Search

Mitochondria are major sites of reactive oxygen species (ROS) production within cells. ROS are important signalling molecules, but excessive production can cause cellular damage and dysfunction. It is therefore crucial to accurately determine when, how and where ROS are produced within mitochondria. Previously, ROS detection involved various chemical probes and fluorescent proteins. These have limitations due to accumulation of the molecules only in the mitochondrial matrix, or the need for a new protein to be expressed for every different species. We report dynamic H2O2 flux changes within all mitochondrial sub-compartments with striking spatial resolution. We combined specific targeting of self-labeling proteins with novel H2O2-reactive probes. The approach is broad-ranging and flexible, with the same expressed proteins loadable with different dyes and sensors. It provides a framework for concomitant analysis of other chemical species, beyond ROS, whose dynamics within mitochondria are yet unknown, without needing to engineer new proteins.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

Mitochondria are rich in multi-protein assemblies that are usually dedicated to one function. In this issue of Cell Reports, Horten et al.1 describe a 3-nanometer megacomplex in the mitochondrial inner membrane, which serves multiple functions integrating mitochondria biogenesis and metabolism.

Mitochondrial membrane proteins play an essential role in all major mitochondrial functions. The respiratory complexes of the inner membrane are key for the generation of energy. The carrier proteins for the influx/efflux of essential metabolites to/from the matrix. Many other inner membrane proteins play critical roles in the import and processing of nuclear encoded proteins (∼99% of all mitochondrial proteins). The outer membrane provides another lipidic barrier to nuclear-encoded protein translocation and is home to many proteins involved in the import process, maintenance of ionic balance, as well as the assembly of outer membrane components. While many aspects of the import and assembly pathways of mitochondrial membrane proteins have been elucidated, many open questions remain, especially surrounding the assembly of the respiratory complexes where certain highly hydrophobic subunits are encoded by the mitochondrial DNA and synthesised and inserted into the membrane from the matrix side. This review will examine the various assembly pathways for inner and outer mitochondrial membrane proteins while discussing the most recent structural and biochemical data examining the biogenesis process.

Mitochondria are double-membrane organelles that contain their own genome, the mitochondrial DNA (mtDNA), and reminiscent of its endosymbiotic origin. Mitochondria are responsible for cellular respiration via the function of the electron oxidative phosphorylation system (OXPHOS), located in the mitochondrial inner membrane and composed of the four electron transport chain (ETC) enzymes (complexes I-IV), and the ATP synthase (complex V). Even though the mtDNA encodes essential OXPHOS components, the large majority of the structural subunits and additional biogenetical factors (more than seventy proteins) are encoded in the nucleus and translated in the cytoplasm. To incorporate these proteins and the rest of the mitochondrial proteome, mitochondria have evolved varied, and sophisticated import machineries that specifically target proteins to the different compartments defined by the two membranes. The intermembrane space (IMS) contains a high number of cysteine-rich proteins, which are mostly imported via the MIA40 oxidative folding system, dependent on the reduction, and oxidation of key Cys residues. Several of these proteins are structural components or assembly factors necessary for the correct maturation and function of the ETC complexes. Interestingly, many of these proteins are involved in the metalation of the active redox centers of complex IV, the terminal oxidase of the mitochondrial ETC. Due to their function in oxygen reduction, mitochondria are the main generators of reactive oxygen species (ROS), on both sides of the inner membrane, i.e., in the matrix and the IMS. ROS generation is important due to their role as signaling molecules, but an excessive production is detrimental due to unwanted oxidation reactions that impact on the function of different types of biomolecules contained in mitochondria. Therefore, the maintenance of the redox balance in the IMS is essential for mitochondrial function. In this review, we will discuss the role that redox regulation plays in the maintenance of IMS homeostasis as well as how mitochondrial ROS generation may be a key regulatory factor for ETC biogenesis, especially for complex IV.

The mitochondrial intermembrane space (IMS) is the most constricted sub-mitochondrial compartment, housing only about 5% of the mitochondrial proteome, and yet is endowed with the largest variability of protein import mechanisms. In this review, we summarize our current knowledge of the major IMS import pathway based on the oxidative protein folding pathway and discuss the stunning variability of other IMS protein import pathways. As IMS-localized proteins only have to cross the outer mitochondrial membrane, they do not require energy sources like ATP hydrolysis in the mitochondrial matrix or the inner membrane electrochemical potential which are critical for import into the matrix or insertion into the inner membrane. We also explore several atypical IMS import pathways that are still not very well understood and are guided by poorly defined or completely unknown targeting peptides. Importantly, many of the IMS proteins are linked to several human diseases, and it is therefore crucial to understand how they reach their normal site of function in the IMS. In the final part of this review, we discuss current understanding of how such IMS protein underpin a large spectrum of human disorders.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

Thiol peroxidases are conserved hydrogen peroxide scavenging and signaling molecules that contain redox-active cysteine residues. We show here that Gpx3, the major H2O2 sensor in yeast, is present in the mitochondrial intermembrane space (IMS), where it serves a compartment-specific role in oxidative metabolism. The IMS-localized Gpx3 contains an 18-amino acid N-terminally extended form encoded from a non-AUG codon. This acts as a mitochondrial targeting signal in a pathway independent of the hitherto known IMS-import pathways. Mitochondrial Gpx3 interacts with the Mia40 oxidoreductase in a redox-dependent manner and promotes efficient Mia40-dependent oxidative protein folding. We show that cells lacking Gpx3 have aberrant mitochondrial morphology, defective protein import capacity, and lower inner membrane potential, all of which can be rescued by expression of a mitochondrial-only form of Gpx3. Together, our data reveal a novel role for Gpx3 in mitochondrial redox regulation and protein homeostasis.

Mitochondria are critical for several cellular functions as they control metabolism, cell physiology, and cell death. The mitochondrial proteome consists of around 1500 proteins, the vast majority of which (about 99% of them) are encoded by nuclear genes, with only 13 polypeptides in human cells encoded by mitochondrial DNA. Therefore, it is critical for all the mitochondrial proteins that are nuclear-encoded to be targeted precisely and sorted specifically to their site of action inside mitochondria. These processes of targeting and sorting are catalysed by protein translocases that operate in each one of the mitochondrial sub-compartments. The main protein import pathway for the intermembrane space (IMS) recognises proteins that are cysteine-rich, and it is the only import pathway that chemically modifies the imported precursors by introducing disulphide bonds to them. In this manner, the precursors are trapped in the IMS in a folded state. The key component of this pathway is Mia40 (called CHCHD4 in human cells), which itself contains cysteine motifs and is subject to redox regulation. In this review, we detail the basic components of the MIA pathway and the disulphide relay mechanism that underpins the electron transfer reaction along the oxidative folding mechanism. Then, we discuss the key protein modulators of this pathway and how they are interlinked to the small redox-active molecules that critically affect the redox state in the IMS. We present also evidence that the mitochondrial redox processes that are linked to iron–sulfur clusters biogenesis and calcium homeostasis coalesce in the IMS at the MIA machinery. The fact that the MIA machinery and several of its interactors and substrates are linked to a variety of common human diseases connected to mitochondrial dysfunction highlight the potential of redox processes in the IMS as a promising new target for developing new treatments for some of the most complex and devastating human diseases.

Evidence is accumulating that unrelated species have independently evolved the same way of importing proteins in their mitochondria.

AbstractDisulfide bond formation is a crucial step for oxidative folding and necessary for the acquisition of a protein's native conformation. Introduction of disulfide bonds is catalyzed in specialized subcellular compartments and requires the coordinated action of specific enzymes. The intermembrane space of mitochondria has recently been found to harbor a dedicated machinery that promotes the oxidative folding of substrate proteins by shuttling disulfide bonds. The newly identified oxidative pathway consists of the redox-regulated receptor Mia40 and the sulfhydryl oxidase Erv1. Proteins destined to the intermembrane space are trapped by a disulfide relay mechanism that involves an electron cascade from the incoming substrate to Mia40, then on to Erv1, and finally to molecular oxygen via cytochrome c. This thiol–disulfide exchange mechanism is essential for the import and for maintaining the structural stability of the incoming precursors. In this review we describe the mechanistic parameters that define the interaction and oxidation of the substrate proteins in light of the recent publications in the mitochondrial oxidative folding field. Antioxid. Redox Signal.13, 1189–1204. [ABSTRACT FROM AUTHOR]