix side in a pairwise fashion. The sorting pathway of pOxa1p is suggested to combine the pathways of general import into the matrix with a bacterial-type export process. We postulate that at least two different sorting pathways exist in mitochondria for polytopic inner membrane proteins, the evolutionarily novel pathway for members of the ADP/ATP carrier family and a conserved Oxa1p-type pathway.     

Biogenesis of cytochrome c in Neurospora crassa. Synthesis of apocytochrome c, transfer to mitochondria and conversion to Holocytochrome c.

1. Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. 2. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. 3. Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labeling kinetics do not subbest a precursor role of microsomal apocytochrome c or holocytochrome c. 4. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. 5. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. 6. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The problems of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.     

Biosynthesis of mitochondrial porin and insertion into the outer mitochondrial membrane of Neurospora crassa

Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell-free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4 degrees C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane.     

Mitochondrial import of the ADP/ATP carrier protein in Saccharomyces cerevisiae. Sequences required for receptor binding and membrane translocation

The ADP/ATP carrier of yeast (309 amino acids) is an abundant transmembrane protein of the mitochondrial inner membrane whose import involves well-defined steps (Pfanner, N., and Neupert, W. (1987) J. Biol. Chem. 262, 7528-7536). Analysis of the in vitro import of gene fusion products containing ADP/ATP carrier (AAC) sequences at the amino terminus and mouse dihydrofolate reductase (DHFR) at the carboxyl terminus indicates that the first 72 amino acids of the soluble carrier protein, a hydrophilic region of the protein, are not by themselves sufficient for initial binding to the AAC receptor on the mitochondrial surface. However, an AAC-DHFR gene fusion containing the first 111 residues of the ADP/ATP carrier protein exhibited binding to mitochondria at low temperature (2 degrees C) and internalization at 25 degrees C to a mitochondrial space protected from proteinase K in the same manner as the wild-type ADP/ATP carrier protein. The AAC-DHFR protein, in contrast to the wild-type AAC protein imported into mitochondria under optimal conditions, remained extractable at alkaline pH and appeared to be blocked at an intermediate step in the AAC import pathway. Based on its extraction properties, this AAC-DHFR hybrid is proposed to be associated with a proteinaceous component of the import apparatus within mitochondria. These data indicate that the import determinants for the AAC protein are not located at its extreme amino terminus and that protein determinants distal to the first 111 residues of the carrier may be necessary to move the protein beyond the alkali-extractable step in the biogenesis of a functional AAC protein.