Identification of Tim40 that mediates protein sorting to the mitochondrial intermembrane space

Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria, and sorted to one of the four mitochondrial subcompartments. Here we identified a new inner membrane protein, Tim40, that mediates sorting of small Tim proteins to the intermembrane space. Tim40 is essential for yeast cell growth, and its function in vivo requires six conserved Cys residues but not anchoring of the protein to the inner membrane by its N-terminal hydrophobic segment. Depletion of Tim40 impairs the import of small Tim proteins into mitochondria both in vivo and in vitro. In wild-type mitochondria, Tim40 forms a translocation intermediate with small Tim proteins prior to their assembly in the intermembrane space in vitro. These results suggest the essential role of Tim40 in sorting/assembly of small Tim proteins.     

Import of proteins into mitochondria. Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells

The import of cytochrome b2 and cytochrome c peroxidase into mitochondria was investigated by pulse-chase experiments with intact yeast cells combined with subcellular fractionation. Import and processing of the precursors of these intermembrane space proteins is blocked by uncouplers of oxidative phosphorylation, indicating that an "energized" inner membrane is required. Cytochrome b2 is processed in two steps. The first step involves energy-dependent transport across both mitochondrial membranes and cleavage by a matrix-located protease to yield an intermediate which is smaller than the precursor, but larger than the mature protein. The second step involves conversion of the intermediate to the mature form. Whereas the precursor and the mature form are soluble, the intermediate is membrane-bound and exposed to the intermembrane space. The maturation of cytochrome c peroxidase is much slower than that of cytochrome b2. Proteolytic processing rather than import is rate-limiting since cytochrome c peroxidase precursor labeled during a 3-min pulse is already found attached to the outer face of the mitochondrial inner membrane. Import of cytochrome b2 and probably also of cytochrome c peroxidase thus involves energy-dependent transport to the matrix and cleavage by a matrix-localized protease. Maturation of cytochrome b2 proceeds in the sequence: soluble precursor leads to membrane-bound intermediate form leads to soluble mature form.     

Mdm35p imports Ups proteins into the mitochondrial intermembrane space by functional complex formation

Ups1p, Ups2p, and Ups3p are three homologous proteins that control phospholipid metabolism in the mitochondrial intermembrane space (IMS). The Ups proteins are atypical IMS proteins in that they lack the two major IMS‐targeting signals, bipartite presequences and cysteine motifs. Here, we show that Ups protein import is mediated by another IMS protein, Mdm35p. In vitro import assays show that import of Ups proteins requires Mdm35p. Loss of Mdm35p led to a decrease in steady state levels of Ups proteins in mitochondria. In addition, mdm35Δ cells displayed a similar phenotype to ups1Δups2Δups3Δ cells. Interestingly, unlike typical import machineries, Mdm35p associated stably with Ups proteins at a steady state after import. Demonstrating that Mdm35p is a functional component of Ups–Mdm35p complexes, restoration of Ups protein levels in mdm35Δ mitochondria failed to restore phospholipid metabolism. These findings provide a novel mechanism in which the formation of functional protein complexes drives mitochondrial protein import.     

The MIA system for protein import into the mitochondrial intermembrane space

When thinking of the mitochondrial intermembrane space we envisage a small compartment that is bordered by the mitochondrial outer and inner membranes. Despite this somewhat simplified perception the intermembrane space has remained a central focus in mitochondrial biology. This compartment accommodates many proteinaceous factors that play critical roles in mitochondrial and cellular metabolism, including the regulation of programmed cell death and energy conversion. The mechanism by which intermembrane space proteins are transported into the organelle and folded remained largely unknown until recently. In pursuit of the answer to this question a novel machinery, the Mitochondrial Intermembrane Space Assembly machinery, exploiting a unique regulated thiol-disulfide exchange mechanism has been revealed. This exciting discovery has not only put in place novel concepts for the biogenesis of intermembrane space precursors but also raises important implications on the mechanisms involved in the generation and transfer of disulfide bonds.     

Chaperoning through the mitochondrial intermembrane space

The first high-resolution structure of a mitochondrial translocase complex, the Tim9-Tim10 chaperone, is reported by Webb et al. (2006) in a recent issue of Molecular Cell, providing important insight in the transport of hydrophobic proteins through the aqueous intermembrane space and the mechanisms of protein 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.     

Shuttle mission in the mitochondrial intermembrane space

Lipid trafficking is essential for biogenesis and maintenance of eukaryotic organelles. In this issue of The EMBO Journal, Saita et al ( 2018 ) revealed that proteolytic processing by the rhomboid protease PARL in the mitochondrial inner membrane facilitates partitioning of START domain‐containing protein STARD7 to the cytosol and mitochondrial intermembrane space. STARD7 in the mitochondrial intermembrane space functions as a lipid transfer protein to shuttle phosphatidylcholine from the outer membrane to the inner membrane.     

Cytosolic thioredoxin system facilitates the import of mitochondrial small Tim proteins

Thiol‐disulphide redox regulation has a key role during the biogenesis of mitochondrial intermembrane space (IMS) proteins. Only the Cys‐reduced form of precursor proteins can be imported into mitochondria, which is followed by disulphide bond formation in the mitochondrial IMS. In contrast to the wealth of knowledge on the oxidation process inside mitochondria, little is known about how precursors are maintained in an import‐competent form in the cytosol. Here we provide the first evidence that the cytosolic thioredoxin system is required to maintain the IMS small Tim proteins in reduced forms and facilitate their mitochondrial import during respiratory growth.     

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.     

Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria

Import of in vitro-synthesized cytochrome b2 (a soluble intermembrane space enzyme) was studied wih isolated yeast mitochondria. Import requires an electrochemical gradient across the inner membrane and is accompanied by cleavage of the precursor to the corresponding mature form. This conversion proceeds via an intermediate form of cytochrome b2, which can be detected as a transient species when mitochondria are incubated with the cytochrome b2 precursor for short times or at low temperatures. Conversion of the precursor to the intermediate form is energy-dependent and catalyzed by an o-phenanthroline-sensitive protease located in the soluble matrix. The intermediate is subsequently converted to mature cytochrome b2 in a reaction which is o-phenanthroline-insensitive and requires neither an energized inner membrane nor a soluble component of the intermembrane space. Whereas mature cytochrome b2 is soluble, the intermediate formed by isolated mitochondria is membrane-bound and exposed to the intermembrane space. The same intermediate is detected as a transient species during cytochrome b2 maturation in intact yeast cells (Reid, G. A., Yonetani, T., and Schatz, G (1982) J. Biol. Chem. 257, 13068-13074). The in vitro studies reported here suggest that a part of the cytochrome b2 precursor polypeptide chain is transported to the matrix where it is cleaved to a membrane-bound intermediate form by the same protease that processes polypeptides destined for the matrix space or for the inner membrane. In a second reaction, the cytochrome b2 intermediate is converted to mature cytochrome b2 which is released into the intermembrane space. The binding of heme is not necessary for converting the intermediate to the mature polypeptide.     

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins

Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.     

Mitochondrial intermembrane space proteins in apoptosis process

Mitochondria, despite their function in cellular energy metabolism, play an important role in the apoptotic signaling pathways. These organelles in response to the death signal undergo changes resulting in the release of proteins which are essential to conduct apoptosis via mitochondrial pathway. This article is focused on the properties and functions of apoptogenic proteins released from the mitochondrial intermembrane space, i.e., caspases, cytochrome c, Smac/DIABLO, serine protease Omi/HtrA2, AIF and endonuclease G.     

Two intermembrane space TIM complexes interact with different domains of Tim23p during its import into mitochondria

Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p-Tim13p and Tim9p-Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p-Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH(2)-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p-Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p-Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.     

Redox regulation of protein folding in the mitochondrial intermembrane space

Protein translocation pathways to the mitochondrial matrix and inner membrane have been well characterized. However, translocation into the intermembrane space, which was thought to be simply a modification of the traditional translocation pathways, is complex. The mechanism by which a subset of intermembrane space proteins, those with disulfide bonds, are translocated has been largely unknown until recently. Specifically, the intermembrane space proteins with disulfide bonds are imported via the mitochondrial intermembrane space assembly (MIA) pathway. Substrates are imported via a disulfide exchange relay with two components Mia40 and Erv1. This new breakthrough has resulted in novel concepts for assembly of proteins in the intermembrane space, suggesting that this compartment may be similar to that of the endoplasmic reticulum and the prokaryotic periplasm. As a better understanding of this pathway emerges, new paradigms for thiol-disulfide exchange mechanisms may be developed. Given that the intermembrane space is important for disease processes including apoptosis and neurodegeneration, new roles in regulation by oxidation-reduction chemistry seem likely to be relevant.     

Successive translocation into and out of the mitochondrial matrix: targeting of proteins to the intermembrane space by a bipartite signal peptide

We investigated the import and sorting pathways of cytochrome b2 and cytochrome c1, which are functionally located in the intermembrane space of mitochondria. Both proteins are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps upon import. The precursors are first translocated across both mitochondrial membranes via contact sites into the matrix. Processing by the matrix peptidase leads to intermediate-sized forms, which are subsequently redirected across the inner membrane. The second proteolytic processing occurs in the intermembrane space. We conclude that the hydrophobic stretches in the presequences of the intermediate-sized forms do not stop transfer across the inner membrane, but rather act as transport signals to direct export from the matrix into the intermembrane space.     

Deleterious role of superoxide dismutase in the mitochondrial intermembrane space

This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1(-/-)) did not. Also, lymphocytes isolated from SOD1(-/-) mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.     

Polycations induce the release of soluble intermembrane mitochondrial proteins

The release of proapoptotic proteins from the intermembrane space of mitochondria is an early critical step in many pathways to apoptosis. Induction of the mitochondrial permeability transition pore (PTP) was suggested to be the mechanism of the release of soluble mitochondrial intermembrane proteins (SIMP) in apoptosis. However, several studies suggested that proapoptotic proteins (e.g. Bax and Bid) can induce the release of SIMP (e.g. cytochrome c (cyt c) and adenylate kinase 2 (AK2)) in vivo and in vitro independent of PTP. We have found that a number of structurally diverse polycations, such as aliphatic polyamines (e.g. spermine and to a lesser extent spermidine), aminoglycosides (e.g. streptomycin, gentamicin and neomycin), and cytotoxic peptides (e.g. melittin), induce the release of SIMP from liver mitochondria, in vitro. All the polycations released AK2 together with cyt c, suggesting that rupture of the outer membrane is a common mechanism of cyt c release by these polycations. Several polycations (e.g. spermine, spermidine and neomycin) induced SIMP release without inducing significant swelling, and this release was not inhibited significantly by the PTP inhibitor cyclosporin. In contrast, under the same conditions, streptomycin and melittin induced swelling and SIMP release that was inhibited strongly by cyclosporin. Gentamicin-induced swelling and release of SIMP were partially inhibited by cyclosporin. The affinity of polyamines to the anionic phospholipids of the mitochondrial membranes (spermine=neomycin>gentamicin>streptomycin=spermidine) correlated roughly with their ability to induce PTP-independent release of SIMP, which suggests that the binding of polycations to the anionic phospholipids of the outer mitochondrial membrane facilitates the rupture of this membrane. However, some polycations facilitated the induction of PTP, possibly by binding to cardiolipin on the inner membrane. This dual mechanism may be relevant to the induction of SIMP release in apoptosis.     

Binding of magnesium by proteins of the mitochondrial intermembrane compartment

The intermembrane compartment of rat liver mitochondria contains high molecular weight compounds, most likely proteins, complexing magnesium ions. Compound/s/ of about 150 000 daltons has a low affinity /K_d 0.37 mM/ and a high binding capacity of 300 ng atoms Mg/mg protein; compound/s/ of about 100 000 daltons is characterized by a very high affinity towards magnesium /K_d of the order of magnitude of 0.001 mM/ and a low capacity of about 20 ng atoms Mg/mg protein.