The mitochondrial proteome comprises ~1000 (yeast)-1500 (human) different proteins, which are distributed into four different subcompartments. The sublocalization of these proteins within the ...organelle in most cases remains poorly defined. Here we describe an integrated approach combining stable isotope labeling, various protein enrichment and extraction strategies and quantitative mass spectrometry to produce a quantitative map of submitochondrial protein distribution in S. cerevisiae. This quantitative landscape enables a proteome-wide classification of 986 proteins into soluble, peripheral, and integral mitochondrial membrane proteins, and the assignment of 818 proteins into the four subcompartments: outer membrane, inner membrane, intermembrane space, or matrix. We also identified 206 proteins that were not previously annotated as localized to mitochondria. Furthermore, the protease Prd1, misannotated as intermembrane space protein, could be re-assigned and characterized as a presequence peptide degrading enzyme in the matrix.Protein localization plays an important role in the regulation of cellular physiology. Here the authors use an integrated proteomics approach to localize proteins to the mitochondria and provide a detailed map of their specific localization within the organelle.
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.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
A study in this issue provides the first molecular understanding of the mitochondrial‐peroxisomal link in peroxisomal disorders.
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.
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•Impaired presequence processing leads to precursor aggregation inside mitochondria•Intramitochondrial precursor aggregates trigger early transcriptional stress response•Relocalization of nuclear transcription factor Rox1 to mitochondria ensures survival•Mitochondrial Rox1 maintains mitochondrial genome expression upon early mtUPR
N-terminal presequences direct cytosolic precursor proteins to mitochondria. Poveda-Huertes et al. show that impaired presequence cleavage leads to proteotoxic aggregates inside mitochondria that trigger an early mtUPR-like stress response. Relocalization of the nuclear transcription factor Rox1 to mitochondria allows maintenance of mtDNA expression ensuring proteostasis and survival upon early mtUPR.
Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that ...protein dephosphorylation may be broadly important for calibrating mitochondrial activities. To explore this, we deleted the poorly characterized matrix phosphatase Pptc7 from mice using CRISPR-Cas9 technology. Strikingly, Pptc7
mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7
tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Among these, we identify the protein translocase complex subunit Timm50 as a putative Pptc7 substrate whose phosphorylation reduces import activity. We further find that phosphorylation within or near the mitochondrial targeting sequences of multiple proteins could disrupt their import rates and matrix processing. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.
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.
The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer ...membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.
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.
►We present an overview of mitochondrial preprotein import and presequence processing. ►We review recent developments in the field of mitochondrial presequence processing. ►We highlight a novel mitochondrial protein quality control mechanism.
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.
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