Protein Translocation into the Intermembrane Space and Matrix of Mitochondria: Mechanisms and Driving Forces

Backes, Sandra; Herrmann, Johannes M.

doi:10.3389/fmolb.2017.00083

Cited by 83 publications

(79 citation statements)

References 102 publications

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“…The IMS environment was characterized using a "bipartite presequence" from two IMS proteins: OPA1 (MTS-OPA1) and apoptosis inducing factor AIFM1 (MTS-AIFM1). A "bipartite presequence" is a feature of a subset of IMS proteins that contain an MTS, followed by a cleavable stop-transfer sequence, which, upon cleavage, results in "soluble" IMS protein (Backes and Herrmann, 2017). Our IMS-BirA* baits identified 120 proximity interactors, of which 112 (93%) were annotated as mitochondrial proteins by either Mitocarta 2.0 or Gene Ontology Cellular Component (GOCC) (Supplementary Fig.…”

Section: Definition Of the Mitochondrial Matrix And Ims Bioid Environmentioning

confidence: 99%

A high-density human mitochondrial proximity interaction network

Antonická

Lin

Janer

et al. 2020

Preprint

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We used BioID, a proximity-dependent biotinylation assay, to interrogate 100 mitochondrial baits from all mitochondrial sub-compartments to create a high resolution human mitochondrial proximity interaction network. We identified 1465 proteins, producing 15626 unique high confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions, and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments, and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles. This proximity network will be a valuable resource for exploring the biology of uncharacterized mitochondrial proteins, the interactions of mitochondria with other cellular organelles, and will provide a framework to interpret alterations in sub-mitochondrial environments associated with mitochondrial disease. Bullet points• We created a high resolution human mitochondrial protein proximity map using BioID• Bait-bait analysis showed that the map has sub-compartment resolution and correlation analysis of preys identified functional clusters and assigned proteins to specific modules• We identified isoforms of matrix and IMS proteins with multiple cellular localizations and an endonuclease that localizes to both the matrix and the OMM • OMM baits showed specific interactions with non-mitochondrial proteins reflecting organellar contact sites and protein dual localization 2013). Mitochondrial diseases due to deficiencies in the oxidative phosphorylation machinery are amongst the most common inherited metabolic disorders (Frazier et al., 2019;Nunnari and Suomalainen, 2012). While nearly 300 causal genes have now been identified, the molecular basis for the extraordinary tissue specificity associated with these diseases remains an enduring mystery.Mitochondria are double-membraned organelles, whose ultrastructure has been investigated for decades. The outer membrane (OMM), which serves as a platform for molecules involved in the regulation of innate immunity and for regulation of apoptosis, is permeable to metabolites and small proteins, and it forms close contacts with the endoplasmic reticulum (ER) for the exchange of lipids and calcium (Csordas et al., 2018). The impermeable inner membrane (IMM) is composed of invaginations called cristae that contain the complexes of the oxidative phosphorylation system. The intermembrane space (IMS) comprises both the space formed...

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Section: Definition Of the Mitochondrial Matrix And Ims Bioid Environmentioning

confidence: 99%

A high-density human mitochondrial proximity interaction network

Antonická

Lin

Janer

et al. 2020

Preprint

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“…CHCHD4 provides an import and oxidoreductase-mediated protein folding function along with the sulfhydryl oxidase GFER (ALR/Erv1) as a key part of the disulphide relay system (DRS) within the mitochondrial IMS [5][6][7]. As such, CHCHD4 controls the import of a number of mitochondrial proteins that contain a twin-CX 9 C or twin-CX 3 C motif [8][9][10].…”

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confidence: 99%

CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain

Thomas

Stephen

Esposito

et al. 2019

Preprint

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BACKGROUND: Tumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) is critical for maintaining intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers, correlates with increased tumour progression and poor patient survival.RESULTS: Here, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I-V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS). CONCLUSIONS:Our study highlights an important role for CHCHD4 in regulating tumour cell metabolism, and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology. BACKGROUNDMetabolic reprogramming and altered mitochondrial metabolism is a feature of cancer, and thus has become an attractive area for therapeutic exploitation [1,2]. Given the importance of mitochondria in controlling normal physiological processes, understanding how mitochondrial metabolism underlies tumorigenesis is important for ascertaining its therapeutic potential in cancer.Previously, we discovered the essential redox-sensitive mitochondrial intermembrane space (IMS) protein CHCHD4, is a critical for regulating intracellular oxygen consumption rate and metabolic responses to low oxygen (hypoxia) in tumour cells [3,4]. Overexpression of Thomas et al. 3CHCHD4 in human cancers significantly correlates with the hypoxia gene signature, tumour progression, disease recurrence and poor patient survival [3].CHCHD4 provides an import and oxidoreductase-mediated protein folding function along with the sulfhydryl oxidase GFER (ALR/Erv1) as a key part of the disulphide relay system (DRS) within the mitochondrial IMS [5][6][7]. As such, CHCHD4 controls the import of a number of mitochondrial proteins that contain a twin-CX 9 C or twin-CX 3 C motif [8][9][10].Additio...

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“…Based on these data, it was suggested that ferredoxin transport occurs by means of a Brownian ratchet mechanism in which FusC acts as a periplasmic anchor to facilitate translocation of ferredoxin across the OM via the lumen of FusA 12,15 . Similar mechanisms have been postulated to account for mitochondrial protein uptake, whereby cytoplasmically synthesised proteins are translocated via the TOM and TIM23 complexes 16,17 . As such, this would represent a hitherto unexpected evolutionary link between mitochondrial and plastid protein import and bacterial protein import via the Fus and other postulated protein uptake systems.…”

Section: Introductionmentioning

confidence: 71%

FusB energises import across the outer membrane through direct interaction with its ferredoxin substrate

Wojnowska

Walker

2019

Preprint

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We recently described an example of protein import into Gram-negative bacteria for nutrient acquisition whereby phytopathogenic Pectobacterium spp. are able to import ferredoxin into the periplasm for proteolytic processing and iron release via the ferredoxin uptake system. Although the outer membrane ferredoxin receptor FusA and the periplasmic ferredoxin processing protease, FusC, have been identified, the mechanistic basis of ferredoxin import is poorly understood. Based on structural analysis of the FusC-ferredoxin complex, an additional role for FusC in direct mediation of protein import has been suggested.In this model, FusC acts as a periplasmic anchor that enables ferredoxin to traverse the outer membrane via FusA using a Brownian ratchet mechanism, akin to that proposed for the transport of mitochondrial proteins across the mitochondrial outer membrane. In this work we demonstrate that FusC does not play a role in protein import and show that, instead, protein translocation across the outer membrane is dependent on the TonB-like protein FusB. In contrast to the loss of FusC, loss of FusB or FusA abolishes ferredoxin transport to the periplasm, demonstrating that FusA and FusB work in concert to transport ferredoxin across the outer membrane. Interestingly, in addition to interaction with the TonB-box region of FusA, FusB also forms a complex with the ferredoxin substrate, with complex formation required for substrate transport. These data suggest that ferredoxin transport requires energy transduction from the cytoplasmic membrane via FusB for both removal of the FusA plug domain and for substrate translocation through the lumen of the FusA barrel.Protein translocation occurs by direct interaction with the an N-terminal intrinsically unstructured region of the toxin that, similar to the TBDRs, carries a TonB-binding motif 10 .We recently demonstrated that TBDR-mediated iron acquisition from the iron-sulphur cluster containing protein ferredoxin represents an unprecedented example of protein translocation into the bacterial cell for nutrient acquision 11 . Ferredoxin binding at the cell surface is mediated by the TBDR FusA and, following transport of intact ferredoxin into the periplasm, the substrate is subjected to proteolytic processing by the M16 protease FusC 11,12 .Cleavage by FusC results in release of the iron-sulphur cluster and is required for effective iron acquisition from ferredoxin by Pectobacterium. Together with the genes encoding FusA and FusC, the Fus operon contains two additional genes, with fusB encoding a TonBhomologue and fusD a putative ABC transporter 12 . Interestingly, the M-type pectocins M1 and M2, which we have previously described, parasitise the ferredoxin uptake system through an N-terminal ferredoxin domain that is highly homologous to plant ferredoxins 13,14 .

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Protein Translocation into the Intermembrane Space and Matrix of Mitochondria: Mechanisms and Driving Forces

Cited by 83 publications

References 102 publications

A high-density human mitochondrial proximity interaction network

A high-density human mitochondrial proximity interaction network

CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain

FusB energises import across the outer membrane through direct interaction with its ferredoxin substrate

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