OMA1 protease eliminates arrested protein import intermediates upon mitochondrial depolarization.

Most mitochondrial proteins originate from the cytosol and require transport into the organelle. Such precursor proteins must be unfolded to pass through translocation channels in mitochondrial membranes. Misfolding of transported proteins can result in their arrest and translocation failure. Arrested proteins block further import, disturbing mitochondrial functions and cellular proteostasis. Cellular responses to translocation failure have been defined in yeast. We developed the cell line-based translocase clogging model to discover molecular mechanisms that resolve failed import events in humans. The mechanism we uncover differs significantly from these described in fungi, where ATPase-driven extraction of blocked protein is directly coupled with proteasomal processing. We found human cells to rely primarily on mitochondrial factors to clear translocation channel blockage. The mitochondrial membrane depolarization triggered proteolytic cleavage of the stalled protein, which involved mitochondrial protease OMA1. The cleavage allowed releasing the protein fragment that blocked the translocase. The released fragment was further cleared in the cytosol by VCP/p97 and the proteasome.

The consequence of ATP synthase dimer angle on mitochondrial morphology studied by cryo-electron tomography.

Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans and show that the dimer angle differs from previously determined structures. The consequences of this species-specific difference at the dimer interface were investigated by comparing C. elegans and S. cerevisiae mitochondrial morphology. We reveal that C. elegans has a larger ATP synthase dimer angle with more lamellar (flatter) cristae when compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling. A comparison of our C. elegans model to an existing S. cerevisiae structure reveals the presence of extensions and rearrangements in C. elegans subunits associated with maintaining the dimer interface. We speculate that increasing dimer angles could provide an advantage for species that inhabit variable-oxygen environments by forming flatter more energetically efficient cristae.

Mechanisms of stress management in mitochondrial protein import.

Mitochondria are vital to the functions of eukaryotic cells. Most mitochondrial proteins are transported into the organelle following their synthesis by cytoplasmic ribosomes. However, precise protein targeting is complex because the two diverse lipid membranes encase mitochondria. Efficient protein translocation across membranes and accurate sorting to specific sub-compartments require the cooperation of multiple factors. Any failure in mitochondrial protein import can disrupt organelle fitness. Proteins intended for mitochondria make up a significant portion of all proteins produced in the cytosol. Therefore, import defects causing their mislocalization can significantly stress cellular protein homeostasis. Recognition of this phenomenon has increased interest in molecular mechanisms that respond to import-related stress and restore proteostasis, which is the focus of this review. Significantly, disruptions in protein homeostasis link strongly to the pathology of several degenerative disorders highly relevant in ageing societies. A comprehensive understanding of protein import quality control will allow harnessing this machinery in therapeutic approaches.

Cytosolic Quality Control of Mitochondrial Protein Precursors-The Early Stages of the Organelle Biogenesis.

With few exceptions, proteins that constitute the proteome of mitochondria originate outside of this organelle in precursor forms. Such protein precursors follow dedicated transportation paths to reach specific parts of mitochondria, where they complete their maturation and perform their functions. Mitochondrial precursor targeting and import pathways are essential to maintain proper mitochondrial function and cell survival, thus are tightly controlled at each stage. Mechanisms that sustain protein homeostasis of the cytosol play a vital role in the quality control of proteins targeted to the organelle. Starting from their synthesis, precursors are constantly chaperoned and guided to reduce the risk of premature folding, erroneous interactions, or protein damage. The ubiquitin-proteasome system provides proteolytic control that is not restricted to defective proteins but also regulates the supply of precursors to the organelle. Recent discoveries provide evidence that stress caused by the mislocalization of mitochondrial proteins may contribute to disease development. Precursors are not only subject to regulation but also modulate cytosolic machinery. Here we provide an overview of the cellular pathways that are involved in precursor maintenance and guidance at the early cytosolic stages of mitochondrial biogenesis. Moreover, we follow the circumstances in which mitochondrial protein import deregulation disturbs the cellular balance, carefully looking for rescue paths that can restore proteostasis.

Motor recruitment to the TIM23 channel's lateral gate restricts polypeptide release into the inner membrane.

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) facilitates anterograde precursor transport into the matrix and lateral release of precursors with stop-transfer signal into the membrane (sorting). Sorting requires precursor exit from the translocation channel into the lipid phase through the lateral gate of the TIM23 complex. How the two transport modes are regulated and balanced against each other is unknown. Here we show that the import motor J-protein Pam18, which is essential for matrix import, controls lateral protein release into the lipid bilayer. Constitutively translocase-associated Pam18 obstructs lateral precursor transport. Concomitantly, Mgr2, implicated in precursor quality control, is displaced from the translocase. We conclude that during motor-dependent matrix protein transport, the transmembrane segment of Pam18 closes the lateral gate to promote anterograde polypeptide movement. This finding explains why a motor-free form of the translocase facilitates the lateral movement of precursors with a stop-transfer signal.

Determinants of the cytosolic turnover of mitochondrial intermembrane space proteins.

BACKGROUND: The proteome of mitochondria comprises mostly proteins that originate as precursors in the cytosol. Before import into the organelle, such proteins are exposed to cytosolic quality control mechanisms. Multiple lines of evidence indicate a significant contribution of the major cytosolic protein degradation machinery, the ubiquitin-proteasome system, to the quality control of mitochondrial proteins. Proteins that are directed to the mitochondrial intermembrane space (IMS) exemplify an entire class of mitochondrial proteins regulated by proteasomal degradation. However, little is known about how these proteins are selected for degradation. RESULTS: The present study revealed the heterogeneous cytosolic stability of IMS proteins. Using a screening approach, we found that different cytosolic factors are responsible for the degradation of specific IMS proteins, with no single common factor involved in the degradation of all IMS proteins. We found that the Cox12 protein is rapidly degraded when localized to the cytosol, thus providing a sensitive experimental model. Using Cox12, we found that lysine residues but not conserved cysteine residues are among the degron features important for protein ubiquitination. We observed the redundancy of ubiquitination components, with significant roles of Ubc4 E2 ubiquitin-conjugating enzyme and Rsp5 E3 ubiquitin ligase. The amount of ubiquitinated Cox12 was inversely related to mitochondrial import efficiency. Importantly, we found that precursor protein ubiquitination blocks its import into mitochondria. CONCLUSIONS: The present study confirms the involvement of ubiquitin-proteasome system in the quality control of mitochondrial IMS proteins in the cytosol. Notably, ubiquitination of IMS proteins prohibits their import into mitochondria. Therefore, ubiquitination directly affects the availability of precursor proteins for organelle biogenesis. Importantly, despite their structural similarities, IMS proteins are not selected for degradation in a uniform way. Instead, specific IMS proteins rely on discrete components of the ubiquitination machinery to mediate their clearance by the proteasome.

Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography.

We employed electron cryo-tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation-arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.

Control of mitochondrial biogenesis and function by the ubiquitin-proteasome system.

Mitochondria are pivotal organelles in eukaryotic cells. The complex proteome of mitochondria comprises proteins that are encoded by nuclear and mitochondrial genomes. The biogenesis of mitochondrial proteins requires their transport in an unfolded state with a high risk of misfolding. The mislocalization of mitochondrial proteins is deleterious to the cell. The electron transport chain in mitochondria is a source of reactive oxygen species that damage proteins. Mitochondrial dysfunction is linked to many pathological conditions and, together with the loss of cellular protein homeostasis (proteostasis), are hallmarks of ageing and ageing-related degeneration diseases. The pathogenesis of neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, has been associated with mitochondrial and proteostasis failure. Thus, mitochondrial proteins require sophisticated surveillance mechanisms. Although mitochondria form a proteasome-exclusive compartment, multiple lines of evidence indicate a crucial role for the cytosolic ubiquitin-proteasome system (UPS) in the quality control of mitochondrial proteins. The proteasome affects mitochondrial proteins at stages of their biogenesis and maturity. The effects of the UPS go beyond the removal of damaged proteins and include the adjustment of mitochondrial proteome composition, the regulation of organelle dynamics and the protection of cellular homeostasis against mitochondrial failure. In turn, mitochondrial activity and mitochondrial dysfunction adjust the activity of the UPS, with implications at the cellular level.

Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol.

Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction.

Retro-translocation of mitochondrial intermembrane space proteins.

The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.

A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the intermembrane space via the redox-driven mitochondrial intermembrane space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

The ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins.

Mitochondrial precursor proteins are synthesized in the cytosol and subsequently imported into mitochondria. The import of mitochondrial intermembrane space proteins is coupled with their oxidative folding and governed by the mitochondrial intermembrane space import and assembly (MIA) pathway. The cytosolic steps that precede mitochondrial import are not well understood. We identified a role for the ubiquitin-proteasome system in the biogenesis of intermembrane space proteins. Interestingly, the function of the ubiquitin-proteasome system is not restricted to conditions of mitochondrial protein import failure. The ubiquitin-proteasome system persistently removes a fraction of intermembrane space proteins under physiological conditions, acting as a negative regulator in the biogenesis of this class of proteins. Thus, the ubiquitin-proteasome system plays an important role in determining the levels of proteins targeted to the intermembrane space of mitochondria.

In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins.

The intermembrane space of mitochondria accommodates the essential mitochondrial intermembrane space assembly (MIA) machinery that catalyzes oxidative folding of proteins. The disulfide bond formation pathway is based on a relay of reactions involving disulfide transfer from the sulfhydryl oxidase Erv1 to Mia40 and from Mia40 to substrate proteins. However, the substrates of the MIA typically contain two disulfide bonds. It was unclear what the mechanisms are that ensure that proteins are released from Mia40 in a fully oxidized form. In this work, we dissect the stage of the oxidative folding relay, in which Mia40 binds to its substrate. We identify dynamics of the Mia40-substrate intermediate complex. Our experiments performed in a native environment, both in organello and in vivo, show that Erv1 directly participates in Mia40-substrate complex dynamics by forming a ternary complex. Thus Mia40 in cooperation with Erv1 promotes the formation of two disulfide bonds in the substrate protein, ensuring the efficiency of oxidative folding in the intermembrane space of mitochondria.

The MIA pathway: a tight bond between protein transport and oxidative folding in mitochondria.

Many newly synthesized proteins obtain disulfide bonds in the bacterial periplasm, the endoplasmic reticulum (ER) and the mitochondrial intermembrane space. The acquisition of disulfide bonds is critical for the folding, assembly and activity of these proteins. Spontaneous oxidation of thiol groups is inefficient in vivo, therefore cells have developed machineries that catalyse the oxidation of substrate proteins. The identification of the machinery that mediates this process in the intermembrane space of mitochondria, known as MIA (mitochondrial intermembrane space assembly), provided a unique mechanism of protein transport. The MIA machinery introduces disulfide bonds into incoming intermembrane space precursors and thus tightly couples the process of precursor translocation to precursor oxidation. We discuss our current understanding of the MIA pathway and the mechanisms that oversee thiol-exchange reactions in mitochondria.

A high density consensus map of rye (Secale cereale L.) based on DArT markers.

BACKGROUND: Rye (Secale cereale L.) is an economically important crop, exhibiting unique features such as outstanding resistance to biotic and abiotic stresses and high nutrient use efficiency. This species presents a challenge to geneticists and breeders due to its large genome containing a high proportion of repetitive sequences, self incompatibility, severe inbreeding depression and tissue culture recalcitrance. The genomic resources currently available for rye are underdeveloped in comparison with other crops of similar economic importance. The aim of this study was to create a highly saturated, multilocus linkage map of rye via consensus mapping, based on Diversity Arrays Technology (DArT) markers. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant inbred lines (RILs) from 5 populations (564 in total) were genotyped using DArT markers and subjected to linkage analysis using Join Map 4.0 and Multipoint Consensus 2.2 software. A consensus map was constructed using a total of 9703 segregating markers. The average chromosome map length ranged from 199.9 cM (2R) to 251.4 cM (4R) and the average map density was 1.1 cM. The integrated map comprised 4048 loci with the number of markers per chromosome ranging from 454 for 7R to 805 for 4R. In comparison with previously published studies on rye, this represents an eight-fold increase in the number of loci placed on a consensus map and a more than two-fold increase in the number of genetically mapped DArT markers. CONCLUSIONS/SIGNIFICANCE: Through the careful choice of marker type, mapping populations and the use of software packages implementing powerful algorithms for map order optimization, we produced a valuable resource for rye and triticale genomics and breeding, which provides an excellent starting point for more in-depth studies on rye genome organization.

Comprehensive analysis of the palindromic motif TCTCGCGAGA: a regulatory element of the HNRNPK promoter.

Definitive identification of promoters, their cis-regulatory motifs, and their trans-acting proteins requires experimental analysis. To define the HNRNPK promoter and its cognate DNA-protein interactions, we performed a comprehensive study combining experimental approaches, including luciferase reporter gene assays, chromatin immunoprecipitations (ChIP), electrophoretic mobility shift assays (EMSA), and mass spectrometry (MS). We discovered that out of the four potential HNRNPK promoters tested, the one containing the palindromic motif TCTCGCGAGA exhibited the highest activity in a reporter system assay. Although further EMSA and MS analyses, performed to uncover the identity of the palindrome-binding transcription factor, did identify a complex of DNA-binding proteins, neither method unambiguously identified the pertinent direct trans-acting protein(s). ChIP revealed similar chromatin states at the promoters with the palindromic motif and at housekeeping gene promoters. A ChIP survey showed significantly higher recruitment of PARP1, a protein identified by MS as ubiquitously attached to DNA probes, within heterochromatin sites. Computational analyses indicated that this palindrome displays features that mark nucleosome boundaries, causing the surrounding DNA landscape to be constitutively open. Our strategy of diverse approaches facilitated the direct characterization of various molecular properties of HNRNPK promoter bearing the palindromic motif TCTCGCGAGA, despite the obstacles that accompany in vitro methods.

Limited clinical relevance of mitochondrial DNA mutation and gene expression analyses in ovarian cancer.

BACKGROUND: In recent years, numerous studies have investigated somatic mutations in mitochondrial DNA in various tumours. The observed high mutation rates might reflect mitochondrial deregulation; consequently, mutation analyses could be clinically relevant. The purpose of this study was to determine if mutations in the mitochondrial D-loop region and/or the level of mitochondrial gene expression could influence the clinical course of human ovarian carcinomas. METHODS: We sequenced a 1320-base-pair DNA fragment of the mitochondrial genome (position 16,000-750) in 54 cancer samples and in 44 corresponding germline control samples. In addition, six transcripts (MT-ATP6, MT-CO1, MT-CYB, MT-ND1, MT-ND6, and MT-RNR1) were quantified in 62 cancer tissues by real-time RT-PCR. RESULTS: Somatic mutations in the D-loop sequence were found in 57% of ovarian cancers. Univariate analysis showed no association between mitochondrial DNA mutation status or mitochondrial gene expression and any of the examined clinicopathologic parameters. A multivariate logistic regression model revealed that the expression of the mitochondrial gene RNR1 might be used as a predictor of tumour sensitivity to chemotherapy. CONCLUSION: In contrast to many previously published papers, our study indicates rather limited clinical relevance of mitochondrial molecular analyses in ovarian carcinomas. These discrepancies in the clinical utility of mitochondrial molecular tests in ovarian cancer require additional large, well-designed validation studies.

Expression of genes encoding mitochondrial proteins can distinguish nonalcoholic steatosis from steatohepatitis.

In patients without substantial alcohol use, triglyceride accumulation in the liver can lead to nonalcoholic fatty liver disease (NAFLD) that may progress to nonalcoholic steatohepatitis (NASH). The differential diagnosis between NAFLD and NASH can be accomplished only by morphological examination. Although the relationship between mitochondrial dysfunction and the progression of liver pathologic changes has been described, the exact mechanisms initiating primary liver steatosis and its progression to NASH are unknown. We selected 16 genes encoding mitochondrial proteins which expression was compared by quantitative RT-PCR in liver tissue samples taken from patients with NAFLD and NASH. We found that 6 of the 16 examined genes were differentially expressed in NAFLD versus NASH patients. The expression of hepatic HK1, UCP2, ME2, and ME3 appeared to be higher in NASH than in NAFLD patients, whereas HMGCS2 and hnRNPK expression was lower in NASH patients. Although the severity of liver morphological injury in the spectrum of NAFLD-NASH may be defined at the molecular level, expression of these selected 6 genes cannot be used as a molecular marker aiding histological examination. Moreover, it is still unclear whether these differences in hepatic gene expression profiles truly reflect the progression of morphological abnormalities or rather indicate various metabolic and hormonal states in patients with different degrees of fatty liver disease.

Molecular defense mechanisms of Barrett's metaplasia estimated by an integrative genomics.

Barrett's esophagus is characterized by the replacement of squamous epithelium with specialized intestinal metaplastic mucosa. The exact mechanisms of initiation and development of Barrett's metaplasia remain unknown, but a hypothesis of "successful adaptation" against noxious reflux components has been proposed. To search for the repertoire of adaptation mechanisms of Barrett's metaplasia, we employed high-throughput functional genomic and proteomic methods that defined the molecular background of metaplastic mucosa resistance to reflux. Transcriptional profiling was established for 23 pairs of esophageal squamous epithelium and Barrett's metaplasia tissue samples using Affymetrix U133A 2.0 GeneChips and validated by quantitative real-time polymerase chain reaction. Differences in protein composition were assessed by electrophoretic and mass-spectrometry-based methods. Among 2,822 genes differentially expressed between Barrett's metaplasia and squamous epithelium, we observed significantly overexpressed metaplastic mucosa genes that encode cytokines and growth factors, constituents of extracellular matrix, basement membrane and tight junctions, and proteins involved in prostaglandin and phosphoinositol metabolism, nitric oxide production, and bioenergetics. Their expression likely reflects defense and repair responses of metaplastic mucosa, whereas overexpression of genes encoding heat shock proteins and several protein kinases in squamous epithelium may reflect lower resistance of normal esophageal epithelium than Barrett's metaplasia to reflux components. Despite the methodological and interpretative difficulties in data analyses discussed in this paper, our studies confirm that Barrett's metaplasia may be regarded as a specific microevolution allowing for accumulation of mucosal morphological and physiological changes that better protect against reflux injury.