MitoStores: stress-induced aggregation of mitochondrial proteins.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS.

Mitochondrial biogenesis and functions depend on the import and assembly of more than 1000 proteins that are made as precursors on cytosolic ribosomes. The majority of these precursor proteins are transported from the ribosome to the translocase of the outer membrane (TOM complex), which constitutes the main entry site for mitochondrial precursors. The transient localization of mitochondrial precursor proteins in the cytosol represents a major burden for cellular proteostasis since these proteins can aggregate and accumulate in different cellular compartments, causing proteotoxic stress. Inside mitochondria, protein translocases sort the precursor proteins into the mitochondrial subcompartments-outer and inner membrane, the intermembrane space and matrix. The imported proteins have to be folded and efficiently assembled into functional protein complexes. Molecular chaperones such as Hsp70 monitor these processes to minimize proteotoxic stress. J-domain proteins stimulate the ATPase activity of Hsp70 and recruit the chaperones to their clients in the biogenesis of mitochondrial proteins. They ensure protein targeting to mitochondria, drive protein import into mitochondria, as well as folding and assembly of mitochondrial proteins. Here, we summarize the emerging view of how J-domain proteins guide mitochondrial precursor proteins from their synthesis in the cytosol until their folding into a mature protein and assembly into protein complexes in mitochondria.

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

As the primary cellular source of ATP, mitochondria form a vital bioenergetic, metabolic and signaling hub. Despite the presence of mitochondrial DNA, almost all mitochondrial proteins (99%) are nuclear encoded and are imported from the cytosol. Protein translocases in mitochondrial membranes are therefore essential for correct protein targeting and localisation. Mitochondrial dysfunction is implicated in ageing, as well as a growing number of human pathologies, including certain cancers, genetically inherited syndromes and neurodegenerative disorders such as Alzheimer's disease. The biogenesis of mitochondrial proteins and import sites are therefore important factors that determine organelle functionality. The broad aim of this research is to understand how protein import is correctly orchestrated and regulated by innovative approaches focussed on state‐of‐the‐art electron cryo‐tomography (cryoET). This technique is the method of choice for the study of proteins or complexes in situ (Figure 1). Samples are preserved by cryo‐fixation, imaged in the electron microscope, and protein structures can be determined by subtomogram averaging (StA). The majority of precursor proteins (preproteins) are actively imported into mitochondria from the cytosol through the T ranslocase of the O uter M embrane (TOM) complex, which forms a common entry gate for proteins that are subsequently targeted to various locations. For decades, it has been known that proteins can be imported into mitochondria post‐translationally, after synthesis on ribosomes in the cytosol or in vitro .However, information regarding the organisation and distribution of mitochondrial protein import sites was sparse. To image active post‐translational protein import in situ, we previously devised a new labelling strategy in which mitochondrial‐targeted preproteins were arrested as two‐membrane‐spanning intermediates through both the TOM complex andthe T ranslocase of the I nner M embrane (TIM23) complex concurrently (Gold, V. A. M. et al. (2014) Nat Commun 5, 4129). This work provided the first views of mitochondrial proteins in the act of import and enabled direct visualization of the number and location of active complexes for the first time (Figure 1). However, information regarding the cytosolic stage of mitochondrial precursor protein import and targeting was still lacking. Most recently, we have developed a new method to investigate the cytosolic stage of mitochondrial protein targeting and import by biochemical techniques, cryo‐ET and StA. This reveals unprecedented details regarding different targeting pathways, shedding light on the interaction between importing proteins and their corresponding membrane‐bound translocons. The consequences of these different modes of targeting and import site redistribution will be discussed in the context of mitochondrial protein biogenesis. Legend to Figure 1 Top panel: mitochondria embedded in a layer of amorphous ice are imaged in the electron microscope. Incremental tilts of the sample yield a series of projections from different viewing angles. The relative orientations of the mitochondrion (brown) and the macromolecular complex of interest (red) vary depending on the projection angle. Bottom panel: a 0° tilt projection image of a S. cerevisiae mitochondrion. op panel: a three‐dimensional tomogram is reconstructed from the two‐dimensional image series by computational back‐projection. The molecular complex of interest is indicated (red, boxed). Bottom panel: a slice through a reconstructed tomogram of the mitochondrion shown in step 1. Labelled importing proteins (black spheres on the outer membrane are indicated (red arrowhead). Top left and bottom panels: surface volume rendering is used to place complexes of interest back into three‐dimensional space in order to visualize their distribution in a native‐like context. Labelled preproteins (black spheres) are shown on the mitochondrial outer membrane (green). The inner membrane (blue) and crista membranes (yellow) are also shown. Top right panel: a slice through the reconstructed tomogram of a labelled preprotein (black sphere, red arrowhead) engaged in mitochondrial import.

Missense mutations in the nuclear-encoded adenine nucleotide translocase 1 (Ant1) cause the protein to clog the mitochondrial protein import pathway, to severely inhibit cell growth in yeast, and to cause neurodegeneration and myopathy in mice that phenocopy ANT1-induced human disease.

All mitochondrial precursor proteins studied so far are recognized initially at the surface of the organelle by the translocase of the outer membrane (TOM complex). Precursors of beta-barrel proteins are transferred further to another complex in the outer membrane that mediates their topogenesis (TOB complex). Tob55 is an essential component of the TOB complex in that it constitutes the core element of the protein-conducting pore. The other two components of the TOB complex are Tob38, which builds a functional TOB core complex with Tob55, and Mas37, a peripheral member of the complex. We have investigated the biogenesis of the TOB complex. Reduced insertion of the Tob55 precursor in the absence of Tom20 and Tom70 argues for initial recognition of the precursor of Tob55 by the import receptors. Next, it is transferred through the import channel formed by Tom40. Variants of the latter protein influenced the insertion of Tob55. Assembly of newly synthesized Tob55 into preexisting TOB complexes, as analyzed by blue native gel electrophoresis, depended on Tob38 but did not require Mas37. Surprisingly, both the association of Mas37 precursor with mitochondria and its assembly into the TOB complex were not affected by mutation in the TOM complex. Mas37 assembled directly with the TOB core complex. Hence, the biogenesis of Mas37 represents a novel import pathway of mitochondrial proteins.

Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.

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.

Bisphenol-A inhibits mitochondrial biogenesis via impairment of GFER mediated mitochondrial protein import in the rat brain hippocampus

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early-onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin-/PINK1-mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.

Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein.

In this report, we summarize recent findings about a role of the outer membrane metabolite channel VDAC/porin in protein import into mitochondria. Mitochondria fulfill key functions for cellular energy metabolism. Their biogenesis involves the import of about 1000 different proteins that are produced as precursors on cytosolic ribosomes. The translocase of the outer membrane (TOM complex) forms the entry gate for mitochondrial precursor proteins. Dedicated protein translocases sort the preproteins into the different mitochondrial subcompartments. While protein transport pathways are analyzed to some detail, only little is known about regulatory mechanisms that fine-tune protein import upon metabolic signaling. Recently, a dual role of the voltage-dependent anion channel (VDAC), also termed porin, in mitochondrial protein biogenesis was reported. First, VDAC/porin promotes as a coupling factor import of carrier proteins into the inner membrane. Second, VDAC/porin regulates the formation of the TOM complex. Thus, the major metabolite channel in the outer membrane VDAC/porin connects protein import to mitochondrial metabolism.

Proteins of the mitochondrial outer membrane are synthesized as precursors on cytosolic ribosomes and sorted via internal targeting sequences to mitochondria. Two different types of integral outer membrane proteins exist: proteins with a transmembrane β-barrel and proteins embedded by a single or multiple α-helices. The import pathways of these two types of membrane proteins differ fundamentally. Precursors of β-barrel proteins are first imported across the outer membrane via the translocase of the outer membrane (TOM complex). The TOM complex is coupled to the sorting and assembly machinery (SAM complex), which catalyzes folding and membrane insertion of these precursors. The mitochondrial import machinery (MIM complex) promotes import of proteins with multiple α-helical membrane spans. Depending on the topology precursors of proteins with a single α-helical membrane anchor are imported via several distinct routes. We summarize current models and open questions of biogenesis of mitochondrial outer membrane proteins and discuss the impact of malfunctions of protein sorting on the development of diseases.

Any PhD student or postdoc, who scrambles to write and submit a publication before getting scooped, can testify that science is a fast‐moving endeavor. Given our limited time and the ever‐increasing pace with which scientific studies are published, few students and postdocs—and PIs as a matter of fact—have time to keep up with the literature. “Reading” a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. Notwithstanding these constraints, I would argue that the scientific literature is a treasure trove of information and ideas that go beyond contemporary papers to include classical publications. However, it can be difficult to motivate students and postdocs to read old landmark publications that reported groundbreaking discoveries and opened up new avenues of research. Many think that these papers are of merely historic interest and have little to contribute to scientists expected to use cutting‐edge methods to produce high‐impact publications. > Reading a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. However, classic papers still have a lot to offer. For once, they withstood the test of the time, as the reported results have shown to be correct and reproducible. This is unfortunately not the case for many high‐impact publications today, which, owing to the hype of selling it to the highest impact factor journal, just tell a cool story that in reality may be much less clear‐cut than reported. Furthermore, classic scientific publications often impress by a conceptual clarity and in many cases simplicity and thereby offer valuable lessons of how best science should be practiced. These are qualities I miss in many of today's papers that contain exceedingly large data sets …

The mitochondrial intermembrane space (IMS) contains an essential machinery for protein import and assembly (MIA). Biogenesis of IMS proteins involves a disulfide relay between precursor proteins, the cysteine-rich IMS protein Mia40 and the sulfhydryl oxidase Erv1. How precursor proteins are specifically directed to the IMS has remained unknown. Here we systematically analyzed the role of cysteine residues in the biogenesis of the essential IMS chaperone complex Tim9-Tim10. Although each of the four cysteines of Tim9, as well as of Tim10, is required for assembly of the chaperone complex, only the most amino-terminal cysteine residue of each precursor is critical for translocation across the outer membrane and interaction with Mia40. Mia40 selectively recognizes cysteine-containing IMS proteins in a site-specific manner in organello and in vitro. Our results indicate that Mia40 acts as a trans receptor in the biogenesis of mitochondrial IMS proteins.