Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.

PRAT Proteins Operate in Organellar Protein Import and Export in Arabidopsis thaliana.

Chloroplasts need to import preproteins and amino acids from the cytosol during their light-induced differentiation. Similarly, chloroplasts have to export organic matter including proteins and amino acids during leaf senescence. Members of the PRAT (preprotein and amino acid transporter) family are candidate transporters for both processes. Here, we defined the role of two small PRAT gene families, At4g26670 and At5g55510 (HP20 subfamily) versus At3g49560 and At5g24650 (HP30 subfamily) during greening of etiolated plants and during leaf senescence. Using a combination of reverse genetics, protein biochemistry and physiological tools, evidence was obtained for a role of chloroplast HP20, HP30 and HP30-2 in protein, but not amino acid, import into chloroplasts. HP20, HP30 and HP30-2 form larger complexes involved in the uptake of transit sequence-less cytosolic precursors. In addition, we identified a fraction of HP30-2 in mitochondria where it served a similar function as found for chloroplasts and operated in the uptake of transit sequence-less cytosolic precursor proteins. By contrast, HP22 was found to act in the export of proteins from chloroplasts during leaf senescence, and thus its role is entirely different from that of its orthologue, HP20. HP22 is part of a unique protein complex in the envelope of senescing chloroplasts that comprises at least 11 proteins and contains with HP65b (At5g55220) a protein that is related to the bacterial trigger factor chaperone. An ortholog of HP65b exists in the cyanobacterium Synechocystis and has previously been implicated in protein secretion. Whereas plants depleted of either HP22 or HP65b or even both were increasingly delayed in leaf senescence and retained much longer stromal chloroplast constituents than wild-type plants, HP22 overexpressors showed premature leaf senescence that was associated with accelerated losses of stromal chloroplast proteins. Together, our results identify the PRAT protein family as a unique system for importing and exporting proteins from chloroplasts.

tRNA-Dependent Import of a Transit Sequence-Less Aminoacyl-tRNA Synthetase (LeuRS2) into the Mitochondria of Arabidopsis.

Aminoacyl-tRNA synthetases (AaRS) charge tRNAs with amino acids for protein translation. In plants, cytoplasmic, mitochondrial, and chloroplast AaRS exist that are all coded for by nuclear genes and must be imported from the cytosol. In addition, only a few of the mitochondrial tRNAs needed for translation are encoded in mitochondrial DNA. Despite considerable progress made over the last few years, still little is known how the bulk of cytosolic AaRS and respective tRNAs are transported into mitochondria. Here, we report the identification of a protein complex that ties AaRS and tRNA import into the mitochondria of Arabidopsis thaliana. Using leucyl-tRNA synthetase 2 (LeuRS2) as a model for a mitochondrial signal peptide (MSP)-less precursor, a ≈30 kDa protein was identified that interacts with LeuRS2 during import. The protein identified is identical with a previously characterized mitochondrial protein designated HP30-2 (encoded by At3g49560) that contains a sterile alpha motif (SAM) similar to that found in RNA binding proteins. HP30-2 is part of a larger protein complex that contains with TIM22, TIM8, TIM9 and TIM10 four previously identified components of the translocase for MSP-less precursors. Lack of HP30-2 perturbed mitochondrial biogenesis and function and caused seedling lethality during greening, suggesting an essential role of HP30-2 in planta.

Mitochondrial dysfunction in sepsis is associated with diminished intramitochondrial TFAM despite its increased cellular expression.

Sepsis is characterized by a dysregulated immune response, metabolic derangements and bioenergetic failure. These alterations are closely associated with a profound and persisting mitochondrial dysfunction. This however occurs despite increased expression of the nuclear-encoded transcription factor A (TFAM) that normally supports mitochondrial biogenesis and functional recovery. Since this paradox may relate to an altered intracellular distribution of TFAM in sepsis, we tested the hypothesis that enhanced extramitochondrial TFAM expression does not translate into increased intramitochondrial TFAM abundance. Accordingly, we prospectively analyzed PBMCs both from septic patients (n = 10) and lipopolysaccharide stimulated PBMCs from healthy volunteers (n = 20). Extramitochondrial TFAM protein expression in sepsis patients was 1.8-fold greater compared to controls (p = 0.001), whereas intramitochondrial TFAM abundance was approximate 80% less (p < 0.001). This was accompanied by lower mitochondrial DNA copy numbers (p < 0.001), mtND1 expression (p < 0.001) and cellular ATP content (p < 0.001) in sepsis patients. These findings were mirrored in lipopolysaccharide stimulated PBMCs taken from healthy volunteers. Furthermore, TFAM-TFB2M protein interaction within the human mitochondrial core transcription initiation complex, was 74% lower in septic patients (p < 0.001). In conclusion, our findings, which demonstrate a diminished mitochondrial TFAM abundance in sepsis and endotoxemia, may help to explain the paradox of lacking bioenergetic recovery despite enhanced TFAM expression.

The Mitochondrial Outer Membrane Protein Tom70-Mediator in Protein Traffic, Membrane Contact Sites and Innate Immunity.

Tom70 is a versatile adaptor protein of 70 kDa anchored in the outer membrane of mitochondria in metazoa, fungi and amoeba. The tertiary structure was resolved for the Tom70 of yeast, showing 26 α-helices, most of them participating in the formation of 11 tetratricopeptide repeat (TPR) motifs. Tom70 serves as a docking site for cytosolic chaperone proteins and co-chaperones and is thereby involved in the uptake of newly synthesized chaperone-bound proteins in mitochondrial biogenesis. In yeast, Tom70 additionally mediates ER-mitochondria contacts via binding to sterol transporter Lam6/Ltc1. In mammalian cells, TOM70 promotes endoplasmic reticulum (ER) to mitochondria Ca2+ transfer by association with the inositol-1,4,5-triphosphate receptor type 3 (IP3R3). TOM70 is specifically targeted by the Bcl-2-related protein MCL-1 that acts as an anti-apoptotic protein in macrophages infected by intracellular pathogens, but also in many cancer cells. By participating in the recruitment of PINK1 and the E3 ubiquitin ligase Parkin, TOM70 can be implicated in the development of Parkinson's disease. TOM70 acts as receptor of the mitochondrial antiviral-signaling protein (MAVS) and thereby participates in the corresponding system of innate immunity against viral infections. The protein encoded by Orf9b in the genome of SARS-CoV-2 binds to TOM70, probably compromising the synthesis of type I interferons.

The selectivity filter of the mitochondrial protein import machinery.

BACKGROUND: The uptake of newly synthesized nuclear-encoded mitochondrial proteins from the cytosol is mediated by a complex of mitochondrial outer membrane proteins comprising a central pore-forming component and associated receptor proteins. Distinct fractions of proteins initially bind to the receptor proteins and are subsequently transferred to the pore-forming component for import. The aim of this study was the identification of the decisive elements of this machinery that determine the specific selection of the proteins that should be imported. RESULTS: We identified the essential internal targeting signal of the members of the mitochondrial metabolite carrier proteins, the largest protein family of the mitochondria, and we investigated the specific recognition of this signal by the protein import machinery at the mitochondrial outer surface. We found that the outer membrane import receptors facilitated the uptake of these proteins, and we identified the corresponding binding site, marked by cysteine C141 in the receptor protein Tom70. However, in tests both in vivo and in vitro, the import receptors were neither necessary nor sufficient for specific recognition of the targeting signals. Although these signals are unrelated to the amino-terminal presequences that mediate the targeting of other mitochondrial preproteins, they were found to resemble presequences in their strict dependence on a content of positively charged residues as a prerequisite of interactions with the import pore. CONCLUSIONS: The general import pore of the mitochondrial outer membrane appears to represent not only the central channel of protein translocation but also to form the decisive general selectivity filter in the uptake of the newly synthesized mitochondrial proteins.

Mimivirus-Encoded Nucleotide Translocator VMC1 Targets the Mitochondrial Inner Membrane.

Mimivirus (Acanthamoeba polyphaga mimivirus) was the first giant DNA virus identified in an amoeba species. Its genome contains at least 979 genes. One of these, L276, encodes a nucleotide translocator with similarities to mitochondrial metabolite carriers, provisionally named viral mitochondrial carrier 1 (VMC1). In this study, we investigated the intracellular distribution of VMC1 upon expression in HeLa cells and in the yeast Saccharomyces cerevisiae. We found that VMC1 is specifically targeted to mitochondria and to the inner mitochondrial membrane. Newly synthesized VMC1 binds to the mitochondrial outer-membrane protein Tom70 and translocates through the import channel formed by the ß-barrel protein Tom40. Derivatization of the four cysteine residues inside Tom40 by N-ethylmaleimide caused a delay in translocation but not a complete occlusion. Cell viability was not reduced by VMC1. Neither the mitochondrial membrane potential nor the intracellular production of reactive oxygen species was affected. Similar to endogenous metabolite carriers, mimivirus-encoded VMC1 appears to act as a specific translocator in the mitochondrial inner membrane. Due to its permeability for deoxyribonucleotides, VMC1 confers to the mitochondria an opportunity to contribute nucleotides for the replication of the large DNA genome of the virus.

Cytotoxic stress induces transfer of mitochondria-associated human endogenous retroviral RNA and proteins between cancer cells.

About 8 % of the human genome consists of human endogenous retroviruses (HERVs), which are relicts of ancient exogenous retroviral infections incurred during evolution. Although the majority of HERVs have functional gene defects or epigenetic modifications, many of them are still able to produce retroviral proteins that have been proposed to be involved in cellular transformation and cancer development. We found that, in chemo-resistant U87RETO glioblastoma cells, cytotoxic stress induced by etoposide promotes accumulation and large-scale fission of mitochondria, associated with the detection of HERV-WE1 (syncytin-1) and HERV-FRD1 (syncytin-2) in these organelles. In addition, mitochondrial preparations also contained the corresponding receptors, i.e. ASCT2 and MFSD2. We clearly demonstrated that mitochondria associated with HERV-proteins were shuttled between adjacent cancer cells not only via tunneling tubes, but also by direct cellular uptake across the cell membrane. Furthermore, anti-syncytin-1 and anti-syncytin-2 antibodies were able to specifically block this direct cellular uptake of mitochondria even more than antibodies targeting the cognate receptors. Here, we suggest that the association of mitochondria with syncytin-1/syncytin-2 together with their respective receptors could represent a novel mechanism of cell-to-cell transfer. In chemotherapy-refractory cancer cells, this might open up attractive avenues to novel mitochondria-targeting therapies.

Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1.

Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion.

Helicobacter pylori VacA: a new perspective on an invasive chloride channel.

The vacuolating cytotoxin VacA, a polypeptide of about 88 kDa, is one of the major virulence factors of Helicobacter pylori. VacA essentially acts as an invasive chloride channel targeting mitochondria. The results of recent studies open a new perspective on the mechanisms by which VacA causes loss of the mitochondrial membrane potential, mitochondrial fragmentation, formation of reactive oxygen species, autophagy, cell death and gastric cancer.

Helicobacter pylori vacuolating toxin A and apoptosis.

VacA, the vacuolating cytotoxin A of Helicobacter pylori, induces apoptosis in epithelial cells of the gastic mucosa and in leukocytes. VacA is released by the bacteria as a protein of 88 kDa. At the outer surface of host cells, it binds to the sphingomyelin of lipid rafts. At least partially, binding to the cells is facilitated by different receptor proteins. VacA is internalized by a clathrin-independent mechanism and initially accumulates in GPI-anchored proteins-enriched early endosomal compartments. Together with early endosomes, VacA is distributed inside the cells. Most of the VacA is eventually contained in the membranes of vacuoles. VacA assembles in hexameric oligomers forming an anion channel of low conductivity with a preference for chloride ions. In parallel, a significant fraction of VacA can be transferred from endosomes to mitochondria in a process involving direct endosome-mitochondria juxtaposition. Inside the mitochondria, VacA accumulates in the mitochondrial inner membrane, probably forming similar chloride channels as observed in the vacuoles. Import into mitochondria is mediated by the hydrophobic N-terminus of VacA. Apoptosis is triggered by loss of the mitochondrial membrane potential, recruitment of Bax and Bak, and release of cytochrome c.

A phosphodiesterase 2A isoform localized to mitochondria regulates respiration.

Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.

Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

Targeting of Helicobacter pylori VacA to mitochondria.

One of the major virulence factors of Helicobacter pylori is the vacuolating toxin vaca. It has been known for a long time that the toxin enters host cells by endocytosis. On the other hand there is ample evidence that vaca is able to trigger apoptosis and this effect has been attributed in part to interactions with mitochondria. However, for 10 years it was difficult to reconcile the obvious accumulation of vaca in endosomes with mitochondrial targeting. The accessibility of the mitochondria to the toxin was enigmatic. In our new study, we investigated the activities of p34, the toxic subunit of vaca, in more detail. We found that the p34 N-terminus carries a unique targeting sequence for import into mitochondria and for insertion into the mitochondrial inner membrane. By forming an anion channel in this membrane, the toxin has the ability to interfere directly with mitochondrial functions. Taking into account additional results from independent studies, we discuss the implications of our findings with respect to intracellular traffic, the remarkable possibility of a direct transfer of VacA from endosomes to mitochondria and vaca-dependent cell death.

Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m)). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m). The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m) loss and apoptosis, demonstrating that dissipation of DeltaPsi(m) is a requirement for cell death caused by neisserial infection.

Transmembrane domain length of viral K+ channels is a signal for mitochondria targeting.

K(+) channels operate in the plasma membrane and in membranes of organelles including mitochondria. The mechanisms and topogenic information for their differential synthesis and targeting is unknown. This article describes 2 similar viral K(+) channels that are differentially sorted; one protein (Kesv) is imported by the Tom complex into the mitochondria, the other (Kcv) to the plasma membrane. By creating chimeras we discovered that mitochondrial sorting of Kesv depends on a hierarchical combination of N- and C-terminal signals. Crucial is the length of the second transmembrane domain; extending its C terminus by > or = 2 hydrophobic amino acids redirects Kesv from the mitochondrial to the plasma membrane. Activity of Kesv in the plasma membrane is detected electrically or by yeast rescue assays only after this shift in sorting. Hence only minor structural alterations in a transmembrane domain are sufficient to switch sorting of a K(+) channel between the plasma membrane and mitochondria.

Role of gamma-subunit N- and C-termini in assembly of the mitochondrial ATP synthase in yeast.

The gamma-subunit is required for the assembly of ATP synthases and plays a crucial role in their catalytic activity. We stepwise shortened the N-terminus and the C-terminus of the gamma-subunit in the mitochondrial ATP synthase of yeast and investigated the relevance of these segments in the assembly of the enzyme and in the growth of the cells. We found that a deletion of 9 residues at the N-terminus or 20 residues at the C-terminus still allowed efficient import of the subunit into mitochondria; however, the assembly of both monomeric and dimeric holoenzymes was partially impaired. gamma-Subunits lacking 13 N-terminal residues or 30 C-terminal residues were not assembled. Yeast strains expressing either of the truncated gamma-subunits did not grow on non-fermentable carbon sources, indicating that non-assembled parts of the ATP synthase accumulated and impaired essential mitochondrial functions.

Biogenesis of yeast dicarboxylate carrier: the carrier signature facilitates translocation across the mitochondrial outer membrane.

A family of related carrier proteins mediates the exchange of metabolites across the mitochondrial inner membrane. The carrier signature Px[D/E]xx[K/R] is a highly conserved sequence motif in all members of this family. To determine its function in the biogenesis of carrier proteins, we used the dicarboxylate carrier (DIC) of yeast as a model protein. We found that the carrier signature was dispensable in binding of the newly synthesized protein to the import receptor Tom70, but that it was specifically required for efficient translocation across the mitochondrial outer membrane. To determine the relevance of individual amino acid residues of the carrier signature in the transport activity of the protein, we exchanged defined residues with alanine and reconstituted the mutant proteins in vitro. Substitution of the carrier signature in helix H1 reduced the transport activity for [(33)P]-phosphate by approximately 90% and an additional substitution of the carrier signature in helix H5 blocked the transport activity completely. We conclude that the carrier signature of the dicarboxylate carrier is involved both in the biogenesis and in the transport activity of the functional protein.

Protein targeting to mitochondria of Saccharomyces cerevisiae and Neurospora crassa: in vitro and in vivo studies.

Most studies on the biogenesis of mitochondrial proteins have been carried out using fungal mitochondria as a model system. In particular, baker's yeast, Saccharomyces cerevisiae, combines several experimental advantages, allowing both genetic and biochemical approaches and thus a combination of investigations in vivo and in vitro. However, the red bread mold Neurospora crassa has also been an important research tool. Isolated mitochondria can be used from both organisms for import experiments in a reconstituted system, using radiolabeled precursor proteins synthesized in reticulocyte lysate or purified preproteins. Assays are available for studies on the import pathways and localization of mitochondrial proteins and for the characterization of the components of the protein import machinery.

Separation of proteins by blue native electrophoresis.

Blue native gel electrophoresis is a native electrophoresis method that can be used for molecular weight determination for most soluble protein complexes as well as for most membrane proteins. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can be used in a second dimension to resolve the complexes into their subunits. The method has been extensively used for the analysis of the respiratory chain complexes, for the determination of intermediates of mitochondrial protein import, and for the identification of the composition of the protein import machinery for mitochondria and chloroplasts. Here we describe the basic method and some applications in the research of mitochondrial protein import.