Mim1 functions in an oligomeric form to facilitate the integration of Tom20 into the mitochondrial outer membrane

The translocase of the outer mitochondrial membrane (TOM) complex is the general entry site into the organelle for newly synthesized proteins. Despite its central role in the biogenesis of mitochondria, the assembly process of this complex is not completely understood. Mim1 (mitochondrial import protein 1) is a mitochondrial outer membrane protein with an undefined role in the assembly of the TOM complex. The protein is composed of an N-terminal cytosolic domain, a central putative transmembrane segment (TMS) and a C-terminal domain facing the intermembrane space. Here we show that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane. We further investigated what the structural characteristics allowing Mim1 to fulfil its function are. The N- and C-terminal domains of Mim1 are crucial neither for the function of the protein nor for its biogenesis. Thus, the TMS of Mim1 is the minimal functional domain of the protein. We show that Mim1 forms homo-oligomeric structures via its TMS, which contains two helix-dimerization GXXXG motifs. Mim1 with mutated GXXXG motifs did not form oligomeric structures and was inactive. With all these data taken together, we propose that the homo-oligomerization of Mim1 allows it to fulfil its function in promoting the integration of Tom20 into the mitochondrial outer membrane.     

The Membrane Insertase Oxa1 Is Required for Efficient Import of Carrier Proteins into Mitochondria

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady‐state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.     

Mitochondrial outer membrane permeabilization during apoptosis: The role of mitochondrial fission

Mitochondria continually fuse and divide to yield a dynamic interconnected network throughout the cell. During apoptosis, concomitantly with permeabilization of the mitochondrial outer membrane (MOMP) and cytochrome c release, mitochondria undergo massive fission. This results in the formation of small, round organelles that tend to aggregate around the nucleus. Under some circumstances, preceding their fission, mitochondria tend to elongate and to hyperfuse, a process that is interpreted as a cell defense mechanism. Since many years, there is a controversy surrounding the physiological relevance of mitochondrial fragmentation in apoptosis. In this review, we present recent advances in this field, describe the mechanisms that underlie this process, and discuss how they could cooperate with Bax to trigger MOMP and cytochrome c release. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Comparison of the TIM and TOM channel activities of the mitochondrial protein import complexes

Water-filled channels are central to the process of translocating proteins since they provide aqueous pathways through the hydrophobic environment of membranes. The Tom and Tim complexes translocate precursors across the mitochondrial outer and inner membranes, respectively, and contain channels referred to as TOM and TIM (previously called PSC and MCC). In this study, little differences were revealed from a direct comparison of the single channel properties of the TOM and TIM channels of yeast mitochondria. As they perform similar functions in translocating proteins across membranes, it is not surprising that both channels are high conductance, voltage-dependent channels that are slightly cation selective. Reconstituted TIM and TOM channel activities are not modified by deletion of the outer membrane channel VDAC, but are similarly affected by signal sequence peptides.     

Evolution of mitochondrial protein biogenesis

Mitochondria and the nucleus are key features that distinguish eukaryotic cells from prokaryotic cells. Mitochondria originated from a bacterium that was endosymbiotically taken up by another cell more than a billion years ago. Subsequently, most mitochondrial genes were transferred and integrated into the host cell's genome, making the evolution of pathways for specific import of mitochondrial proteins necessary. The mitochondrial protein translocation machineries are composed of numerous subunits. Interestingly, many of these subunits are at least in part derived from bacterial proteins, although only few of them functioned in bacterial protein translocation. We propose that the primitive α-proteobacterium, which was once taken up by the eukaryote ancestor cell, contained a number of components that were utilized for the generation of mitochondrial import machineries. Many bacterial components of seemingly unrelated pathways were integrated to form the modern cooperative mitochondria-specific protein translocation system.     

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.     

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.     

Identification of a novel member of yeast mitochondrial Hsp70-associated motor and chaperone proteins that facilitates protein translocation across the inner membrane

Here, we report the identification of yeast 15-kD Tim15/Zim17, a new member of mitochondrial Hsp70 (mtHsp70)-associated motor and chaperone (MMC) proteins. The 15-kD MMC protein is a peripheral inner membrane protein with a zinc-finger motif. Depletion of the 15-kD protein led to impaired import of presequence-containing proteins into the matrix in vivo and in vitro. Overexpression of the 15-kD protein rescued the functional defects of mtHsp70 in ssc1-3 cells, and a fusion protein containing the 15-kD protein physically interacts with purified mtHsp70. Tim15/Zim17 therefore cooperates with mtHsp70 to facilitate import of presequence-containing proteins into the matrix.     

A dynamic machinery for import of mitochondrial precursor proteins

Mitochondria contain approximately 1000 different proteins, which are located in four different compartments, outer membrane, inner membrane, intermembrane space and matrix. The vast majority of these proteins has to be imported from the cytosol. Therefore, sophisticated molecular machineries have evolved that mediate protein translocation across or insertion into mitochondrial membranes and subsequent assembly into multi-subunit complexes. While the initial entry of virtually all mitochondrial proteins is mediated by the general import pore of the outer membrane, at least four different downstream pathways are dedicated to import and assembly of proteins into a specific compartment.     

Cryo-electron microscopy structure of a yeast mitochondrial preprotein translocase

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for proteins imported into mitochondria. We determined the structure of the native, unstained ∼ 550-kDa core-Tom20 complex from Saccharomycescerevisiae by cryo-electron microscopy at 18-Å resolution. The complex is triangular, measuring 145 Å on edge, and has near-3-fold symmetry. Its bulk is made up of three globular ∼ 50-Å domains. Three elliptical pores on the c-face merge into one central ∼ 70-Å cavity with a cage-like assembly on the opposite t-face. Nitrilotriacetic acid-gold labeling indicates that three Tom22 subunits in the TOM complex are located at the perimeter of the complex near the interface of the globular domains. We assign Tom22, which controls complex assembly, to three peripheral protrusions on the c-face, while the Tom20 subunit is tentatively assigned to the central protrusion on this surface. Based on our three-dimensional map, we propose a model of transient interactions and functional dynamics of the TOM assembly.     

Novel mitochondrial intermembrane space proteins as substrates of the MIA import pathway

Mitochondria consist of four compartments, the outer membrane, intermembrane space (IMS), inner membrane and the matrix. Most mitochondrial proteins are synthesized as precursors in the cytosol and have to be imported into these compartments. While the protein import machineries of the outer membrane, inner membrane and matrix have been investigated in detail, a specific mitochondrial machinery for import and assembly of IMS proteins, termed MIA, was identified only recently. To date, only a very small number of substrate proteins of the MIA pathway have been identified. The substrates contain characteristic cysteine motifs, either a twin Cx(3)C or a twin Cx(9)C motif. The largest MIA substrates known possess a molecular mass of 11 kDa, implying that this new import pathway has a very small size limit. Here, we have compiled a list of Saccharomyces cerevisiae proteins with a twin Cx(9)C motif and identified three IMS proteins that were previously localized to incorrect cellular compartments by tagging approaches. Mdm35, Mic14 (YDR031w) and Mic17 (YMR002w) require the two essential subunits, Mia40 and Erv1, of the MIA machinery for their localization in the mitochondrial IMS. With a molecular mass of 14 kDa and 17 kDa, respectively, Mic14 and Mic17 are larger than the known MIA substrates. Remarkably, the precursor of Erv1 itself is imported via the MIA pathway. As Erv1 has a molecular mass of 22 kDa and a twin Cx(2)C motif, this study demonstrates that the MIA pathway can transport substrates that are twice as large as the substrates known to date and is not limited to proteins with twin Cx(3)C or Cx(9)C motifs. However, tagging of MIA substrates can interfere with their subcellular localization, indicating that the proper localization of mitochondrial IMS proteins requires the characterization of the authentic untagged proteins.     

Distinctive biochemistry in the trypanosome mitochondrial intermembrane space suggests a model for stepwise evolution of the MIA pathway for import of cysteine-rich proteins

Mia40-dependent disulphide bond exchange is used by animals, yeast, and probably plants for import of small, cysteine-rich proteins into the mitochondrial intermembrane space (IMS). During import, electrons are transferred from the imported substrate to Mia40 then, via the sulphydryl oxidase Erv1, into the respiratory chain. Curiously, however, there are protozoa which contain substrates for Mia40-dependent import, but lack Mia40. There are also organisms where Erv1 is present in the absence of respiratory chain components. In accommodating these and other relevant observations pertaining to mitochondrial cell biology, we hypothesise that the ancestral IMS import pathway for disulphide-bonded proteins required only Erv1 (but not Mia40) and identify parasites in which O(2) is the likely physiological oxidant for Erv1.     

Bax inserts into the mitochondrial outer membrane by different mechanisms

Bax insertion into the mitochondrial outer membrane is essential for the implementation of apoptosis. However, little is known about the first stage of Bax integration into the mitochondrial outer membrane. We have recently shown that TOM22, a mitochondrial outer membrane receptor, is important for insertion, although other reports have suggested that only mitochondrial lipids are involved in this process. Here, we show that monomers, but not dimers, of Bax require the presence of TOM22 and TOM40 to integrate into mitochondria. In addition we show that once inserted into the membrane, Bax can act as a receptor for cytosolic Bax.     

Biogenesis of mitochondria: dual role of Tom7 in modulating assembly of the preprotein translocase of the outer membrane

Biogenesis of the translocase of the outer mitochondrial membrane (TOM complex) involves the assembly of the central β-barrel forming protein Tom40 with six different subunits that are embedded in the membrane via α-helical transmembrane segments. The sorting and assembly machinery (SAM complex) of the outer membrane plays a central role in this process. The SAM complex mediates the membrane integration of β-barrel precursor proteins including Tom40. The small Tom proteins Tom5 and Tom6 associate with the precursor of Tom40 at the SAM complex at an early stage of the assembly process and play a stimulatory role in the formation of the mature TOM complex. A fraction of the SAM components interacts with the outer membrane protein mitochondrial distribution and morphology protein 10 (Mdm10) to form the SAM-Mdm10 machinery; however, different views exist on the function of the SAM-Mdm10 complex. We report here that the third small Tom protein, Tom7, plays an inhibitory role at two distinct steps in the biogenesis of the TOM complex. First, Tom7 plays an antagonistic role to Tom5 and Tom6 at the early stage of Tom40 assembly at the SAM complex. Second, Tom7 interacts with Mdm10 that is not bound to the SAM complex, and thus promotes dissociation of the SAM-Mdm10 complex. Since the SAM-Mdm10 complex is required for the biogenesis of Tom22, Tom7 delays the assembly of Tom22 with Tom40 at a late stage of assembly of the TOM complex. Thus, Tom7 modulates the biogenesis of topologically different proteins, the β-barrel forming protein Tom40 and Tom22 that contains a transmembrane α-helix.     

Msp1p is an intermembrane space dynamin-related protein that mediates mitochondrial fusion in a Dnm1p-dependent manner in S. pombe

Mitochondrial morphology is controlled by large GTPases, such as Msp1p, whose action on mitochondrial membranes is not yet understood. The sub-mitochondrial localization of Msp1p, the subject of ongoing controversies, was found to be within the intermembrane space. Overexpression of Msp1p led to aggregation of the mitochondrial network, while its downregulation resulted in fragmentation of this network. Mutations affecting the integrity of the Msp1p GTPase function had a dominant phenotype and induced mitochondrial fragmentation followed by mitochondrial DNA loss and cell death. These effects were not observed in cells deleted for Dnm1p, an actor in mitochondrial fission, suggesting that Msp1p is involved in the fusion of mitochondria.     

C1orf163/RESA1 Is a Novel Mitochondrial Intermembrane Space Protein Connected to Respiratory Chain Assembly

Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60 kDa and 150 kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1).     

The mechanosensitive channel protein MscL is targeted by the SRP to the novel YidC membrane insertion pathway of Escherichia coli

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypo-osmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle (SRP) for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon. Mutants affected in the Sec-components are competent for MscL assembly. Translocation of the periplasmic domain was detected using a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The modification of a single cysteine residue at position 68 indicated its translocation across the inner membrane. From these in vivo experiments, it is concluded that the electrical chemical membrane potential is not necessary for membrane insertion of MscL. However, depletion of the membrane insertase YidC inhibits translocation of the protein across the membrane. We show here that YidC is essential for efficient membrane insertion of the MscL protein. YidC is a component of a recently identified membrane insertion pathway that is evolutionarily conserved in bacteria, mitochondria and chloroplasts.     

Mitochondrial import of Omi: the definitive role of the putative transmembrane region and multiple processing sites in the amino-terminal segment

The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.