Active unfolding of precursor proteins during mitochondrial protein import.

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import.     

Functions of outer membrane receptors in mitochondrial protein import

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins and are imported into mitochondria. The targeting signals for mitochondria are encoded in the presequences or in the mature parts of the precursor proteins, and are decoded by the receptor sites in the translocator complex in the mitochondrial outer membrane. The recently determined NMR structure of the general import receptor Tom20 in a complex with a presequence peptide reveals that, although the amphiphilicity and positive charges of the presequence is essential for the import ability of the presequence, Tom20 recognizes only the amphiphilicity, but not the positive charges. This leads to a new model that different features associated with the mitochondrial targeting sequence of the precursor protein can be recognized by the mitochondrial protein import system in different steps during the import.     

Transport of proteins into yeast mitochondria

The amino-terminal sequences of several imported mitochondrial precursor proteins have been shown to contain all the information required for transport to and sorting within mitochondria. Proteins transported into the matrix contain a matrix-targeting sequence. Proteins destined for other submitochondrial compartments contain, in addition, an intramitochondrial sorting sequence. The sorting sequence in the cytochrome c1 presequence is a stop-transport sequence for the inner mitochondrial membrane. Proteins containing cleavable presequences can reach the intermembrane space by either of two pathways: (1) Part of the presequence is transported into the matrix; the attached protein, however, is transported across the outer but not the inner membrane (eg, the cytochrome c1 presequence). (2) The precursor is first transported into the matrix; part of the presequence is then removed, and the protein is reexported across the inner membrane (eg, the precursor of the iron-sulphur protein of the cytochrome bc1 complex). Matrix-targeting sequences lack primary amino acid sequence homology, but they share structural characteristics. Many DNA sequences in a genome can potentially encode a matrix-targeting sequence. These sequences become active if positioned upstream of a protein coding sequence. Artificial matrix-targeting sequences include synthetic presequences consisting of only a few different amino acids, a known amphiphilic helix found inside a cytosolic protein, and the presequence of an imported chloroplast protein. Transport of proteins across mitochrondrial membranes requires a membrane potential, ATP, and a 45-kd protein of the mitochondrial outer membrane. The ATP requirement for import is correlated with a stable structure in the imported precursor molecule. We suggest that transmembrane transport of a stably folded precursor requires an ATP-dependent unfolding of the precursor protein.     

Binding of a mitochondrial presequence to natural and artificial membranes: role of surface potential.

The binding of a synthetic mitochondrial presequence to large, negatively charged, unilamellar vesicles and to unenergized yeast mitochondria has been measured. The presequence, which corresponds to the amino-terminal 25 residues of the yeast cytochrome oxidase subunit IV precursor, was labeled with a fluorescent probe and used to examine the importance of the surface potentials of membranes on the interactions with the presequence. Binding of the fluorescent presequence to the membranes was determined by measuring a decrease in the fluorescence emission of the bound presequence. Binding both to the vesicles and to the mitochondria could be described as a simple partitioning of the presequence between the aqueous and lipid phases. The partitioning was found to depend on the ionic strength of the medium, and the Gouy-Chapman theory could be used to describe the partitioning at various ionic strengths. Application of the theory allowed the determination of an apparent charge on the presequence (+2.31 +/- 0.25), salt-independent apparent partition coefficients for vesicles (99 +/- 84 M-1) and for unenergized mitochondria (14.5 +/- 3.6 L g-1), and an estimated charge density for the mitochondrial outer membrane (-0.0124 +/- 0.0016 C m-2). This study shows that electrostatic effects are significant for the binding of a mitochondrial presequence both to lipid vesicles and to mitochondria, the natural target membrane of the presequence. The accumulation of positively charged presequences at the negative mitochondrial surface and the subsequent partitioning of the presequences directly into the mitochondrial outer membrane probably represent early steps in the translocation of precursor proteins into mitochondria.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Mitochondrial protein import

Most mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes and are subsequently imported into mitochondria. Many precursors carry amino-terminal presequences which contain information for their targeting to mitochondria. In several cases, targeting and sorting information is also contained in non-amino-terminal portions of the precursor protein. Nucleoside triphosphates are required to keep precursors in an import-competent (unfolded) conformation. The precursors bind to specific receptor proteins on the mitochondrial surface and interact with a general insertion protein (GIP) in the outer membrane. The initial interaction of the precursor with the inner membrane requires the mitochondrial membrane potential (delta psi) and occurs at contact sites between outer and inner membranes. Completion of translocation into the inner membrane or matrix is independent of delta psi. The presequences are cleaved off by the processing peptidase in the mitochondrial matrix. In several cases, a second proteolytic processing event is performed in either the matrix or in the intermembrane space. Other modifications can occur such as the addition of prosthetic groups (e.g., heme or Fe/S clusters). Some precursors of proteins of the intermembrane space or the outer surface of the inner membrane are retranslocated from the matrix space across the inner membrane to their functional destination ('conservative sorting'). Finally, many proteins are assembled in multi-subunit complexes. Exceptions to this general import pathway are known. Precursors of outer membrane proteins are transported directly into the outer membrane in a receptor-dependent manner. The precursor of cytochrome c is directly translocated across the outer membrane and thereby reaches the intermembrane space. In addition to the general sequence of events which occurs during mitochondrial protein import, current research focuses on the molecules themselves that are involved in these processes.     

Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

Direct interaction of mitochondrial targeting presequences with purified components of the TIM23 protein complex

Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.     

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.     

Investigations on the in vitro import ability of mitochondrial precursor proteins synthesized in wheat germ transcription-translation extract

Mitochondrial precursor proteins synthesized in rabbit reticulocyte lysate (RRL) are readily imported into mitochondria, whereas the same precursors synthesized in wheat germ extract (WGE) fail to be imported. We have investigated factors that render import incompetence from WGE. A precursor that does not require addition of extramitochondrial ATP for import, the F(A)d ATP synthase subunit, is imported from WGE. Import of chimeric constructs between precursors of the F(A)d protein and alternative oxidase (AOX) with switched presequences revealed that the mature domain of the F(A)d precursor defines the import competence in WGE as only the construct containing the presequence of AOX and mature portion of F(A)d (pAOX-mF(A)d) could be imported. Import competence of F(A)d and pAOX-mF(A)d correlated with solubility of these precursors in WGE, however, solubilization of import-incompetent precursors with urea did not restore import competence. Addition of RRL to WGE-synthesized precursors did not stimulate import but addition of WGE to the RRL-synthesized precursors or to the over-expressed mitochondrial precursor derived from the F1beta ATP synthase precursor inhibited import into mitochondria. The dual-targeted glutathione reductase precursor synthesized in WGE was imported into chloroplasts, but not into mitochondria. Antibodies against the 14-3-3 guidance complex characterized for chloroplast targeting were able to immunoprecipitate all of the precursors tested except the F(A)d ATP synthase precursor. Our results point to the conclusion that the import incompetence of WGE-synthesized mitochondrial precursors is not presequence dependent and is a result of interaction of WGE inhibitory factors with the mature portion of precursor proteins.     

Peptide library approach with a disulfide tether to refine the Tom20 recognition motif in mitochondrial presequences

Many mitochondrial matrix and inner-membrane proteins are synthesized in the cytosol as precursor proteins with an N-terminal presequence, and are imported into the mitochondria. Although no distinct sequence homology has been found among mitochondrial presequences, Tom20, a general import receptor in the outer mitohcondrial membrane, binds to presequences, and distinguishes mitochondrial proteins from non-mitochonrial proteins. The recently determined structure of the cytosolic domain of Tom20 (DeltaTom20) in a complex with the presequence of rat aldehyde dehydrogenase (ALDH) showed that a short stretch of the presequence forms an amphiphilic helix, and its hydrophobic surface interacts with the hydrophobic-binding groove of Tom20. The following NMR analyses revealed a common five-residue pattern for Tom20 binding in five different presequences. To refine the common amino acid motif for the recognition by Tom20, we introduced a new peptide library approach in this study: we prepared a mixture of ALDH presequence variants, tethered these peptides to DeltaTom20 in a competitive manner by an intermolecular disulfide bond, and determined the relative affinities by MALDI-TOF mass spectrometry. We successfully deduced a refined, common motif for the recognition by Tom20, and found that the segment consisting of residues 14-20 of the ALDH presequence was locally optimized in the sequence space, with respect to Tom20 binding.     

Mitochondrial protein import

Most polypeptides of mitochondria are imported from the cytosol. Precursor proteins contain targeting and sorting information, often in the form of amino-terminal presequences. Precursors first bind to receptors in the outer membrane. Two putative import receptors have been identified: a 19-kilodalton protein (MOM19) inNeurospora mitochondria, and a 70-kilodalton protein (MAS70) in yeast. Some precursors integrate directly into the outer membrane, but the majority are translocated through one or both membranes. This process requires an electrochemical potential across the inner membrane. Import appears to occur through a hydrophilic pore, although the inner and outer membranes may contain functionally separate translocation machineries. In yeast, a 42-kilodalton protein (ISP42) probably forms part of the outer membrane channel. After import, precursors interact with chaperonin ATPases in the matrix. Presequences then are removed by the matrix protease. Finally, some proteins are retranslocated across the inner membrane to the intermembrane space.     

Targeting and insertion of nuclear-encoded preproteins into the mitochondrial outer membrane

Most mitochondrial proteins are synthesized in the cytosol as preproteins with a cleavable presequence and are delivered to the import receptors on the mitochondria by cytoplasmic import factors. The proteins are then imported to the intramitochondrial compartments by the import systems of the outer and inner membranes, TOM and TIM. Mitochondrial outer membrane proteins are synthesized without a cleavable presequence and most of them contain hydrophobic transmembrane domains, which, in conjunction with the flanking segments, function as the mitochondria import signals. Some of the proteins are inserted into the outer membrane by the TOM machinery; the import signal probably arrests further translocation and is released from the translocation channel to the lipid bilayer. The other proteins are inserted into the membrane by a novel pathway independent of the TOM machinery. This article reviews recent developments in the biogenesis of mitochondrial outer membrane proteins.     

The cleavable presequence is not essential for import and assembly of the phosphate carrier of mammalian mitochondria but enhances the specificity and efficiency of import.

The phosphate carrier (PiC) of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to other members of the mitochondrial family of inner membrane carrier proteins. The precursor of PiC is efficiently imported, proteolytically processed, and correctly assembled in isolated mitochondria. Here we report that a presequence-deficient PiC was imported with an efficiency of about 50% as compared with the authentic precursor of PiC. This mature-sized PiC was correctly assembled, demonstrating that the presequence is not essential for the assembly pathway. We found the following functions for the PiC presequence. (i) The presequence by itself was able to target a passenger protein to mitochondria with a low efficiency, suggesting that the mammalian PiC contains multiple targeting signals, the more efficient one(s) present in the mature protein part. (ii) Deletion of the presequence allowed a more efficient heterologous import of mammalian PiC into mitochondria from Saccharomyces cerevisiae and Neurospora crassa, indicating an important role of the presequence in determining the specificity of PiC import. (iii) Import of the presequence-deficient PiC required a higher membrane potential across the inner membrane than that of the presequence-carrying form. Therefore, the presequence also enhances the translocation of PiC into the inner membrane.     

A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23.

Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.     

NMR identification of the Tom20 binding segment in mitochondrial presequences

Many mitochondrial proteins are synthesized in the cytosol as precursors with N-terminal presequences, and are imported into mitochondria with the aid of translocator protein complexes containing presequence-binding proteins. Tom20, a receptor protein which functions in an early step of the mitochondrial protein import, recognizes presequences with divergent amino acid sequences. Here, we report the identification of the segments involved in binding to Tom20 in mitochondrial presequences. We monitored the chemical shift perturbation of the NMR signals of five different 15N-labeled presequence peptides by the addition of the cytosolic receptor domain of rat or yeast Tom20. The perturbed segments occupy different positions, either near the N terminus or at the C terminus, in the presequences. Spin label experiments revealed that this is not due to different orientations of the presequence peptides bound to Tom20. The results presented here will offer a starting point to perform detailed analyses of Tom20-binding elements by systematic amino acid replacements.     

蛋白质跨线粒体膜运送的研究进展

线粒体拥有约1000种蛋白质,其中98％以上系由细胞核编码,在细胞质核糖体上以前体形式合成,之后再运至线粒体,经跨膜运送并分选定位于各部分。现对定位于外膜、基质和内膜的蛋白质的运送途径的研究进展作一扼要介绍。脱血红素细胞色素c是细胞色素c的前体,它不含导肽,对其转运的研究概况也作了评述。     

Gene expression of D-beta-hydroxybutyrate dehydrogenase. II. Incorporation of pre-BDH into mitochondria

beta-hydroxybutyrate dehydrogenase (BDH), a major protein located in the inner mitochondrial membrane is encoded, as most of mitochondrial proteins, in the nuclear genome. It is synthetized on the free polysomes and post-translationally imported into the mitochondria. The neosynthesized protein is a higher molecular weight precursor. The presequence is cleaved by the matrix protease to give the mature protein. The translocation across the mitochondrial membranes needs energy. The results also indicate that cytosolic factors with low molecular weight are essential in the recognition of precursor by mitochondria and to sort out newly synthetized nuclear encoded mitochondrial proteins from others nuclear encoded proteins.     

Biogenesis of eel liver citrate carrier (CIC): negative charges can substitute for positive charges in the presequence

A family of structurally related carrier proteins mediates the flux of metabolites across the mitochondrial inner membrane. Differently from most other mitochondrial proteins, members of the carrier family are synthesized without an amino-terminal targeting sequence. However, in some mammalian and plant species, representatives were identified that carry a positively charged presequence. To obtain data on a carrier protein from lower vertebrates, we determined the primary structure of eel mitochondrial citrate carrier (CIC) and investigated its import pathway into the target organelle. The protein carries a cleavable presequence of 20 amino acids, including two positively charged residues. The cleavage site is recognized by a magnesium-dependent peptidase in the intermembrane space. The presequence is dispensable both for targeting and translocation, but prior to import into mitochondria, significantly increases the solubility of the precursor protein. This effect is completely retained if the positive charges are exchanged with negative charges. Following this observation, we found that several carrier proteins appear to carry non-cleavable presequences that may similarly act as charged intramolecular chaperones.