Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria.

Mitochondria contain two distinct protein import systems, one in the outer and the other in the inner membrane. These systems can act independently of one another in submitochondrial fractions of if a protein is transported to the outer membrane or to the intermembrane space. It has been proposed that the two systems associate reversibly when a protein is transported across both membranes, but this hypothesis has remained unproven. In order to address this question, we have checked whether antibodies against a subunit of one system can co-immunoprecipitate subunits of the other system. We find that the two systems associate stably if a matrix-targeted precursor is arrested during import; no association is seen in the absence of a stuck precursor. These experiments provide direct evidence that protein import into the mitochondrial matrix is mediated by the reversible interaction of the two translocation systems.     

Import of bacterial pathogenicity factors into mitochondria

Recent research on the mechanism underlying the interaction of bacterial pathogens with their host has shifted the focus to secreted microbial proteins affecting the physiology and innate immune response of the target cell. These proteins either traverse the plasma membrane via specific entry pathways involving host cell receptors or are directly injected via bacterial secretion systems into the host cell, where they frequently target mitochondria. The import routes of bacterial proteins are mostly unknown, whereas the effect of mitochondrial targeting by these proteins has been investigated in detail. For a number of them, classical leader sequences recognized by the mitochondrial protein import machinery have been identified. Bacterial outer membrane beta-barrel proteins can also be recognized and imported by mitochondrial transporters. Besides an obvious importance in pathogenicity, understanding import of bacterial proteins into mitochondria has a highly relevant evolutionary aspect, considering the endosymbiotic, proteobacterial origin of mitochondria. The review covers the current knowledge on the mitochondrial targeting and import of bacterial pathogenicity factors.     

The First Steps of Protein Import into Mitochondria

Protein import into mitochondria is initiated by the recognition and binding of precursor proteins by import components in the cytosol, on the mitochondrial surface, and in the mitochondrial outer membrane. Following their synthesis on cytoplasmic ribosomes, some precursor proteins interact with molecular chaperones in the cytosol which function in maintaining the precursor protein in an import-competent state and may also aid in the delivery of the precursor to the mitochondria. A multisubunit protein import receptor then recognises and binds precursor proteins before feeding them into the outer membrane import site. Some proteins are sorted from the import site into the outer membrane, but most precursor proteins travel through the outer membrane import site into the mitochondria, where the later steps of protein import take place.     

The C-terminal region of TIM17 links the outer and inner mitochondrial membranes in Arabidopsis and is essential for protein import

The translocase of the inner membrane 17 (AtTIM17-2) protein from Arabidopsis has been shown to link the outer and inner mitochondrial membranes. This was demonstrated by several approaches: (i) In vitro organelle import assays indicated the imported AtTIM17-2 protein remained protease accessible in the outer membrane when inserted into the inner membrane. (ii) N-terminal and C-terminal tagging indicated that it was the C-terminal region that was located in the outer membrane. (iii) Antibodies raised to the C-terminal 100 amino acids recognize a 31-kDa protein from purified mitochondria, but cross-reactivity was abolished when mitochondria were protease-treated to remove outer membrane-exposed proteins. Antibodies to AtTIM17-2 inhibited import of proteins via the general import pathway into outer membrane-ruptured mitochondria, but did not inhibit protein import via the carrier import pathway. Together these results indicate that the C-terminal region of AtTIM17-2 is exposed on the outer surface of the outer membrane, and the C-terminal region is essential for protein import into mitochondria.     

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.     

Type V secretion: From biogenesis to biotechnology

The two membranes of Gram-negative bacteria contain protein machines that have a general function in their assembly. To interact with the extra-cellular milieu, Gram-negatives target proteins to their cell surface and beyond. Many specialized secretion systems have evolved with dedicated translocation machines that either span the entire cell envelope or localize to the outer membrane. The latter act in concert with inner-membrane transport systems (i.e. Sec or Tat). Secretion via the Type V secretion system follows a two-step mechanism that appears relatively simple. Proteins secreted via this pathway are important for the Gram-negative life-style, either as virulence factors for pathogens or by contributing to the survival of non-invasive environmental species. Furthermore, this system appears well suited for the secretion of biotechnologically relevant proteins. In this review we focus on the biogenesis and application of two Type V subtypes, the autotransporters and two-partner secretion (TPS) systems. For translocation across the outer membrane the autotransporters require the assistance of the Bam complex that also plays a generic role in the assembly of outer membrane proteins. The TPS systems do use a dedicated translocator, but this protein shows resemblance to BamA, the major component of the Bam complex. Interestingly, both the mechanistic and more applied studies on these systems have provided a better understanding of the secretion mechanism and the biogenesis of outer membrane proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Multispan mitochondrial outer membrane protein Ugo1 follows a unique Mim1-dependent import pathway

The mitochondrial outer membrane (MOM) harbors several multispan proteins that execute various functions. Despite their importance, the mechanisms by which these proteins are recognized and inserted into the outer membrane remain largely unclear. In this paper, we address this issue using yeast mitochondria and the multispan protein Ugo1. Using a specific insertion assay and analysis by native gel electrophoresis, we show that the import receptor Tom70, but not its partner Tom20, is involved in the initial recognition of the Ugo1 precursor. Surprisingly, the import pore formed by the translocase of the outer membrane complex appears not to be required for the insertion process. Conversely, the multifunctional outer membrane protein mitochondrial import 1 (Mim1) plays a central role in mediating the insertion of Ugo1. Collectively, these results suggest that Ugo1 is inserted into the MOM by a novel pathway in which Tom70 and Mim1 contribute to the efficiency and selectivity of the process.     

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase.     

Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts.

The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.     

Insertion of OEP14 into the outer envelope membrane is mediated by proteinaceous components of chloroplasts

Most chloroplastic outer envelope membrane proteins are synthesized in the cytosol at their mature size without a cleavable targeting signal. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. The insertion has been assumed to be mediated by a spontaneous mechanism or by interaction solely with the lipid components of the outer membrane. However, we show here that insertion of an outer membrane protein requires some trypsin-sensitive and some N-ethylmaleimide-sensitive components of chloroplasts. Association and insertion of the outer membrane protein are saturable and compete with the import of another outer membrane protein. These data suggest that import of chloroplastic outer membrane proteins occurs at specific proteinaceous sites on chloroplasts.     

Protein translocation pathways of the mitochondrion

The biogenesis of mitochondria depends on the coordinated import of precursor proteins from the cytosol coupled with the export of mitochondrially coded proteins from the matrix to the inner membrane. The mitochondria contain an elaborate network of protein translocases in the outer and inner membrane along with a battery of chaperones and processing enzymes in the matrix and intermembrane space to mediate protein translocation. A mitochondrial protein, often with an amino-terminal targeting sequence, is escorted through the cytosol by chaperones to the TOM complex (translocase of the outer membrane). After crossing the outer membrane, the import pathway diverges; however, one of two TIM complexes (translocase of inner membrane) is generally utilized. This review is focused on the later stages of protein import after the outer membrane has been crossed. An accompanying paper by Lithgow reviews the early stages of protein translocation.     

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Protein secretion in the absence of ATP: the autotransporter,two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (Review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In Gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these ‘self-sufficient’ pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Functional definition of outer membrane proteins involved in preprotein import into mitochondria

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.     

Type I secretion and multidrug efflux: transport through the TolC channel-tunnel

The crystal structure of TolC from Escherichia coli was recently determined to 2.1-A resolution and shows a unique type of channel architecture: a 12-stranded beta-barrel spans the outer membrane and is attached to a long alpha-helical channel that penetrates far into the periplasm. The structure suggests a mechanism for its role in secretion of proteins and in efflux of toxic small molecules. The TolC export pathway is compared with several import pathways of gram-negative bacteria where the outer membrane protein structures are also known.     

The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of β-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.     

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.     

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane.     

Biogenesis of the dicarboxylate carrier (DIC): translocation across the mitochondrial outer membrane and subsequent release from the TOM channel are membrane potential-independent

The mitochondrial inner membrane of Saccharomyces cerevisiae contains a group of homologous carrier proteins that mediate the exchange of several metabolites. The members of this protein family are synthesized in the cytosol and reach their final topology after translocation across the mitochondrial outer membrane. Using the ADP/ATP carrier (AAC) as a model protein, previous studies have established four distinct steps of the import pathway (stages I-IV). In the absence of the mitochondrial membrane potential (deltapsi), the AAC accumulates at the inner surface of the outer membrane (stage IIIa) and remains bound to the outer membrane import channel. Only in the presence of the membrane potential, can a complex of small Tim proteins mediate transfer of the AAC to the inner membrane. In this study, we characterized the import pathway of the dicarboxylate carrier (DIC). Different from the AAC, the DIC showed complete deltapsi-independent translocation across the outer membrane, release from the import pore, and mainly accumulated in a soluble state in the intermembrane space, thus defining a new translocation intermediate (stage III*). The DIC should be a suitable model protein for the characterization of deltapsi-independent functions of the intermembrane space Tim proteins.     

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.