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1.
EMBO J ; 43(5): 754-779, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38287189

ABSTRACT

Tank-binding kinase 1 (TBK1) is a Ser/Thr kinase that is involved in many intracellular processes, such as innate immunity, cell cycle, and apoptosis. TBK1 is also important for phosphorylating the autophagy adaptors that mediate the selective autophagic removal of damaged mitochondria. However, the mechanism by which PINK1-Parkin-mediated mitophagy activates TBK1 remains largely unknown. Here, we show that the autophagy adaptor optineurin (OPTN) provides a unique platform for TBK1 activation. Both the OPTN-ubiquitin and the OPTN-pre-autophagosomal structure (PAS) interaction axes facilitate assembly of the OPTN-TBK1 complex at a contact sites between damaged mitochondria and the autophagosome formation sites. At this assembly point, a positive feedback loop for TBK1 activation is initiated that accelerates hetero-autophosphorylation of the protein. Expression of monobodies engineered here to bind OPTN impaired OPTN accumulation at contact sites, as well as the subsequent activation of TBK1, thereby inhibiting mitochondrial degradation. Taken together, these data show that a positive and reciprocal relationship between OPTN and TBK1 initiates autophagosome biogenesis on damaged mitochondria.


Subject(s)
Cell Cycle Proteins , Membrane Transport Proteins , Mitochondrial Membranes , Mitophagy , Humans , Autophagy/physiology , Cell Cycle Proteins/metabolism , HeLa Cells , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Protein Serine-Threonine Kinases/metabolism
2.
Proc Natl Acad Sci U S A ; 121(2): e2306454120, 2024 Jan 09.
Article in English | MEDLINE | ID: mdl-38170752

ABSTRACT

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.


Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Hexokinase , Hexokinase/genetics , Hexokinase/metabolism , Prospective Studies , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Mitochondria/metabolism , Lysosomes/metabolism , Protein Kinases/metabolism , Cellular Senescence/genetics , Homeostasis , Autophagy/genetics
3.
J Biol Chem ; : 107476, 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38879013

ABSTRACT

DJ-1, a causative gene for hereditary recessive Parkinsonism, is evolutionarily conserved across eukaryotes and prokaryotes. Structural analyses of DJ-1 and its homologs suggested the 106th Cys is a nucleophilic cysteine functioning as the catalytic center of hydratase or hydrolase activity. Indeed, DJ-1 and its homologs can convert highly electrophilic α-oxoaldehydes such as methylglyoxal into α-hydroxy acids as hydratase in vitro, and oxidation-dependent ester hydrolase (esterase) activity has also been reported for DJ-1. The mechanism underlying such plural activities, however, has not been fully characterized. To address this knowledge gap, we conducted a series of biochemical assays assessing the enzymatic activity of DJ-1 and its homologs. We found no evidence for esterase activity in any of the Escherichia coli DJ-1 homologs. Furthermore, contrary to previous reports, we found that oxidation inactivated rather than facilitated DJ-1 esterase activity. The E. coli DJ-1 homolog HchA possesses phenylglyoxalase and methylglyoxalase activities but lacks esterase activity. Since evolutionary trace analysis identified the 186th H as a candidate residue involved in functional differentiation between HchA and DJ-1, we focused on H186 of HchA and found that an esterase activity was acquired by H186A mutation. Introduction of reverse mutations into the equivalent position in DJ-1 (A107H) selectively eliminated its esterase activity without compromising α-oxoaldehyde hydratase activity. The obtained results suggest that differences in the amino acid sequences near the active site contributed to acquisition of esterase activity in vitro, and provide an important clue to the origin and significance of DJ-1 esterase activity.

4.
EMBO J ; 40(3): e104705, 2021 02 01.
Article in English | MEDLINE | ID: mdl-33438778

ABSTRACT

Degradation of mitochondria via a selective form of autophagy, named mitophagy, is a fundamental mechanism conserved from yeast to humans that regulates mitochondrial quality and quantity control. Mitophagy is promoted via specific mitochondrial outer membrane receptors, or ubiquitin molecules conjugated to proteins on the mitochondrial surface leading to the formation of autophagosomes surrounding mitochondria. Mitophagy-mediated elimination of mitochondria plays an important role in many processes including early embryonic development, cell differentiation, inflammation, and apoptosis. Recent advances in analyzing mitophagy in vivo also reveal high rates of steady-state mitochondrial turnover in diverse cell types, highlighting the intracellular housekeeping role of mitophagy. Defects in mitophagy are associated with various pathological conditions such as neurodegeneration, heart failure, cancer, and aging, further underscoring the biological relevance. Here, we review our current molecular understanding of mitophagy, and its physiological implications, and discuss how multiple mitophagy pathways coordinately modulate mitochondrial fitness and populations.


Subject(s)
Gene Regulatory Networks , Mitochondria/physiology , Animals , Autophagy-Related Proteins/metabolism , Fungi/metabolism , Humans , Mitochondrial Proteins/metabolism , Mitophagy
5.
Cell ; 142(3): 362-3, 2010 Aug 06.
Article in English | MEDLINE | ID: mdl-20691896

ABSTRACT

Although mitochondrial biogenesis requires the import of specific RNAs, the pathways and cellular machineries involved are only poorly understood. Wang et al. (2010) now find that polynucleotide phosphorylase in the intermembrane space of mammalian mitochondria facilitates import of several RNAs into the mitochondrial matrix.

6.
J Biol Chem ; 299(2): 102822, 2023 02.
Article in English | MEDLINE | ID: mdl-36563856

ABSTRACT

RING-between RING (RBR)-type ubiquitin (Ub) ligases (E3s) such as Parkin receive Ub from Ub-conjugating enzymes (E2s) in response to ligase activation. However, the specific E2s that transfer Ub to each RBR-type ligase are largely unknown because of insufficient methods for monitoring their interaction. To address this problem, we have developed a method that detects intracellular interactions between E2s and activated Parkin. Fluorescent homotetramer Azami-Green fused with E2 and oligomeric Ash (Assembly helper) fused with Parkin form a liquid-liquid phase separation (LLPS) in cells only when E2 and Parkin interact. Using this method, we identified multiple E2s interacting with activated Parkin on damaged mitochondria during mitophagy. Combined with in vitro ubiquitination assays and bioinformatics, these findings revealed an underlying consensus sequence for E2 interactions with activated Parkin. Application of this method to other RBR-type E3s including HOIP, HHARI, and TRIAD1 revealed that HOIP forms an LLPS with its substrate NEMO in response to a proinflammatory cytokine and that HHARI and TRIAD1 form a cytosolic LLPS independent of Ub-like protein NEDD8. Since an E2-E3 interaction is a prerequisite for RBR-type E3 activation and subsequent substrate ubiquitination, the method we have established here can be an in-cell tool to elucidate the potentially novel mechanisms involved in RBR-type E3s.


Subject(s)
Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligases , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/isolation & purification , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/isolation & purification , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Protein Binding , Mitophagy , Mitochondria/metabolism , Mitochondria/pathology , I-kappa B Kinase/metabolism
7.
J Cell Sci ; 134(22)2021 11 15.
Article in English | MEDLINE | ID: mdl-34676411

ABSTRACT

Diverse genes associated with familial Parkinson's disease (familial Parkinsonism) have been implicated in mitochondrial quality control. One such gene, PARK7 encodes the protein DJ-1, pathogenic mutations of which trigger its translocation from the cytosol to the mitochondrial matrix. The translocation of steady-state cytosolic proteins like DJ-1 to the mitochondrial matrix upon missense mutations is rare, and the underlying mechanism remains to be elucidated. Here, we show that the protein unfolding associated with various DJ-1 mutations drives its import into the mitochondrial matrix. Increasing the structural stability of these DJ-1 mutants restores cytosolic localization. Mechanistically, we show that a reduction in the structural stability of DJ-1 exposes a cryptic N-terminal mitochondrial-targeting signal (MTS), including Leu10, which promotes DJ-1 import into the mitochondrial matrix for subsequent degradation. Our work describes a novel cellular mechanism for targeting a destabilized cytosolic protein to the mitochondria for degradation.


Subject(s)
Parkinson Disease , Humans , Mitochondria/genetics , Parkinson Disease/genetics
8.
EMBO Rep ; 20(12): e47728, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31602805

ABSTRACT

Ubiquitylation of outer mitochondrial membrane (OMM) proteins is closely related to the onset of familial Parkinson's disease. Typically, a reduction in the mitochondrial membrane potential results in Parkin-mediated ubiquitylation of OMM proteins, which are then targeted for proteasomal and mitophagic degradation. The role of ubiquitylation of OMM proteins with non-degradative fates, however, remains poorly understood. In this study, we find that the mitochondrial E3 ubiquitin ligase MITOL/March5 translocates from depolarized mitochondria to peroxisomes following mitophagy stimulation. This unusual redistribution is mediated by peroxins (peroxisomal biogenesis factors) Pex3/16 and requires the E3 ligase activity of Parkin, which ubiquitylates K268 in the MITOL C-terminus, essential for p97/VCP-dependent mitochondrial extraction of MITOL. These findings imply that ubiquitylation directs peroxisomal translocation of MITOL upon mitophagy stimulation and reveal a novel role for ubiquitin as a sorting signal that allows certain specialized proteins to escape from damaged mitochondria.


Subject(s)
Membrane Proteins/metabolism , Mitochondria/metabolism , Peroxisomes/metabolism , Ubiquitin-Protein Ligases/metabolism , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Membrane Proteins/chemistry , Mitophagy , Peroxins/metabolism , Protein Transport , Ubiquitin-Protein Ligases/chemistry , Ubiquitination , Valosin Containing Protein/metabolism
9.
J Biol Chem ; 294(26): 10300-10314, 2019 06 28.
Article in English | MEDLINE | ID: mdl-31110043

ABSTRACT

PINK1 (PARK6) and PARKIN (PARK2) are causal genes of recessive familial Parkinson's disease. Parkin is a ubiquitin ligase E3 that conjugates ubiquitin to impaired mitochondrial proteins for organelle degradation. PINK1, a Ser/Thr kinase that accumulates only on impaired mitochondria, phosphorylates two authentic substrates, the ubiquitin-like domain of Parkin and ubiquitin. Our group and others have revealed that both the subcellular localization and ligase activity of Parkin are regulated through interactions with phosphorylated ubiquitin. Once PINK1 localizes on impaired mitochondria, PINK1-catalyzed phosphoubiquitin recruits and activates Parkin. Parkin then supplies a ubiquitin chain to PINK1 for phosphorylation. The amplified ubiquitin functions as a signal for the sequestration and degradation of the damaged mitochondria. Although a bewildering variety of Parkin substrates have been reported, the basis for Parkin substrate specificity remains poorly understood. Moreover, the mechanism underlying initial activation and translocation of Parkin onto mitochondria remains unclear, because the presence of ubiquitin on impaired mitochondria is thought to be a prerequisite for the initial PINK1 phosphorylation process. Here, we show that artificial mitochondria-targeted proteins are ubiquitylated by Parkin, suggesting that substrate specificity of Parkin is not determined by its amino acid sequence. Moreover, recruitment and activation of Parkin are delayed following depletion of the mitochondrial E3, MITOL/March5. We propose a model in which the initial step in Parkin recruitment and activation requires protein ubiquitylation by MITOL/March5 with subsequent PINK1-mediated phosphorylation. Because PINK1 and Parkin amplify the ubiquitin signal via a positive feedback loop, the low substrate specificity of Parkin might facilitate this amplification process.


Subject(s)
Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , HeLa Cells , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Mitochondria/genetics , Mitochondrial Proteins/genetics , Phosphorylation , Protein Kinases/genetics , RNA, Small Interfering/genetics , Substrate Specificity , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/genetics , Ubiquitination
10.
Genes Cells ; 23(10): 822-827, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30273445

ABSTRACT

The 1st International Mitochondria Meeting for Young Scientists (International YoungMito 2018) was held at Hotel Co-op Inn Kyoto in Kyoto, Japan, from 20 to 22 April 2018. The meeting was attended by 130 mitochondrial researchers from 15 countries. International YoungMito 2018 was the first international mitochondria meeting held in Japan organized by and for young mitochondrial researchers. Over the 3-day period, there were 28 oral presentations including two keynote lectures, 20 presentations from invited speakers, and six short talks selected from abstract submissions. Many different topics were covered including quality control pathways acting against mitochondrial stresses, mitochondrial dynamics, protein/lipid transport, cristae organization, respiration/ATP synthesis, mtDNA maintenance, mitochondrial disease models, and pharmacological approaches. In addition, we had 64 posters, a number which represented almost half of all attendees. Thanks to the cutting-edge information and high-quality unpublished data that were presented, there were many lively discussions during oral and poster sessions that continued into the coffee breaks, lunchtime, and nighttime discussions. The 1st international YoungMito meeting was successful in promoting intellectual exchange among all participants, facilitating collaborations beyond national boundaries, and closed with great success. It was a great pleasure that many participants were looking forward to the next YoungMito meeting.


Subject(s)
Mitochondria , Humans , Japan , Research
11.
Nature ; 504(7479): 291-5, 2013 Dec 12.
Article in English | MEDLINE | ID: mdl-24270810

ABSTRACT

An increasing body of evidence points to mitochondrial dysfunction as a contributor to the molecular pathogenesis of neurodegenerative diseases such as Parkinson's disease. Recent studies of the Parkinson's disease associated genes PINK1 (ref. 2) and parkin (PARK2, ref. 3) indicate that they may act in a quality control pathway preventing the accumulation of dysfunctional mitochondria. Here we elucidate regulators that have an impact on parkin translocation to damaged mitochondria with genome-wide small interfering RNA (siRNA) screens coupled to high-content microscopy. Screening yielded gene candidates involved in diverse cellular processes that were subsequently validated in low-throughput assays. This led to characterization of TOMM7 as essential for stabilizing PINK1 on the outer mitochondrial membrane following mitochondrial damage. We also discovered that HSPA1L (HSP70 family member) and BAG4 have mutually opposing roles in the regulation of parkin translocation. The screens revealed that SIAH3, found to localize to mitochondria, inhibits PINK1 accumulation after mitochondrial insult, reducing parkin translocation. Overall, our screens provide a rich resource to understand mitochondrial quality control.


Subject(s)
Genome, Human/genetics , Mitophagy , RNA Interference , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , HCT116 Cells , HEK293 Cells , HSP70 Heat-Shock Proteins/metabolism , HeLa Cells , Humans , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/metabolism , Multigene Family/genetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Protein Kinases/metabolism , Protein Transport , RNA, Small Interfering/analysis , RNA, Small Interfering/genetics , Reproducibility of Results
12.
EMBO Rep ; 17(3): 300-16, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26882551

ABSTRACT

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early-onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin-/PINK1-mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.


Subject(s)
Autophagy , Mitochondria/metabolism , Mitophagy , Parkinson Disease/metabolism , Ubiquitination , Animals , Humans , Protein Kinases/genetics , Protein Kinases/metabolism
13.
Genes Cells ; 21(7): 772-88, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27270837

ABSTRACT

DJ-1 has been identified as a gene responsible for recessive familial Parkinson's disease (familial Parkinsonism), which is caused by a mutation in the PARK7 locus. Consistent with the inferred correlation between Parkinson's disease and mitochondrial impairment, mitochondrial localization of DJ-1 and its implied role in mitochondrial quality control have been reported. However, the mechanism by which DJ-1 affects mitochondrial function remains poorly defined, and the mitochondrial localization of DJ-1 is still controversial. Here, we show the mitochondrial matrix localization of various pathogenic and artificial DJ-1 mutants by multiple independent experimental approaches including cellular fractionation, proteinase K protection assays, and specific immunocytochemistry. Localization of various DJ-1 mutants to the matrix is dependent on the membrane potential and translocase activity in both the outer and the inner membranes. Nevertheless, DJ-1 possesses neither an amino-terminal alpha-helix nor a predictable matrix-targeting signal, and a post-translocation processing-derived molecular weight change is not observed. In fact, wild-type DJ-1 does not show any evidence of mitochondrial localization at all. Such a mode of matrix localization of DJ-1 is difficult to explain by conventional mechanisms and implies a unique matrix import mechanism for DJ-1 mutants.


Subject(s)
Membrane Potential, Mitochondrial/genetics , Mutant Proteins/genetics , Parkinson Disease/genetics , Protein Deglycase DJ-1/genetics , Humans , Mitochondria/genetics , Mitochondrial Membranes/chemistry , Mutant Proteins/isolation & purification , Mutation , Parkinson Disease/pathology , Protein Deglycase DJ-1/chemistry , Protein Deglycase DJ-1/isolation & purification
14.
J Biol Chem ; 290(42): 25199-211, 2015 Oct 16.
Article in English | MEDLINE | ID: mdl-26260794

ABSTRACT

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser(65) by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity.


Subject(s)
Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Binding Sites , HeLa Cells , Humans , Phosphorylation , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/chemistry
15.
EMBO J ; 30(6): 1003-11, 2011 Mar 16.
Article in English | MEDLINE | ID: mdl-21326212

ABSTRACT

While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.


Subject(s)
Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Membrane Proteins/chemistry , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/chemistry , Models, Biological , Molecular Sequence Data , Protein Structure, Secondary
16.
Proc Natl Acad Sci U S A ; 108(37): 15179-83, 2011 Sep 13.
Article in English | MEDLINE | ID: mdl-21896724

ABSTRACT

Mitochondrial protein import requires cooperation of the machineries called translocators in the outer and inner mitochondrial membranes. Here we analyze the interactions of Tom22, a multifunctional subunit of the outer membrane translocator TOM40 complex, with other translocator subunits such as Tom20, Tom40, and Tim50 and with substrate precursor proteins at a spatial resolution of the amino acid residue by in vivo and in organello site-specific photocross-linking. Changes in cross-linking patterns caused by excess substrate precursor proteins or presequence peptides indicate how the cytosolic receptor domain of Tom22 accepts substrate proteins and how the intermembrane space domain of Tom22 transfers them to Tim50 of the inner-membrane translocator.


Subject(s)
Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Proteins/metabolism , Protein Interaction Mapping , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Cross-Linking Reagents/metabolism , Cytosol/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Proteins/chemistry , Molecular Sequence Data , Peptides/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/chemistry
17.
J Biochem ; 175(3): 217-219, 2024 Mar 04.
Article in English | MEDLINE | ID: mdl-38156789

ABSTRACT

Mitochondria-endoplasmic reticulum (ER) contact sites in mammals provide platforms for various reactions, such as calcium signaling, lipid metabolism, organelle dynamics and autophagy. To fulfill these tasks, a number of proteins assemble at the contact sites including MITOL/MARCHF5, a critical mitochondrial ubiquitin ligase. How MITOL regulates mitochondrial function from the contact site, however, has been largely unresolved. Recently, a new role for MITOL in the active transport of phosphatidic acid from the ER to mitochondria was reported. In this commentary, we briefly summarize our current understanding of mitochondria-ER contact sites and discuss the recently elucidated mechanism of MITOL fine-tuning phospholipid transfer activity through ubiquitination.


Subject(s)
Autophagy , Mitochondria , Animals , Calcium Signaling , Ubiquitination , Lipids , Mammals
18.
J Biochem ; 175(5): 487-494, 2024 Apr 29.
Article in English | MEDLINE | ID: mdl-38102729

ABSTRACT

Mitochondria are essential eukaryotic organelles that produce ATP as well as synthesize various macromolecules. They also participate in signalling pathways such as the innate immune response and apoptosis. These diverse functions are performed by >1,000 different mitochondrial proteins. Although mitochondria are continuously exposed to potentially damaging conditions such as reactive oxygen species, proteases/peptidases localized in different mitochondrial subcompartments, termed mitoproteases, maintain mitochondrial quality and integrity. In addition to processing incoming precursors and degrading damaged proteins, mitoproteases also regulate metabolic reactions, mitochondrial protein half-lives and gene transcription. Impaired mitoprotease function is associated with various pathologies. In this review, we highlight recent advances in our understanding of mitochondrial quality control regulated by autophagy, ubiquitin-proteasomes and mitoproteases.


Subject(s)
Mitochondria , Mitochondrial Proteins , Proteolysis , Humans , Mitochondria/metabolism , Animals , Mitochondrial Proteins/metabolism , Autophagy/physiology , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism
19.
Dev Cell ; 58(13): 1189-1205.e11, 2023 07 10.
Article in English | MEDLINE | ID: mdl-37192622

ABSTRACT

In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis.


Subject(s)
Liver Neoplasms , Proteomics , Animals , Humans , Mice , Sequestosome-1 Protein/metabolism , Autophagy , Organelles/metabolism
20.
Biochim Biophys Acta ; 1808(3): 955-70, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20655871

ABSTRACT

Mitochondrial functions rely on precise and efficient transport of 1000-1500 different mitochondrial proteins from the cytosol to appropriate mitochondrial subcompartments. Those mitochondrial protein transport processes are mediated by the dedicated mitochondrial protein import system comprised of translocators in the outer and inner mitochondrial membranes and soluble factors in the cytosol, intermembrane space, and matrix. In the last decade, high-resolution structures of many of the components of the mitochondrial protein import machineries have become available, which has significantly advanced our understanding of the molecular mechanisms of mitochondrial protein transport. Here we review the currently available high-resolution structures of the components of the mitochondrial protein import machineries that afford structural and mechanistic insight into how the mitochondrial import system works. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.


Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Animals , Humans , Protein Transport
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