-
Annual Review of Biochemistry Jun 2017Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized... (Review)
Review
Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics.
Topics: Carrier Proteins; Eukaryotic Cells; Gene Expression; Humans; Isoenzymes; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Organelle Biogenesis; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Precursors; Protein Transport
PubMed: 28301740
DOI: 10.1146/annurev-biochem-060815-014352 -
Nature May 2023Peroxisomes are organelles that carry out β-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction. Among...
Peroxisomes are organelles that carry out β-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction. Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes. Current models postulate a large pore formed by transmembrane proteins; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as 'stickers' in associative polymer models of LLPS. Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP-Pex13 and GFP-Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5-cargo. Our findings lead us to suggest a model in which LLPS of Pex5-cargo with Pex13 and Pex14 results in transient protein transport channels.
Topics: Intracellular Membranes; Membrane Proteins; Peroxins; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes; Phase Transition; Protein Binding; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Intrinsically Disordered Proteins
PubMed: 37165185
DOI: 10.1038/s41586-023-06044-1 -
The EMBO Journal Jul 2023Chloroplasts are plant organelles responsible for photosynthesis and environmental sensing. Most chloroplast proteins are imported from the cytosol through the...
Chloroplasts are plant organelles responsible for photosynthesis and environmental sensing. Most chloroplast proteins are imported from the cytosol through the translocon at the outer envelope membrane of chloroplasts (TOC). Previous work has shown that TOC components are regulated by the ubiquitin-proteasome system (UPS) to control the chloroplast proteome, which is crucial for the organelle's function and plant development. Here, we demonstrate that the TOC apparatus is also subject to K63-linked polyubiquitination and regulation by selective autophagy, potentially promoting plant stress tolerance. We identify NBR1 as a selective autophagy adaptor targeting TOC components, and mediating their relocation into vacuoles for autophagic degradation. Such selective autophagy is shown to control TOC protein levels and chloroplast protein import and to influence photosynthetic activity as well as tolerance to UV-B irradiation and heat stress in Arabidopsis plants. These findings uncover the vital role of selective autophagy in the proteolytic regulation of specific chloroplast proteins, and how dynamic control of chloroplast protein import is critically important for plants to cope with challenging environments.
Topics: Chloroplasts; Plants; Organelles; Protein Transport; Chloroplast Proteins; Arabidopsis; Autophagy; Plant Proteins; Arabidopsis Proteins; Carrier Proteins
PubMed: 37248861
DOI: 10.15252/embj.2022112534 -
Molecular Cell Jan 2022Most mitochondrial proteins are translated in the cytosol and imported into mitochondria. Mutations in the mitochondrial protein import machinery cause human...
Most mitochondrial proteins are translated in the cytosol and imported into mitochondria. Mutations in the mitochondrial protein import machinery cause human pathologies. However, a lack of suitable tools to measure protein uptake across the mitochondrial proteome has prevented the identification of specific proteins affected by import perturbation. Here, we introduce mePROD, a pulsed-SILAC based proteomics approach that includes a booster signal to increase the sensitivity for mitochondrial proteins selectively, enabling global dynamic analysis of endogenous mitochondrial protein uptake in cells. We applied mePROD to determine protein uptake kinetics and examined how inhibitors of mitochondrial import machineries affect protein uptake. Monitoring changes in translation and uptake upon mitochondrial membrane depolarization revealed that protein uptake was extensively modulated by the import and translation machineries via activation of the integrated stress response. Strikingly, uptake changes were not uniform, with subsets of proteins being unaffected or decreased due to changes in translation or import capacity.
Topics: Carbonyl Cyanide m-Chlorophenyl Hydrazone; Electron Transport Complex I; Female; HeLa Cells; Humans; Kinetics; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Proteins; Protein Biosynthesis; Protein Transport; Proteome; Proteomics; Uncoupling Agents
PubMed: 34847359
DOI: 10.1016/j.molcel.2021.11.004 -
Biochemical Society Transactions Oct 2015The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across... (Review)
Review
The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.
Topics: ATP-Binding Cassette Transporters; Adenosine Triphosphatases; Animals; Biocatalysis; Biological Transport, Active; Drug Resistance, Multiple; Humans; Isoenzymes; Models, Molecular; Mutation; Protein Conformation; Protein Isoforms; Protein Structure, Tertiary
PubMed: 26517899
DOI: 10.1042/BST20150047 -
Nature Mar 2017Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many...
Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.
Topics: Cell Line; Cytosol; HSP70 Heat-Shock Proteins; Heat-Shock Response; Homeostasis; Humans; Mitochondria; Mitochondrial Proteins; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein Aggregates; Protein Folding; Protein Refolding; Protein Stability; Protein Transport; Proteins; Proteolysis; Saccharomyces cerevisiae
PubMed: 28241148
DOI: 10.1038/nature21695 -
Nature Sep 2023Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical...
Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.
Topics: Cryoelectron Microscopy; Mitochondrial Precursor Protein Import Complex Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Mitochondria
PubMed: 37344598
DOI: 10.1038/s41586-023-06239-6 -
Science Translational Medicine Jun 2016α-Synuclein accumulation and mitochondrial dysfunction have both been strongly implicated in the pathogenesis of Parkinson's disease (PD), and the two appear to be...
α-Synuclein accumulation and mitochondrial dysfunction have both been strongly implicated in the pathogenesis of Parkinson's disease (PD), and the two appear to be related. Mitochondrial dysfunction leads to accumulation and oligomerization of α-synuclein, and increased levels of α-synuclein cause mitochondrial impairment, but the basis for this bidirectional interaction remains obscure. We now report that certain posttranslationally modified species of α-synuclein bind with high affinity to the TOM20 (translocase of the outer membrane 20) presequence receptor of the mitochondrial protein import machinery. This binding prevented the interaction of TOM20 with its co-receptor, TOM22, and impaired mitochondrial protein import. Consequently, there were deficient mitochondrial respiration, enhanced production of reactive oxygen species, and loss of mitochondrial membrane potential. Examination of postmortem brain tissue from PD patients revealed an aberrant α-synuclein-TOM20 interaction in nigrostriatal dopaminergic neurons that was associated with loss of imported mitochondrial proteins, thereby confirming this pathogenic process in the human disease. Modest knockdown of endogenous α-synuclein was sufficient to maintain mitochondrial protein import in an in vivo model of PD. Furthermore, in in vitro systems, overexpression of TOM20 or a mitochondrial targeting signal peptide had beneficial effects and preserved mitochondrial protein import. This study characterizes a pathogenic mechanism in PD, identifies toxic species of wild-type α-synuclein, and reveals potential new therapeutic strategies for neuroprotection.
Topics: Animals; Membrane Transport Proteins; Mitochondria; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Parkinson Disease; Protein Binding; Protein Transport; Rats; Rats, Mutant Strains; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; alpha-Synuclein
PubMed: 27280685
DOI: 10.1126/scitranslmed.aaf3634 -
Science (New York, N.Y.) Dec 2022Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal proteins are imported from the cytosol in a folded state by the...
Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal proteins are imported from the cytosol in a folded state by the soluble receptor PEX5. How folded cargo crosses the membrane is unknown. Here, we show that peroxisomal import is similar to nuclear transport. The peroxisomal membrane protein PEX13 contains a conserved tyrosine (Y)- and glycine (G)-rich YG domain, which forms a selective phase resembling that formed by phenylalanine-glycine (FG) repeats within nuclear pores. PEX13 resides in the membrane in two orientations that oligomerize and suspend the YG meshwork within the lipid bilayer. Purified YG domains form hydrogels into which PEX5 selectively partitions, by using conserved aromatic amino acid motifs, bringing cargo along. The YG meshwork thus forms an aqueous conduit through which PEX5 delivers folded proteins into peroxisomes.
Topics: Humans; Glycine; Nuclear Pore; Peroxisomes; Protein Transport; Membrane Proteins; Conserved Sequence; Protein Domains; Tyrosine
PubMed: 36520918
DOI: 10.1126/science.adf3971 -
International Journal of Molecular... Mar 2022Human mitochondrial disorders impact tissues with high energetic demands and can be associated with cardiac muscle disease (cardiomyopathy) and early mortality. However,... (Review)
Review
Human mitochondrial disorders impact tissues with high energetic demands and can be associated with cardiac muscle disease (cardiomyopathy) and early mortality. However, the mechanistic link between mitochondrial disease and the development of cardiomyopathy is frequently unclear. In addition, there is often marked phenotypic heterogeneity between patients, even between those with the same genetic variant, which is also not well understood. Several of the mitochondrial cardiomyopathies are related to defects in the maintenance of mitochondrial protein homeostasis, or proteostasis. This essential process involves the importing, sorting, folding and degradation of preproteins into fully functional mature structures inside mitochondria. Disrupted mitochondrial proteostasis interferes with mitochondrial energetics and ATP production, which can directly impact cardiac function. An inability to maintain proteostasis can result in mitochondrial dysfunction and subsequent mitophagy or even apoptosis. We review the known mitochondrial diseases that have been associated with cardiomyopathy and which arise from mutations in genes that are important for mitochondrial proteostasis. Genes discussed include DnaJ heat shock protein family member C19 (), mitochondrial import inner membrane translocase subunit TIM16 (), translocase of the inner mitochondrial membrane 50 (), mitochondrial intermediate peptidase (), X-prolyl-aminopeptidase 3 (), HtraA serine peptidase 2 (), caseinolytic mitochondrial peptidase chaperone subunit B () and heat shock 60-kD protein 1 (HSPD1). The identification and description of disorders with a shared mechanism of disease may provide further insights into the disease process and assist with the identification of potential therapeutics.
Topics: Cardiomyopathies; HSP40 Heat-Shock Proteins; Homeostasis; Humans; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Proteins; Peptide Hydrolases; Protein Transport; Proteostasis
PubMed: 35328774
DOI: 10.3390/ijms23063353