Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation.

The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of ß-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the ß-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring ß-barrel formation in vivo and in organello and demonstrated that the ß-barrel was formed and membrane inserted while the precursor was bound to SAM. ß-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.

Suppressing toxic aggregates: let MIA do it!

Mitochondrial activity is becoming an inherent aspect of cellular protein homeostasis (proteostasis). In this issue, Schlagowski et al (2021) report on the attractive notion that modulating mitochondrial protein import activity stimulates protein aggregate clearance in the cytosol, thereby affecting cytosolic proteostasis and its collapse observed in neurodegenerative diseases.

Proteasome activity contributes to pro-survival response upon mild mitochondrial stress in Caenorhabditis elegans.

Defects in mitochondrial function activate compensatory responses in the cell. Mitochondrial stress that is caused by unfolded proteins inside the organelle induces a transcriptional response (termed the "mitochondrial unfolded protein response" [UPRmt]) that is mediated by activating transcription factor associated with stress 1 (ATFS-1). The UPRmt increases mitochondrial protein quality control. Mitochondrial dysfunction frequently causes defects in the import of proteins, resulting in the accumulation of mitochondrial proteins outside the organelle. In yeast, cells respond to mistargeted mitochondrial proteins by increasing activity of the proteasome in the cytosol (termed the "unfolded protein response activated by mistargeting of proteins" [UPRam]). The presence and relevance of this response in higher eukaryotes is unclear. Here, we demonstrate that defects in mitochondrial protein import in Caenorhabditis elegans lead to proteasome activation and life span extension. Both proteasome activation and life span prolongation partially depend on ATFS-1, despite its lack of influence on proteasomal gene transcription. Importantly, life span prolongation depends on the fully assembled proteasome. Our data provide a link between mitochondrial dysfunction and proteasomal activity and demonstrate its direct relevance to mechanisms that promote longevity.

Importing mitochondrial proteins: machineries and mechanisms.

Most mitochondrial proteins are synthesized on cytosolic ribosomes and must be imported across one or both mitochondrial membranes. There is an amazingly versatile set of machineries and mechanisms, and at least four different pathways, for the importing and sorting of mitochondrial precursor proteins. The translocases that catalyze these processes are highly dynamic machines driven by the membrane potential, ATP, or redox reactions, and they cooperate with molecular chaperones and assembly complexes to direct mitochondrial proteins to their correct destinations. Here, we discuss recent insights into the importing and sorting of mitochondrial proteins and their contributions to mitochondrial biogenesis.

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.

Muscle-derived exophers promote reproductive fitness.

Organismal functionality and reproduction depend on metabolic rewiring and balanced energy resources. However, the crosstalk between organismal homeostasis and fecundity and the associated paracrine signaling mechanisms are still poorly understood. Using Caenorhabditis elegans, we discovered that large extracellular vesicles (known as exophers) previously found to remove damaged subcellular elements in neurons and cardiomyocytes are released by body wall muscles (BWM) to support embryonic growth. Exopher formation (exopheresis) by BWM is sex-specific and a non-cell autonomous process regulated by developing embryos in the uterus. Embryo-derived factors induce the production of exophers that transport yolk proteins produced in the BWM and ultimately deliver them to newly formed oocytes. Consequently, offspring of mothers with a high number of muscle-derived exophers grew faster. We propose that the primary role of muscular exopheresis is to stimulate reproductive capacity, thereby influencing the adaptation of worm populations to the current environmental conditions.

Mitochondrial protein biogenesis in the synapse is supported by local translation.

Synapses are the regions of the neuron that enable the transmission and propagation of action potentials on the cost of high energy consumption and elevated demand for mitochondrial ATP production. The rapid changes in local energetic requirements at dendritic spines imply the role of mitochondria in the maintenance of their homeostasis. Using global proteomic analysis supported with complementary experimental approaches, we show that an essential pool of mitochondrial proteins is locally produced at the synapse indicating that mitochondrial protein biogenesis takes place locally to maintain functional mitochondria in axons and dendrites. Furthermore, we show that stimulation of synaptoneurosomes induces the local synthesis of mitochondrial proteins that are transported to the mitochondria and incorporated into the protein supercomplexes of the respiratory chain. Importantly, in a mouse model of fragile X syndrome, Fmr1 KO mice, a common disease associated with dysregulation of synaptic protein synthesis, we observed altered morphology and respiration rates of synaptic mitochondria. That indicates that the local production of mitochondrial proteins plays an essential role in synaptic functions.

Mgr2 functions as lateral gatekeeper for preprotein sorting in the mitochondrial inner membrane.

The majority of preproteins destined for mitochondria carry N-terminal presequences. The presequence translocase of the inner mitochondrial membrane (TIM23 complex) plays a central role in protein sorting. Preproteins are either translocated through the TIM23 complex into the matrix or are laterally released into the inner membrane. We report that the small hydrophobic protein Mgr2 controls the lateral release of preproteins. Mgr2 interacts with preproteins in transit through the TIM23 complex. Overexpression of Mgr2 delays preprotein release, whereas a lack of Mgr2 promotes preprotein sorting into the inner membrane. Preproteins with a defective inner membrane sorting signal are translocated into the matrix in wild-type mitochondria but are released into the inner membrane in Mgr2-deficient mitochondria. We conclude that Mgr2 functions as a lateral gatekeeper of the mitochondrial presequence translocase, providing quality control for the membrane sorting of preproteins.

Correction: Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish.

[This corrects the article DOI: 10.1371/journal.pgen.1007743.].

Mitochondrial stress-dependent regulation of cellular protein synthesis.

The production of newly synthesized proteins is vital for all cellular functions and is a determinant of cell growth and proliferation. The synthesis of polypeptide chains from mRNA molecules requires sophisticated machineries and mechanisms that need to be tightly regulated, and adjustable to current needs of the cell. Failures in the regulation of translation contribute to the loss of protein homeostasis, which can have deleterious effects on cellular function and organismal health. Unsurprisingly, the regulation of translation appears to be a crucial element in stress response mechanisms. This review provides an overview of mechanisms that modulate cytosolic protein synthesis upon cellular stress, with a focus on the attenuation of translation in response to mitochondrial stress. We then highlight links between mitochondrion-derived reactive oxygen species and the attenuation of reversible cytosolic translation through the oxidation of ribosomal proteins at their cysteine residues. We also discuss emerging concepts of how cellular mechanisms to stress are adapted, including the existence of alternative ribosomes and stress granules, and the regulation of co-translational import upon organelle stress.

The ubiquitin-proteasome system and its crosstalk with mitochondria as therapeutic targets in medicine.

The ubiquitin-proteasome system constitutes a major pathway for protein degradation in the cell. Therefore the crosstalk of this pathway with mitochondria is a major topic with direct relevance to many mitochondrial diseases. Proteasome dysfunction triggers not only protein toxicity, but also mitochondrial dysfunction. The involvement of proteasomes in the regulation of protein transport into mitochondria contributes to an increase in mitochondrial function defects. On the other hand, mitochondrial impairment stimulates reactive oxygen species production, which increases protein damage, and protein misfolding and aggregation leading to proteasome overload. Concurrently, mitochondrial dysfunction compromises cellular ATP production leading to reduced protein ubiquitination and proteasome activity. In this review we discuss the complex relationship and interdependence of the ubiquitin-proteasome system and mitochondria. Furthermore, we describe pharmacological inhibition of proteasome activity as a novel strategy to treat a group of mitochondrial diseases.

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.

Mitochondrial control of cellular protein homeostasis.

Mitochondria are involved in several vital functions of the eukaryotic cell. The majority of mitochondrial proteins are coded by nuclear DNA. Constant import of proteins from the cytosol is a prerequisite for the efficient functioning of the organelle. The protein import into mitochondria is mediated by diverse import pathways and is continuously under watch by quality control systems. However, it is often challenged by both internal and external factors, such as oxidative stress or energy shortage. The impaired protein import and biogenesis leads to the accumulation of mitochondrial precursor proteins in the cytosol and activates several stress response pathways. These defense mechanisms engage a network of processes involving transcription, translation, and protein clearance to restore cellular protein homeostasis. In this review, we provide a comprehensive analysis of various factors and processes contributing to mitochondrial stress caused by protein biogenesis failure and summarize the recovery mechanisms employed by the cell.

Non-canonical translation initiation in yeast generates a cryptic pool of mitochondrial proteins.

Utilization of non-AUG alternative translation start sites is most common in bacteria and viruses, but it has been also reported in other organisms. This phenomenon increases proteome complexity by allowing expression of multiple protein isoforms from a single gene. In Saccharomyces cerevisiae, a few described cases concern proteins that are translated from upstream near-cognate start codons as N-terminally extended variants that localize to mitochondria. Using bioinformatics tools, we provide compelling evidence that in yeast the potential for producing alternative protein isoforms by non-AUG translation initiation is much more prevalent than previously anticipated and may apply to as many as a few thousand proteins. Several hundreds of candidates are predicted to gain a mitochondrial targeting signal (MTS), generating an unrecognized pool of mitochondrial proteins. We confirmed mitochondrial localization of a subset of proteins previously not identified as mitochondrial, whose standard forms do not carry an MTS. Our data highlight the potential of non-canonical translation initiation in expanding the capacity of the mitochondrial proteome and possibly also other cellular features.

Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish.

Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.

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.

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.

Protein trafficking at the crossroads to mitochondria.

Mitochondria are central power stations in the cell, which additionally serve as metabolic hubs for a plethora of anabolic and catabolic processes. The sustained function of mitochondria requires the precisely controlled biogenesis and expression coordination of proteins that originate from the nuclear and mitochondrial genomes. Accuracy of targeting, transport and assembly of mitochondrial proteins is also needed to avoid deleterious effects on protein homeostasis in the cell. Checkpoints of mitochondrial protein transport can serve as signals that provide information about the functional status of the organelles. In this review, we summarize recent advances in our understanding of mitochondrial protein transport and discuss examples that involve communication with the nucleus and cytosol.

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.