An Early mtUPR: Redistribution of the Nuclear Transcription Factor Rox1 to Mitochondria Protects against Intramitochondrial Proteotoxic Aggregates.

The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.

A common evolutionary origin reveals fundamental principles of protein insertases.

Membrane proteins require protein machineries to insert their hydrophobic transmembrane domains (TMDs) into the lipid bilayer. A functional analysis of protein insertases in this issue of PLOS Biology reveals that the fundamental mechanism of membrane protein insertion is universally conserved.

p166 links membrane and intramitochondrial modules of the trypanosomal tripartite attachment complex.

The protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only known TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Whereas the outer membrane region requires four essential subunits for proper TAC function, the inner membrane integral p166, via its interaction with TAC60 and TAC102, would theoretically suffice to bridge the distance between the OM and the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of additional TAC-associated proteins which loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC. However, binding of full length p166 to these TAC-associated proteins alone would not be sufficient to withstand the mechanical load imposed by the segregating basal bodies.

Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability.

Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.

Increased mitochondrial protein import and cardiolipin remodelling upon early mtUPR.

Mitochondrial defects can cause a variety of human diseases and protective mechanisms exist to maintain mitochondrial functionality. Imbalances in mitochondrial proteostasis trigger a transcriptional program, termed mitochondrial unfolded protein response (mtUPR). However, the temporal sequence of events in mtUPR is unclear and the consequences on mitochondrial protein import are controversial. Here, we have quantitatively analyzed all main import pathways into mitochondria after different time spans of mtUPR induction. Kinetic analyses reveal that protein import into all mitochondrial subcompartments strongly increases early upon mtUPR and that this is accompanied by rapid remodelling of the mitochondrial signature lipid cardiolipin. Genetic inactivation of cardiolipin synthesis precluded stimulation of protein import and compromised cellular fitness. At late stages of mtUPR upon sustained stress, mitochondrial protein import efficiency declined. Our work clarifies the enigma of protein import upon mtUPR and identifies sequential mtUPR stages, in which an early increase in protein biogenesis to restore mitochondrial proteostasis is followed by late stages characterized by a decrease in import capacity upon prolonged stress induction.

Mitochondria as emergency landing for abandoned peroxins.

Zellweger spectrum disorder (ZSD) is the most severe peroxisomal biogenesis disorder (PBD). Why ZSD patients not only loose functional peroxisomes but also present with severe mitochondrial dysfunction was a long-standing mystery. In this issue, Nuebel et al (2021) identified that loss of peroxisomes leads to re-routing of peroxisomal proteins to mitochondria, thereby impairing mitochondrial structure and function. The findings provide the first molecular understanding of the mitochondrial-peroxisomal link in ZSD.

Improving Identification of In-organello Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker.

An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.

Mutations in PMPCB Encoding the Catalytic Subunit of the Mitochondrial Presequence Protease Cause Neurodegeneration in Early Childhood.

Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.

COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2.

Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2.

Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease.

Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

The versatility of the mitochondrial presequence processing machinery: cleavage, quality control and turnover.

Mitochondria play a key role in several metabolic and cell biological pathways and have attracted increasing attention due to their implication in life-span, ageing and human diseases. Mitochondrial proteases have a special role in these multiple biological functions, as they are involved in the regulation of various processes, e.g., mitochondrial protein biogenesis and quality control, mitochondrial dynamics, mitophagy and programmed cell death. The mitochondrial presequence processing machinery serves the particular purpose of maturing the majority of incoming precursor proteins by presequence cleavage, to ensure a stable mature protein by trimming of intermediate N-termini and to remove free toxic targeting peptides.

Quantitative Profiling for Substrates of the Mitochondrial Presequence Processing Protease Reveals a Set of Nonsubstrate Proteins Increased upon Proteotoxic Stress.

The majority of mitochondrial preproteins are targeted via N-terminal presequences that are cleaved upon import into the organelle. The essential mitochondrial processing protease (MPP) is assumed to cleave the majority of incoming precursors; however, only a small fraction of mitochondrial precursors have been experimentally analyzed limiting the information on MPP recognition and substrate specificity. Here we present the first systematic approach for identification of authentic MPP substrate proteins using a temperature-sensitive mutant of the MPP subunit Mas1. Inactivation of MPP at nonpermissive temperature leads to accumulation of immature precursors in mitochondria, which were measured by quantitative N-terminal ChaFRADIC. This led to the identification of 66 novel MPP substrates. Deduction of the cleaved presequences determines arginine in position -2 of the cleavage site as a main factor for MPP recognition. Interestingly, a set of nonprocessed proteins was also increased in mas1 mutant mitochondria. Additionally, mas1 mitochondria respond to temperature elevation with an increase in membrane potential and oxygen consumption. These changes might indicate that mas1 cells exert a response to balance the proteotoxic stress induced by MPP dysfunction.

A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis.

Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-X(L) and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms.

TNFα-induced lysosomal membrane permeability is downstream of MOMP and triggered by caspase-mediated NDUFS1 cleavage and ROS formation.

When NF-κB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFα) can induce apoptosis through Bax- and Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFα-induced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we present the novel finding that LMP induced by the addition of TNFα plus cycloheximide (CHX), the release of lysosomal cathepsins and ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent LMP mediated by TNFα plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFα apoptosis signaling.

Intermembrane space proteome of yeast mitochondria.

The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.

Synchronized assembly of the oxidative phosphorylation system controls mitochondrial respiration in yeast.

Control of protein stoichiometry is essential for cell function. Mitochondrial oxidative phosphorylation (OXPHOS) presents a complex stoichiometric challenge as the ratio of the electron transport chain (ETC) and ATP synthase must be tightly controlled, and assembly requires coordinated integration of proteins encoded in the nuclear and mitochondrial genome. How correct OXPHOS stoichiometry is achieved is unknown. We identify the Mitochondrial Regulatory hub for respiratory Assembly (MiRA) platform, which synchronizes ETC and ATP synthase biogenesis in yeast. Molecularly, this is achieved by a stop-and-go mechanism: the uncharacterized protein Mra1 stalls complex IV assembly. Two "Go" signals are required for assembly progression: binding of the complex IV assembly factor Rcf2 and Mra1 interaction with an Atp9-translating mitoribosome induce Mra1 degradation, allowing synchronized maturation of complex IV and the ATP synthase. Failure of the stop-and-go mechanism results in cell death. MiRA controls OXPHOS assembly, ensuring correct stoichiometry of protein machineries encoded by two different genomes.

DYRK1A signalling synchronizes the mitochondrial import pathways for metabolic rewiring.

Mitochondria require an extensive proteome to maintain a variety of metabolic reactions, and changes in cellular demand depend on rapid adaptation of the mitochondrial protein composition. The TOM complex, the organellar entry gate for mitochondrial precursors in the outer membrane, is a target for cytosolic kinases to modulate protein influx. DYRK1A phosphorylation of the carrier import receptor TOM70 at Ser91 enables its efficient docking and thus transfer of precursor proteins to the TOM complex. Here, we probe TOM70 phosphorylation in molecular detail and find that TOM70 is not a CK2 target nor import receptor for MIC19 as previously suggested. Instead, we identify TOM20 as a MIC19 import receptor and show off-target inhibition of the DYRK1A-TOM70 axis with the clinically used CK2 inhibitor CX4945 which activates TOM20-dependent import pathways. Taken together, modulation of DYRK1A signalling adapts the central mitochondrial protein entry gate via synchronization of TOM70- and TOM20-dependent import pathways for metabolic rewiring. Thus, DYRK1A emerges as a cytosolic surveillance kinase to regulate and fine-tune mitochondrial protein biogenesis.

Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55.

We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two S. cerevisiae samples within 10 h of LC-MS, starting from only 50 µg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Δ yeast (30 µg each). Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)-dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule. Using ChaFRADIC we identified 36 icp55 substrates, 14 of which were previously unknown, expanding the set of known icp55 substrates to a total of 52 proteins. Interestingly, a novel substrate, Isa2, is likely processed by Icp55 in two consecutive steps and thus might represent the first example of a triple processing event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful and practicable tool for protease and peptidase research, providing the sensitivity to characterize even samples that can be obtained only in small quantities.

Processing and topology of the yeast mitochondrial phosphatidylserine decarboxylase 1.

The inner mitochondrial membrane plays a crucial role in cellular lipid homeostasis through biosynthesis of the non-bilayer-forming lipids phosphatidylethanolamine and cardiolipin. In the yeast Saccharomyces cerevisiae, the majority of cellular phosphatidylethanolamine is synthesized by the mitochondrial phosphatidylserine decarboxylase 1 (Psd1). The biogenesis of Psd1 involves several processing steps. It was speculated that the Psd1 precursor is sorted into the inner membrane and is subsequently released into the intermembrane space by proteolytic removal of a hydrophobic sorting signal. However, components involved in the maturation of the Psd1 precursor have not been identified. We show that processing of Psd1 involves the action of the mitochondrial processing peptidase and Oct1 and an autocatalytic cleavage at a highly conserved LGST motif yielding the α- and ß-subunit of the enzyme. The Psd1 ß-subunit (Psd1ß) forms the membrane anchor, which binds the intermembrane space-localized α-subunit (Psd1α). Deletion of a transmembrane segment in the ß-subunit results in mislocalization of Psd1 and reduced enzymatic activity. Surprisingly, autocatalytic cleavage does not depend on proper localization to the inner mitochondrial membrane. In summary, membrane integration of Psd1 is crucial for its functionality and for maintenance of mitochondrial lipid homeostasis.

Processing of mitochondrial presequences.

Mitochondrial proteins are synthesized as precursor proteins on either cytosolic or mitochondrial ribosomes. The synthesized precursors from both translation origins possess targeting signals that guide the protein to its final destination in one of the four subcompartments of the organelle. The majority of nuclear-encoded mitochondrial precursors and also mitochondrial-encoded preproteins have an N-terminal presequence that serves as a targeting sequence. Specific presequence peptidases that are found in the matrix, inner membrane and intermembrane space of mitochondria proteolytically remove the signal sequence upon import or sorting. Besides the classical presequence peptidases MPP, IMP and Oct1, several novel proteases have recently been described to possess precursor processing activity, and analysis of their functional relevance revealed a tight connection between precursor processing, mitochondrial dynamics and protein quality control. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.