Zinc-dependent intermembrane space proteins stimulate import of carrier proteins into plant mitochondria

Mitochondrial inner membrane carrier proteins are imported into mitochondria from yeast, fungi and mammals by specific machinery, some components of which are distinct from those utilized by other proteins. Import of two different carriers into plant mitochondria showed that one contains a cleavable presequence which was processed during import, while the other imported in a valinomycin-sensitive manner without processing. Mild osmotic shock of mitochondria released intermembrane space (IMS) components and impaired carrier protein import. Adding back the released IMS proteins as a concentrate in the presence of micromolar ZnCl2 stimulated carrier import into IMS-depleted mitochondria, but did not stimulate import of a non-carrier control precursor protein, the alternative oxidase. Anion-exchange separation of IMS components before addition to IMS-depleted mitochondria revealed a correlation between several 9-10 kDa proteins and stimulation of carrier import. MS/MS sequencing of these proteins identified them as plant homologues of the yeast zinc-finger carrier import components Tim9 and Tim10. Stimulation of import was dependent on either Zn2+ or Cd2+ and inhibited by both N-ethylmalamide (NEM) and a divalent cation chelator, consistent with a functional requirement for a zinc finger protein. This represents direct functional evidence for a distinct carrier import pathway in plant mitochondria, and provides a tool for determining the potential function of other IMS proteins associated with protein import.     

The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery

Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal- anchor sequence. The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment. The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human). Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed. This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts. The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins. In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein- translocation machinery. These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide.     

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.     

Mitochondrial precursor proteins are imported through a hydrophilic membrane environment

We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit beta (F1 beta) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1 beta translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites.     

A MICOS–TIM22 Association Promotes Carrier Import into Human Mitochondria

Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space toward the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the mitochondrial contact site and cristae organizing system (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast mic10Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria, the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane.     

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.     

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.     

The Mitochondrial Protein Import Pathway: Are Precursors Imported through Membrane Channels?

Mitochondrial biogenesis requires the import of hundreds of different proteins from the cytosol. Protein import into mitochondria is a multistep pathway that includes recognition of precursor proteins by machinery both in the cytoplasm and on the mitochondrial surface, translocation of the precursor across one or both mitochondrial membranes, and folding of the protein after its import into the organelle. Over the past several years, many components of the import machinery have been identified using both biochemical and genetic methods. Recently, significant progress has been made determining the function of some of these import proteins. One purpose of this minireview is to summarize our current understanding of the import pathway, and to introduce the topics of the minireviews that will follow. The other goal of this minireview is to discuss recent findings suggesting that proteins are translocated across both the mitochondrial inner and outer membranes through aqueous channels.     

Protein import into and across the mitochondrial inner membrane: role of the TIM23 and TIM22 translocons

Import of nuclear-encoded mitochondrial proteins requires the action of at least two different import machines, called translocons, in the mitochondrial inner membrane (IM). The TIM23 complex mediates the translocation of proteins into the mitochondria matrix, whereas the TIM22 complex is required for the insertion of polytopic proteins into the IM. While the two translocons are distinct and composed of separate subunits, the essential reactions in each complex are carried out by homologous proteins. In addition, the core components of both the TIM23 and TIM22 translocons have been shown to form aqueous pores in the mitochondrial IM. In this review, we summarize what is known about import of proteins across the mitochondrial IM.     

Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane

Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential- and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free of outer membrane components. Third, import into purified inner membrane vesicles is insensitive to antibody against the outer membrane. Thus, while outer membrane components are clearly required in vivo, the inner membrane contains a complete protein translocation system that can operate by itself if the outer membrane barrier is removed.     

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.     

Functional staging of ADP/ATP carrier translocation across the outer mitochondrial membrane.

The ADP/ATP carrier (AAC) is the major representative of the inner membrane carrier proteins of mitochondria that are synthesized without cleavable presequences. The characterization of the import pathway of AAC into mitochondria has mainly depended on an operational staging system. Here, we introduce two approaches for analyzing the import of AAC, blue native electrophoresis and folding-induced translocation arrest, that allow a functional staging of AAC transport across the outer membrane. (i) Blue native electrophoresis permits a direct monitoring of the receptor stage of AAC and its chase into mitochondria. Binding to this stage requires the receptor protein Tom70 but not Tom37 or Tom20. (ii) A fusion protein between AAC and dihydrofolate reductase can be selectively arrested in the general import pore complex of the outer membrane by ligand induced folding of the passenger protein. Cross-linking demonstrates that the arrested preprotein is in close contact not only with several receptors and Tim10 but also with the channel protein Tom40, providing the first direct evidence that cleavable preproteins and carrier preproteins interact with the same outer membrane channel. The staging system presented here permits a molecular dissection of AAC transport across the outer mitochondrial membrane, relates it to functional units of the translocases, and indicates a coordinated and successive cooperation of distinct translocase subcomplexes during transfer of the preprotein.     

The structure of precursor proteins during import into mitochondria

Precursor proteins must be at least partially unfolded during import into mitochondria, but their actual conformation during translocation is not known. Are proteins fully unfolded and threaded through the import machinery amino acid by amino acid, or do they retain some partial structure? The folding pathway of most proteins in vitro contains a partially folded intermediate known as the molten globule state, and it has been suggested that proteins are in the molten globule state during translocation across membranes. Here we show that precursors are normally fully unfolded during import into mitochondria. However, precursors containing residual structure can be imported, if less efficiently.     

Bacterial proteins predisposed for targeting to mitochondria

Mitochondria evolved from an endosymbiotic proteobacterium in a process that required the transfer of genes from the bacterium to the host cell nucleus, and the translocation of proteins thereby made in the host cell cytosol into the internal compartments of the organelle. According to current models for this evolution, two highly improbable events are required to occur simultaneously: creation of a protein translocation machinery to import proteins back into the endosymbiont and creation of targeting sequences on the protein substrates themselves. Using a combination of two independent prediction methods, validated through tests on simulated genomes, we show that at least 5% of proteins encoded by an extant proteobacterium are predisposed for targeting to mitochondria, and propose we that mitochondrial targeting information was preexisting for many proteins of the endosymbiont. We analyzed a family of proteins whose members exist both in bacteria and in mitochondria of eukaryotes and show that the amino-terminal extensions occasionally found in bacterial family members can function as a crude import sequence when the protein is presented to isolated mitochondria. This activity leaves the development of a primitive translocation channel in the outer membrane of the endosymbiont as a single hurdle to initiating the evolution of mitochondria.     

The role of the TIM8-13 complex in the import of Tim23 into mitochondria

Tim8 and Tim13 are non-essential, conserved proteins of the mitochondrial intermembrane space, which are organized in a hetero-oligomeric complex. They are structurally related to Tim9 and Tim10, essential components of the import machinery for mitochondrial carrier proteins. Here we show that the TIM8-13 complex interacts with translocation intermediates of Tim23, which are partially translocated across the outer membrane but not with fully imported or assembled Tim23. The TIM8-13 complex binds to the N-terminal or intermediate domain of Tim23. It traps the incoming precursor in the intermembrane space thereby preventing retrograde translocation. The TIM8-13 complex is strictly required for import of Tim23 under conditions when a low membrane potential exists in the mitochondria. The human homologue of Tim8 is encoded by the DDP1 (deafness/dystonia peptide 1) gene, which is associated with the Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder leading to deafness. It is demonstrated that import of human Tim23 is dependent on a high membrane potential. A mechanism to explain the pathology of MTS is discussed.     

Finding the right organelle. Targeting signals in mitochondrial outer-membrane proteins

The mitochondrial outer membrane contains a diverse set of proteins that includes enzymes, components of the preprotein translocation machinery, pore-forming proteins, regulators of programmed cell death, and those that control the morphology of the organelle. All these proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus, so they are synthesized in the cytosol and contain signals that are essential for their subsequent import into mitochondria. This review summarizes our current knowledge of the signals that target mitochondrial outer-membrane proteins to their correct intracellular location. In addition, the mechanisms by which these signals are decoded by the mitochondria are discussed.     

Mechanisms of protein translocation into mitochondria.

Mitochondrial biogenesis utilizes a complex proteinaceous machinery for the import of cytosolically synthesized preproteins. At least three large multisubunit protein complexes, one in the outer membrane and two in the inner membrane, have been identified. These translocase complexes cooperate with soluble proteins from the cytosol, the intermembrane space and the matrix. The translocation of presequence-containing preproteins through the outer membrane channel includes successive electrostatic interactions of the charged mitochondrial targeting sequence with a chain of import components. Translocation across the inner mitochondrial membrane utilizes the energy of the proton motive force of the inner membrane and the hydrolysis of ATP. The matrix chaperone system of the mitochondrial heat shock protein 70 forms an ATP-dependent import motor by interaction with the polypeptide chain in transit and components of the inner membrane translocase. The precursors of integral inner membrane proteins of the metabolite carrier family interact with newly identified import components of the intermembrane space and are inserted into the inner membrane by a second translocase complex. A comparison of the full set of import components between the yeast Sacccharomyces cerevisiae and the nematode Caenorhabditis elegans demonstrates an evolutionary conservation of most components of the mitochondrial import machinery with a possible greater divergence for the import pathway of the inner membrane carrier proteins.     

The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.     

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.