Intermembrane Space Proteins Research Articles

Peroxisome Proliferator-activated Receptor γ (PPARγ) Mediates a Ski Oncogene-induced Shift from Glycolysis to Oxidative Energy Metabolism

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

Estrogen receptor mediates a distinct mitochondrial unfolded protein response.

Unfolded protein responses (UPRs) of the endoplasmic reticulum and mitochondrial matrix have been described. Here, we show that the accumulation of proteins in the inter-membrane space (IMS) of mitochondria in the breast cancer cell line MCF-7 activates a distinct UPR. Upon IMS stress, overproduction of reactive oxygen species (ROS) and phosphorylation of AKT triggers estrogen receptor (ER) activity, which further upregulates the transcription of the mitochondrial regulator NRF1 and the IMS protease OMI (officially known as HTRA2). Moreover, we demonstrate that the IMS stress-induced UPR culminates in increased proteasome activity. Given our previous report on a proteasome- and OMI-dependent checkpoint that limits the import of IMS proteins, the findings presented in this study suggest that this newly discovered UPR acts as a cytoprotective response to overcome IMS stress.

Dimerization of Smac is crucial for its mitochondrial retention by XIAP subsequent to mitochondrial outer membrane permeabilization

Following the apoptotic permeabilization of the outer mitochondrial membrane, the inter-membrane space protein second mitochondria-derived activator of caspases (Smac) is released into the cytosol. Smac efficiently promotes apoptosis by antagonizing x-linked inhibitor of apoptosis protein (XIAP), an inhibitor of caspases-9, -3, and -7, via a short NH2-terminal inhibitor of apoptosis protein (IAP) binding motif (AVPI). Native Smac dimerizes to form a highly stable and inflexible elongated arch, however, a functional role for this outstretched structure so far remained unknown. Using time-lapse single-cell imaging of DLD-1 and HCT-116 colon cancer cells, we here demonstrate that upon mitochondrial outer membrane permeabilization physiological expression levels of XIAP are sufficient to selectively prolong the release of dimeric but not monomeric Smac. Elevating the expression of XIAP further extended the release duration of dimeric Smac and resulted in the mitochondrial retention of a significant proportion of the Smac pool. In contrast, monomeric Smac was always fully released and the release kinetics were not affected by altered XIAP expression. Our findings therefore indicate that the dimerization of Smac is critical for the XIAP-mediated retention of Smac at or inside the mitochondria.

Effect of Ionic Strength on Bax:TBID Mediated Mitochondrial Outer Membrane Permeabilization (MOMP)

Fluctuations in ionic strength have been documented in apoptosis. While the exact molecular identity of the channels through which proteins permeate across the outer mitochondrial membrane is unclear, it is fairly certain that the channel is large in size and sustained over time. We sought to determine the effect of salt concentration on Bax and tBid in inducing MOMP. While high salt buffer (60mM) facilitated more release of inter-membrane space (IMS) proteins (Adenylate Kinase and Sulfite Oxidase) with Bax and tBid than low salt (5mM HEPES buffer), low salt conditions induced more sustained, real-time permeabilization, as measured by accessibility of exogenous cytochrome c to complex IV. There was a dichotomy between the kinetics of IMS protein release and real-time permeabilization at high salt conditions, while under low salt conditions, they were congruent. The effect of ionic strength on sustained permeabilization is biphasic, with maxima at 10mM. The sustained permeabilization is inducible by diluting the salt concentration to that of low salt, but irreversible by incorporating high salt conditions. This observation is inconsistent with a dynamic channel model sensitive to ionic strength. It seems likely that under low salt conditions, there is a co-operative interaction between Bax molecules in the membrane and those in solution, but the growth of the channel by membrane-bound Bax molecules itself is independent of ionic strength. Supported by a grant from NSF (MCB-0641208).

A novel mitochondrial outer membrane protein, MOMA-1, that affects cristae morphology inCaenorhabditis elegans

Three proteins with similar effects on mitochondrial morphology were identified in an RNA interference (RNAi) screen for mitochondrial abnormalities in Caenorhabditis elegans. One of these is the novel mitochondrial outer membrane protein MOMA-1. The second is the CHCHD3 homologue, CHCH-3, a small intermembrane space protein that may act as a chaperone. The third is a mitofilin homologue, IMMT-1. Mitofilins are inner membrane proteins that control the shapes of cristae. RNAi or mutations in each of these genes change the relatively constant diameters of mitochondria into highly variable diameters, ranging from thin tubes to localized swellings. Neither growth nor brood size of the moma-1, chch-3, or immt-1 single mutants is affected, suggesting that their metabolic functions are normal. However, growth of moma-1 or immt-1 mutants on chch-3(RNAi) leads to withered gonads, a lack of mitochondrial staining, and a dramatic reduction in fecundity, while moma-1; immt-1 double mutants are indistinguishable from single mutants. Mutations in moma-1 and immt-1 also have similar effects on cristae morphology. We conclude that MOMA-1 and IMMT-1 act in the same pathway. It is likely that the observed effects on mitochondrial diameter are an indirect effect of disrupting cristae morphology.

Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation.

Structural Basis for the Disulfide Relay System in the Mitochondrial Intermembrane Space

Mitochondria contain two biological membranes. Although reducing agents can diffuse from the cytosol into the intermembrane space (IMS) between the outer and inner mitochondrial membranes, the IMS has a dedicated disulfide relay system to introduce disulfide bonds into mainly small and soluble proteins. This system consists of two essential proteins, a disulfide carrier Tim40/Mia40 and a flavin-dependent sulfhydryl oxidase Erv1, high-resolution structures that have recently become available. Tim40/Mia40 transfers disulfide bonds to newly imported IMS proteins by dithiol/disulfide exchange reactions involving mixed disulfide intermediates. Tight folding by introduction of disulfide bonds prevents egress of these small IMS proteins, resulting in their selective retention in the compartment. After disulfide transfer from Tim40/Mia40 to substrate proteins, Tim40/Mia40 is reoxidized again by Erv1, which is then oxidized by electron transfer to either cytochrome c or molecular oxygen. Here we review the recent advancement of the knowledge on the mechanism of the disulfide relay system in the mitochondrial IMS, especially shedding light on the structural aspects of its components.

Import, Maturation, and Function of SOD1 and Its Copper Chaperone CCS in the Mitochondrial Intermembrane Space

Cu, Zn, superoxide dismutase (SOD1) is a ubiquitous enzyme localized in multiple cellular compartments, including mitochondria, where it concentrates in the intermembrane space (IMS). Similar to other small IMS proteins, the import and retention of SOD1 in the IMS is linked to its folding and maturation, involving the formation of critical intra- and intermolecular disulfide bonds. Therefore, the cysteine residues of SOD1 play a fundamental role in its IMS localization. IMS import of SOD1 involves its copper chaperone, CCS, whose mitochondrial distribution is regulated by the Mia40/Erv1 disulfide relay system in a redox-dependent manner: CCS promotes SOD1 maturation and retention in the IMS. The function of SOD1 in the IMS is still unknown, but it is plausible that it serves to remove superoxide released from the mitochondrial respiratory chain. Mutations in SOD1 cause familial amyotrophic lateral sclerosis (ALS), whose pathologic features include mitochondrial bioenergetic dysfunction. Mutant SOD1 localization in the IMS is not dictated by oxygen concentration and the Mia40/Erv1 system, but is primarily dependent on aberrant protein folding and aggregation. Mutant SOD1 localization and aggregation in the IMS might cause the mitochondrial abnormalities observed in familial ALS and could play a significant role in disease pathogenesis.

Plasma Membrane Ca2+-ATPase Overexpression Depletes Both Mitochondrial and Endoplasmic Reticulum Ca2+ Stores and Triggers Apoptosis in Insulin-secreting BRIN-BD11 Cells

Ca(2+) may trigger apoptosis in β-cells. Hence, the control of intracellular Ca(2+) may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca(2+)-ATPase (PMCA) overexpression on Ca(2+)-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca(2+)] using a combination of aequorins with different Ca(2+) affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. Overexpression of PMCA decreased [Ca(2+)] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca(2+)] in the cytosol and subcellular heterogeneities in [Ca(2+)] among mitochondria. PMCA overexpression depletes intracellular [Ca(2+)] stores and, despite a decrease in mitochondrial [Ca(2+)], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca(2+) homeostasis that could decrease β-cell apoptosis in diabetes.

Ups delivery to the intermembrane space of mitochondria: a novel affinity-driven protein import pathway

Mitochondria are comprised of two aqueous subcompartments: the matrix and the intermembrane space (IMS). Following their synthesis on cytosolic ribosomes, proteins destined to the matrix are threaded by N‐terminal matrix‐targeting sequences through TOM and TIM translocases in the outer and the inner membrane of the organelle. Proteins of the IMS often lack such presequences, and the principles mediating their uptake into mitochondria are diverse and poorly understood. In this issue, [Potting et al (2010)][1] and [Tamura et al (2010)][2] show that Ups proteins, a group of soluble IMS proteins critical for lipid homeostasis, adsorb to the IMS factor Mdm35 after their translocation across the outer membrane. Mdm35 thereby apparently functions as an intramitochondrial acceptor protein. The existence of affinity‐driven import routes has been proposed before, but Mdm35 represents the first intramitochondrial acceptor protein that is identified. [1]: #ref-9 [2]: #ref-12

Mitochondria and cell death: outer membrane permeabilization and beyond

Mitochondrial outer membrane permeabilization (MOMP) is often required for activation of the caspase proteases that cause apoptotic cell death. Various intermembrane space (IMS) proteins, such as cytochrome c, promote caspase activation following their mitochondrial release. As a consequence, mitochondrial outer membrane integrity is highly controlled, primarily through interactions between pro- and anti-apoptotic members of the B cell lymphoma 2 (BCL-2) protein family. Following MOMP by pro-apoptotic BCL-2-associated X protein (BAX) or BCL-2 antagonist or killer (BAK), additional regulatory mechanisms govern the mitochondrial release of IMS proteins and caspase activity. MOMP typically leads to cell death irrespective of caspase activity by causing a progressive decline in mitochondrial function, although cells can survive this under certain circumstances, which may have pathophysiological consequences.

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, mdm35Delta cells displayed a similar phenotype to ups1Deltaups2Deltaups3Delta 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 mdm35Delta mitochondria failed to restore phospholipid metabolism. These findings provide a novel mechanism in which the formation of functional protein complexes drives mitochondrial protein import.

Mitochondria in cell death

Apoptosis can be thought of as a signalling cascade that results in the death of the cell. Properly executed apoptosis is critically important for both development and homoeostasis of most animals. Accordingly, defects in apoptosis can contribute to the development of autoimmune disorders, neurological diseases and cancer. Broadly speaking, there are two main pathways by which a cell can engage apoptosis: the extrinsic apoptotic pathway and the intrinsic apoptotic pathway. At the centre of the intrinsic apoptotic signalling pathway lies the mitochondrion, which, in addition to its role as the bioenergetic centre of the cell, is also the cell's reservoir of pro-death factors which reside in the mitochondrial IMS (intermembrane space). During intrinsic apoptosis, pores are formed in the OMM (outer mitochondrial membrane) of the mitochondria in a process termed MOMP (mitochondrial outer membrane permeabilization). This allows for the release of IMS proteins; once released during MOMP, some IMS proteins, notably cytochrome c and Smac/DIABLO (Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI), promote caspase activation and subsequent cleavage of structural and regulatory proteins in the cytoplasm and the nucleus, leading to the demise of the cell. MOMP is achieved through the co-ordinated actions of pro-apoptotic members and inhibited by anti-apoptotic members of the Bcl-2 family of proteins. Other aspects of mitochondrial physiology, such as mitochondrial bioenergetics and dynamics, are also involved in processes of cell death that proceed through the mitochondria. Proper regulation of these mitochondrial functions is vitally important for the life and death of the cell and for the organism as a whole.

The Conserved Mitochondrial Twin Cx9C Protein Cmc2 Is a Cmc1 Homologue Essential for Cytochrome c Oxidase Biogenesis

Mitochondrial copper metabolism and delivery to cytochrome c oxidase and mitochondrially localized CuZn-superoxide dismutase (Sod1) requires a growing number of intermembrane space proteins containing a twin Cx(9)C motif. Among them, Cmc1 was recently identified by our group. Here we describe another conserved mitochondrial metallochaperone-like protein, Cmc2, a close homologue of Cmc1, whose function affects both cytochrome c oxidase and Sod1. In the yeast Saccharomyces cerevisiae, Cmc2 localizes to the mitochondrial inner membrane facing the intermembrane space. In the absence of Cmc2, cytochrome c oxidase activity measured spectrophotometrically and cellular respiration measured polarographically are undetectable. Additionally, mutant cmc2 cells display 2-fold increased mitochondrial Sod1 activity, whereas CMC2 overexpression results in Sod1 activity decreased to 60% of wild-type levels. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vice versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. Cmc2 function is conserved from yeast to humans. Human CMC2 localizes to the mitochondria and CMC2 expression knockdown produces cytochrome c oxidase deficiency in Caenorhabditis elegans. We conclude that Cmc1 and Cmc2 have cooperative but nonoverlapping functions in cytochrome c oxidase biogenesis.

Caspase-9 Activation by the Apoptosome Is Not Required for Fas-mediated Apoptosis in Type II Jurkat Cells

Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-x(L) were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-x(L)-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells.

Mammalian Oxa1 protein is useful for assessment of submitochondrial protein localization and mitochondrial membrane integrity

Taking advantage of the unique topology of oxidase assembly 1 (Oxa1) protein, a mitochondrial inner membrane protein with N (intermembrane space)–C (matrix) orientation, we explored the usefulness of the protein as a marker for submitochondrial protein localization. Mammalian Oxa1 protein exhibited different proteolytic patterns depending on mitochondrial membrane integrity, and in mitochondria with a disrupted outer membrane and outer and inner membranes, the proteolytic patterns of Oxa1 protein were consistent with those of mitochondrial intermembrane space and matrix marker proteins, respectively, suggesting that Oxa1 protein, a single molecule, can serve as a versatile submitochondrial localization marker that doubles as a membrane integrity marker.

Mitochondrial ‘kiss-and-run’: interplay between mitochondrial motility and fusion–fission dynamics

Visualizing mitochondrial fusion in real time, we identified two classes of fusion events in mammalian cells. In addition to complete fusion, we observed transient fusion events, wherein two mitochondria came into close apposition, exchanged soluble inter-membrane space and matrix proteins, and re-separated, preserving the original morphology. Transient fusion exhibited rapid kinetics of the sequential and separable mergers of the outer and inner membranes, but allowed only partial exchange of integral membrane proteins. When the inner membrane fusion protein Opa1 level was lowered or was greatly elevated, transient fusions could occur, whereas complete fusions disappeared. Furthermore, transient fusions began from oblique or lateral interactions of mitochondria associated with separate microtubules, whereas complete fusions resulted from longitudinal merging of organelles travelling along a single microtubule. In contrast to complete fusion, transient fusions both required and promoted mitochondrial motility. Transient fusions were also necessary and sufficient to support mitochondrial metabolism. Thus, Opa1 expression and cytoskeletal anchorage govern a novel form of fusion that has a distinct function in mitochondrial maintenance.

Disulphide Bond Formation in the Intermembrane Space of Mitochondria

Proteins of the intermembrane space (IMS) of mitochondria fulfil crucial functions in cellular processes, such as transport of proteins and metal ions, ATP production and apoptotic cell death. All IMS proteins are synthesized in the cytosol and then transported across the mitochondrial outer membrane. A subset of these proteins contains disulphide bonds. For their import into the IMS, they employ a disulphide relay system, made up of two essential proteins, Mia40/Tim40 and the flavin-dependent sulfhydryl-electron transferase Erv1. The disulphide relay system introduces disulphide bonds in substrate proteins triggering their folding. The oxidative folding traps substrates in the IMS and thereby drives their net import into the IMS. Thus, protein import is coupled to oxidative protein folding, maybe providing a first control of protein quality. Here, we review the current knowledge about the Erv1-Mia40 system and address aspects that require further consideration.

Ups1p and Ups2p antagonistically regulate cardiolipin metabolism in mitochondria

Cardiolipin, a unique phospholipid composed of four fatty acid chains, is located mainly in the mitochondrial inner membrane (IM). Cardiolipin is required for the integrity of several protein complexes in the IM, including the TIM23 translocase, a dynamic complex which mediates protein import into the mitochondria through interactions with the import motor presequence translocase–associated motor (PAM). In this study, we report that two homologous intermembrane space proteins, Ups1p and Ups2p, control cardiolipin metabolism and affect the assembly state of TIM23 and its association with PAM in an opposing manner. In ups1Δ mitochondria, cardiolipin levels were decreased, and the TIM23 translocase showed altered conformation and decreased association with PAM, leading to defects in mitochondrial protein import. Strikingly, loss of Ups2p restored normal cardiolipin levels and rescued TIM23 defects in ups1Δ mitochondria. Furthermore, we observed synthetic growth defects in ups mutants in combination with loss of Pam17p, which controls the integrity of PAM. Our findings provide a novel molecular mechanism for the regulation of cardiolipin metabolism.

GABAergic striatal neurons exhibit caspase‐independent, mitochondrially mediated programmed cell death

GABAergic striatal neurons are compromised in basal ganglia pathologies and we analysed how insult nature determined their patterns of injury and recruitment of the intrinsic mitochondrial pathway during programmed cell death (PCD). Stressors affecting targets implicated in striatal neurodegeneration [3-morpholinylsydnoneimine (SIN-1), 3-nitropropionic acid (3-NP), NMDA, 3,5-dihydroxyphenylglycine (DHPG), and staurosporine (STS)] were compared in cultured GABAergic neurons from murine striatum by analyzing the progression of injury and its correlation with mitochondrial involvement, the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Stressors produced PCD exhibiting slow-onset kinetics with time-dependent annexin-V labeling and eventual DNA fragmentation. IMS proteins including cytochrome c were differentially distributed, although stressors except STS produced early redistribution of apoptosis-inducing factor and Omi, suggestive of early recruitment of both caspase-dependent and caspase-independent signaling. In general, Bax mobilization to mitochondria appeared to promote IMS protein redistribution. Caspase 3 activation was prominent after STS, whereas NMDA and SIN-1 produced mainly calpain activation, and 3-NP and DHPG elicited a mixed profile of protease activation. PCD and redistribution of IMS proteins in striatal GABAergic neurons were canonical and insult-dependent, reflecting differential interplay between the caspase cascade and alternate cell death pathways.