Affinage

TIMM50

Mitochondrial import inner membrane translocase subunit TIM50 · UniProt Q3ZCQ8

Length
353 aa
Mass
39.6 kDa
Annotated
2026-04-28
39 papers in source corpus 20 papers cited in narrative 20 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

TIMM50 (Tim50) is an essential subunit of the mitochondrial TIM23 translocase that serves as the primary presequence receptor at the inner membrane, coupling preprotein recognition in the intermembrane space (IMS) with channel gating and import motor activation in the matrix. Its large IMS domain captures preproteins emerging from the TOM complex and directs them to the Tim23 channel via a structurally characterized β-hairpin-mediated interaction, while simultaneously closing the Tim23 pore in the absence of substrate to maintain the inner membrane permeability barrier; presequences antagonize this closure to activate translocation (PMID:12437924, PMID:16763150, PMID:21704637). Tim50 transmits presequence recognition across the membrane through its transmembrane and matrix domains, recruiting the PAM coupling factor Pam17 and stimulating Hsp70-driven import, and cardiolipin modulates the Tim50–Tim23 channel interaction (PMID:32130909, PMID:28879236). Pathogenic TIMM50 variants preferentially impair the TIM23^SORT lateral release pathway, reducing OXPHOS and mitochondrial ribosomal components, and TIMM50 deficiency in neurons decreases respiration, ATP production, and mitochondrial trafficking while increasing aberrant electrical activity (PMID:38828998, PMID:39680434).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 2002 High

    The discovery that Tim50 is an essential TIM23 subunit that contacts preproteins in the IMS and directs them to Tim23 established Tim50 as a previously unknown central component of the preprotein import pathway.

    Evidence Genetic depletion, co-immunoprecipitation, photocrosslinking of translocation intermediates, and in vivo import assays in yeast, reported independently by two labs

    PMID:12437924 PMID:12437925

    Open questions at the time
    • Mechanism of presequence recognition by Tim50 not yet identified
    • No structural information on Tim50
    • How Tim50 regulates the Tim23 channel was unknown
  2. 2004 High

    Characterization of human TIMM50 showed conservation of the Tim23 interaction and revealed an intrinsic phosphatase activity, while establishing that TIMM50 loss sensitizes cells to apoptosis by facilitating cytochrome c release.

    Evidence Co-immunoprecipitation, RNAi knockdown, phosphatase activity assay, cytochrome c release assay in human cells

    PMID:15044455

    Open questions at the time
    • Physiological substrate of phosphatase activity unidentified
    • Whether apoptosis sensitization reflects import defects or a direct barrier-maintenance function was unclear
  3. 2005 Medium

    Identification of a nuclear isoform (Tim50a) that localizes to nuclear speckles and interacts with coilin, snRNPs, and SMN raised the possibility of a non-mitochondrial role in snRNP biogenesis.

    Evidence Subcellular fractionation, co-immunoprecipitation, competition binding, fluorescence microscopy in human cells

    PMID:16008839

    Open questions at the time
    • Functional consequence of Tim50a on snRNP assembly not demonstrated
    • Not independently replicated by other labs
    • Relationship to mitochondrial isoform unclear
  4. 2006 High

    Reconstituted electrophysiology revealed that the Tim50 IMS domain closes the Tim23 channel in the absence of substrate and that presequences antagonize this closure, establishing Tim50 as a gatekeeper that maintains the inner membrane permeability barrier while enabling presequence-regulated channel opening.

    Evidence Purified Tim50 IMS domain applied to reconstituted Tim23 channels; electrophysiology with presequence peptides

    PMID:16763150

    Open questions at the time
    • Structural basis for Tim50-mediated channel closure unknown
    • In vivo relevance of reconstituted gating not yet confirmed
  5. 2008 High

    Direct IMS domain–IMS domain binding between Tim50 and Tim23 was quantified and specific mutations that disrupt this interaction were shown to be lethal, establishing this interaction as essential for import.

    Evidence SPR with purified recombinant IMS domains, chemical cross-linking, mutagenesis with in vivo growth and import assays in yeast

    PMID:19017642

    Open questions at the time
    • Structural determinants at atomic resolution not yet resolved
    • Whether multiple binding surfaces exist was unknown
  6. 2009 High

    Tim50–Tim23 IMS interactions were shown to facilitate not only TOM-to-TIM23 transfer but also a late import step by promoting matrix Hsp70 motor function, revealing that Tim50 coordinates both early and late phases of translocation.

    Evidence Genetic epistasis, co-immunoprecipitation, in vivo import assays with IMS domain mutants in yeast

    PMID:19139266

    Open questions at the time
    • Mechanism by which IMS signals reach the matrix motor was unknown
    • Role of Tim50 transmembrane and matrix domains not addressed
  7. 2011 High

    Two landmark studies established that Tim50 is the primary presequence receptor at the inner membrane—photo-affinity labeling mapped its presequence-binding domain—and the 1.83 Å crystal structure of the IMS domain identified a protruding β-hairpin essential for Tim23 binding.

    Evidence Photo-affinity labeling with mass spectrometric mapping; X-ray crystallography at 1.83 Å with functional mutagenesis in yeast

    PMID:21704637 PMID:22065641

    Open questions at the time
    • Atomic-level view of presequence bound to Tim50 not obtained
    • Conformational dynamics of the β-hairpin during translocation unknown
  8. 2015 Medium

    A second crystal form of Tim50 IMS domain revealed conformational plasticity in the β-hairpin and helix A2 regions, with crystal packing suggesting that presequence recognition involves hydrophobic interactions within the β-hairpin groove.

    Evidence X-ray crystallography at 2.67 Å; structural analysis of crystal contacts

    PMID:26323300

    Open questions at the time
    • Presequence-binding mode inferred from crystal packing rather than a co-crystal structure
    • No mutagenesis validation of the proposed binding groove
  9. 2017 High

    Cardiolipin was identified as a direct modulator of Tim50–Tim23 interaction, with Tim50 binding membranes and specific Tim23 sites in a cardiolipin-dependent manner, adding a lipid regulatory layer to translocase function.

    Evidence In vivo/in vitro interaction assays, nanoscale model membranes, SAXS, molecular dynamics in yeast system

    PMID:28879236

    Open questions at the time
    • Physiological consequence of cardiolipin depletion on Tim50 gating in vivo not fully resolved
    • Structural details of the cardiolipin binding site on Tim50 at atomic resolution unknown
  10. 2018 Medium

    Human TIMM50 was shown to interact directly with cytochrome P450scc (CYP11A1) at the TIM23 translocase, with this interaction required for full SCC enzymatic activity, extending Tim50 function to regulation of steroidogenesis beyond canonical import.

    Evidence Co-immunoprecipitation, mass spectrometry, mutagenesis, Tim50 knockdown in steroidogenic cells

    PMID:30348838

    Open questions at the time
    • Whether this reflects a chaperoning role during import or a stable post-import complex is unclear
    • Not replicated independently
  11. 2019 Medium

    Random mutagenesis identified two distinct surface patches on Tim50 required for Tim23 binding, resolving earlier ambiguity about whether a single or multiple contact sites mediate the interaction.

    Evidence Random mutagenesis, temperature-sensitive mutant analysis, co-immunoprecipitation, in vivo import in yeast

    PMID:30765764

    Open questions at the time
    • No co-crystal of Tim50–Tim23 complex to assign patches at atomic resolution
    • How the two patches coordinate during translocation is unclear
  12. 2020 Medium

    Domain dissection established that Tim50 transmits presequence recognition across the inner membrane: the matrix domain recruits PAM coupling factor Pam17, the IMS domain promotes PAM recruitment to TIM23, and the transmembrane segment stimulates import-driving force, resolving how IMS presequence sensing is mechanistically coupled to matrix motor activation.

    Evidence Domain dissection with functional complementation, genetic analysis, in vivo import in yeast

    PMID:32130909

    Open questions at the time
    • Molecular mechanism of signal transduction across the transmembrane segment unresolved
    • No direct biochemical reconstitution of the Tim50–Pam17 interaction
  13. 2023 Medium

    The two IMS sub-domains of Tim50 (core and PBD) were shown to have distinct essential functions—core domain for presequence binding and TIM23 recruitment, PBD for TOM–TIM23 cooperation—and can complement each other in trans, revealing a modular architecture.

    Evidence Trans-complementation, domain deletion, in vivo import assays in yeast

    PMID:37748811

    Open questions at the time
    • Structural basis for independent function of PBD at TOM–TIM23 interface not determined
    • Whether trans-complementation reflects physiological sub-complex dynamics is unknown
  14. 2024 High

    Three concurrent studies revealed that TIMM50 pathogenic variants specifically impair the TIM23SORT lateral release pathway (reducing OXPHOS and mitoribosomal components), that TIMM50 deficiency in neurons decreases respiration/ATP and disrupts mitochondrial trafficking and electrical activity, and that eIF5A controls Tim50 levels by alleviating ribosome stalling at polyproline stretches in Tim50 mRNA.

    Evidence Quantitative proteomics in patient fibroblasts and CRISPR models; neuronal knockdown with respirometry, imaging, and electrophysiology; ribosome profiling with polyproline deletion rescue in yeast

    PMID:38828998 PMID:39509053 PMID:39680434

    Open questions at the time
    • Relative contribution of TIM23SORT vs. TIM23MOTOR pathway impairment to disease phenotype unknown
    • Whether eIF5A-dependent Tim50 regulation operates in mammalian neurons not tested
    • No therapeutic strategy to restore TIM23SORT function in TIMM50 disease

Open questions

Synthesis pass · forward-looking unresolved questions
  • A high-resolution structure of the full-length Tim50 in complex with Tim23, a co-crystal with bound presequence, and a detailed mechanism for how presequence recognition is transduced across the membrane to activate the PAM motor remain unresolved.
  • No structure of full-length Tim50 or Tim50–Tim23 complex
  • No co-crystal or cryo-EM structure with bound presequence
  • Transmembrane signal transduction mechanism from IMS to matrix unresolved at molecular level
  • Physiological role and substrate of phosphatase activity remain unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 4 GO:0098772 molecular function regulator activity 3 GO:0016787 hydrolase activity 1
Localization
GO:0005739 mitochondrion 10 GO:0005634 nucleus 1
Pathway
R-HSA-9609507 Protein localization 7 R-HSA-392499 Metabolism of proteins 4 R-HSA-1430728 Metabolism 2
Partners
COILINCYP11A1HSD3B2PAM17SMNTIM23
Complex memberships
TIM23 complex

Evidence

Reading pass · 20 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2002 Tim50 is an essential subunit of the TIM23 complex, anchored to the inner mitochondrial membrane with its major domain exposed to the intermembrane space, where it interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits import of preproteins with classical matrix-targeting signals. Genetic depletion, co-immunoprecipitation, in vivo import assays, fractionation Cell High 12437924 12437925
2002 Tim50 interacts with the N-terminal intermembrane space domain of Tim23, and a translocation intermediate accumulated at the TOM complex is crosslinked to Tim50, indicating Tim50 facilitates transfer of translocating proteins from the TOM complex to the TIM23 complex. Site-specific photocrosslinking of translocation intermediates, co-immunoprecipitation Cell High 12437924 12437925
2006 The intermembrane space domain of Tim50 induces the Tim23 channel to close, maintaining the permeability barrier of the mitochondrial inner membrane. Presequences overcome this effect and activate the channel for translocation, establishing a presequence-regulated gating mechanism. Reconstituted channel electrophysiology, in vitro assay with purified IMS domain of Tim50 Science High 16763150
2008 The IMS domains of Tim50 and Tim23 interact directly; specific mutations in Tim23 that abolish Tim50 binding in vitro also destabilize the interaction in vivo and cause defective preprotein import and cell death at elevated temperatures. In vitro reconstitution with purified recombinant IMS domains, chemical cross-linking, surface plasmon resonance, in vivo import assays The Journal of biological chemistry High 19017642
2009 Tim23-Tim50 interactions in the IMS facilitate transfer of precursor proteins from the TOM40 complex to the TIM23 complex, and also facilitate a late step of translocation by promoting motor functions of mitochondrial Hsp70 in the matrix. Genetic epistasis, co-immunoprecipitation, in vivo import assays with IMS domain mutants The Journal of cell biology High 19139266
2011 Tim50 is the primary presequence receptor at the inner membrane; photo-affinity labeling and mass spectrometric mapping identified a presequence-binding domain in Tim50, and targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner. Photo-affinity labeling with engineered presequence probes, mass spectrometric mapping of crosslink sites The Journal of cell biology High 22065641
2011 Crystal structure of the IMS domain of yeast Tim50 resolved to 1.83 Å; a protruding β-hairpin is crucial for interaction with Tim23, providing structural basis for Tim50-Tim23 cooperation in preprotein translocation. X-ray crystallography at 1.83 Å resolution, functional mutagenesis Journal of molecular biology High 21704637
2004 Human TIMM50 is present in a complex with human Tim23, possesses phosphatase activity, and its knockdown by RNAi increases sensitivity to death stimuli by accelerating cytochrome c release from mitochondria. Co-immunoprecipitation, RNAi knockdown, cytochrome c release assay, phosphatase activity assay The Journal of biological chemistry High 15044455
2011 Human TIMM50 interacts with 3β-HSD2 primarily through Tim50's intermembrane space domain binding the N-terminus of 3β-HSD2; this interaction contributes to 3β-HSD2 enzymatic activity and conformational change, and Tim50 knockdown inhibits steroidogenic catalysis without rescuing activity by restoring protein levels alone. Co-immunoprecipitation, mass spectrometry of mitochondrial complexes, density gradient ultracentrifugation, Tim50 knockdown, CD spectroscopy The Journal of biological chemistry Medium 21930695
2015 A second crystal structure of the Tim50 IMS domain [Tim50(164-361)] at 2.67 Å reveals significant conformational plasticity in the β-hairpin and helix A2, and crystal packing shows helix A1 from a neighboring monomer docking into the presequence-binding groove, suggesting Tim50 recognizes presequences via hydrophobic interactions within the β-hairpin. X-ray crystallography at 2.67 Å resolution, structural analysis of crystal packing Acta crystallographica Section F Medium 26323300
2017 Cardiolipin directly modulates the interaction between the soluble receptor domain of Tim50 and the Tim23 channel; Tim50 binds membranes and specific sites on Tim23 in a cardiolipin-dependent manner, and SAXS-based structural analysis of the Tim50 receptor domain combined with molecular dynamics identified structural elements mediating this interaction. In vivo and in vitro interaction assays, nanoscale model membranes, small-angle X-ray scattering (SAXS), molecular dynamics simulations, biophysical measurements Science advances High 28879236
2019 Random mutagenesis of Tim50 identified two distinct surface patches whose mutation impairs interaction with Tim23 and causes defective TIM23-dependent preprotein import, establishing that two regions of Tim50 are required for Tim23 binding. Random mutagenesis, temperature-sensitive mutant analysis, co-immunoprecipitation, in vivo import assays Scientific reports Medium 30765764
2020 Tim50 transmits presequence recognition signals across the inner membrane: the Tim50 matrix domain facilitates recruitment of the PAM coupling factor Pam17, the IMS domain of Tim50 promotes PAM recruitment to TIM23, and the Tim50 transmembrane segment stimulates the matrix-directed import-driving force by PAM, coordinating preprotein recognition with motor activation. Genetic analysis, in vivo import assays, domain dissection with functional complementation Cell reports Medium 32130909
2023 The two IMS domains of Tim50 (core and PBD) have distinct essential roles: the core domain contains the main presequence-binding site and is the main recruitment point to TIM23, while the PBD plays a critical role in cooperation between TOM and TIM23 complexes; the two domains can complement each other in trans. Trans-complementation experiments, in vivo import assays, domain deletion analysis Life science alliance Medium 37748811
2024 Pathogenic variants in TIMM50 specifically reduce laterally released substrates imported via the TIM23SORT pathway; proteins involved in OXPHOS and mitochondrial ultrastructure are enriched in the TIM23SORT substrate pool, providing a biochemical mechanism for the specific defects in TIMM50-associated disease. Quantitative proteomics of patient fibroblasts and CRISPR TIMM50 HEK293 model, pathway analysis Molecular and cellular biology Medium 38828998
2024 eIF5A controls mitochondrial protein import by alleviating ribosome stalling at polyproline sequences in Tim50 mRNA; eIF5A depletion reduces Tim50 levels and causes mitoprotein precursor accumulation and mitochondrial import stress; removal of polyprolines from Tim50 rescues the import stress response. eIF5A depletion in yeast, ribosome profiling, mitochondrial import stress assay, polyproline deletion mutagenesis The Journal of cell biology High 39509053
2024 TIMM50 deficiency in neurons reduces levels of OXPHOS and mitochondrial ribosome complex components, decreases respiration and ATP, causes defective mitochondrial trafficking in neuronal processes, and increases electrical activity correlated with reduced KCNJ10 and KCNA2 potassium channels. TIMM50 knockdown in mouse neurons, proteomics, respirometry, ATP measurement, neuronal imaging, electrophysiology eLife Medium 39680434
2005 A nuclear isoform of Tim50, Tim50a, localizes strictly to the nucleus (enriched in speckles with snRNPs) due to an N-terminal nuclear localization signal, interacts with coilin, snRNPs, and SMN, and competition binding shows coilin competes with Sm proteins and SMN for Tim50a binding sites, suggesting a role in snRNP biogenesis. Subcellular fractionation, co-immunoprecipitation, competition binding assay, fluorescence microscopy BMC cell biology Medium 16008839
2018 Tim50 directly interacts with cytochrome P450 SCC (CYP11A1) via SCC amino acids 141-146 after SCC is imported into the matrix and partially processed, forming a large complex at the TIM23 translocase; Tim50 knockdown or mutation of the SCC-Tim50 interaction site reduces SCC enzymatic activity. Fractionation, mass spectrometry, co-immunoprecipitation, Tim50 knockdown, mutagenesis Molecular and cellular biology Medium 30348838
2025 TIMM50 downregulation is sufficient to trigger cellular senescence through impaired mitochondrial function; TIMM50 expression is regulated by sirtuin1-dependent downregulation of CEBPα (a transcriptional activator of TIMM50); overexpression of TIMM50 slows senescence onset. Multiple senescence models, pathway analysis, overexpression/knockdown, mitochondrial function assays Advanced biology Low 40128440

Source papers

Stage 0 corpus · 39 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2002 The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel. Cell 216 12437924
2002 Tim50 is a subunit of the TIM23 complex that links protein translocation across the outer and inner mitochondrial membranes. Cell 210 12437925
2006 Tim50 maintains the permeability barrier of the mitochondrial inner membrane. Science (New York, N.Y.) 152 16763150
2009 Tim23-Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import. The Journal of cell biology 117 19139266
2004 Tim50, a component of the mitochondrial translocator, regulates mitochondrial integrity and cell death. The Journal of biological chemistry 82 15044455
2011 Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex. The Journal of cell biology 79 22065641
2008 Interaction of Tim23 with Tim50 Is essential for protein translocation by the mitochondrial TIM23 complex. The Journal of biological chemistry 55 19017642
2017 Cardiolipin mediates membrane and channel interactions of the mitochondrial TIM23 protein import complex receptor Tim50. Science advances 53 28879236
2011 Structural basis for the function of Tim50 in the mitochondrial presequence translocase. Journal of molecular biology 41 21704637
2011 Inner mitochondrial translocase Tim50 interacts with 3β-hydroxysteroid dehydrogenase type 2 to regulate adrenal and gonadal steroidogenesis. The Journal of biological chemistry 34 21930695
2007 Involvement of the mitochondrial protein translocator component tim50 in growth, cell proliferation and the modulation of respiration in Drosophila. Genetics 34 17435247
2018 Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions. EMBO molecular medicine 32 30190335
2016 Mitochondrial epileptic encephalopathy, 3-methylglutaconic aciduria and variable complex V deficiency associated with TIMM50 mutations. Clinical genetics 32 27573165
2011 Upregulation of the mitochondrial transport protein, Tim50, by mutant p53 contributes to cell growth and chemoresistance. Archives of biochemistry and biophysics 31 21621504
2012 Tim50 in Trypanosoma brucei possesses a dual specificity phosphatase activity and is critical for mitochondrial protein import. The Journal of biological chemistry 25 23212919
2015 Loss of TIM50 suppresses proliferation and induces apoptosis in breast cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 21 26289846
2019 Mutations in TIMM50 cause severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology. Human mutation 18 31058414
2019 A mutagenesis analysis of Tim50, the major receptor of the TIM23 complex, identifies regions that affect its interaction with Tim23. Scientific reports 15 30765764
2020 Transmembrane Coordination of Preprotein Recognition and Motor Coupling by the Mitochondrial Presequence Receptor Tim50. Cell reports 14 32130909
2011 Arabidopsis mitochondrial protein TIM50 affects hypocotyl cell elongation through intracellular ATP level. Plant science : an international journal of experimental plant biology 13 22195596
2021 Diverse Functions of Tim50, a Component of the Mitochondrial Inner Membrane Protein Translocase. International journal of molecular sciences 12 34360547
2015 The structure of Tim50(164-361) suggests the mechanism by which Tim50 receives mitochondrial presequences. Acta crystallographica. Section F, Structural biology communications 12 26323300
2005 Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis. BMC cell biology 11 16008839
2018 Inner Mitochondrial Translocase Tim50 Is Central in Adrenal and Testicular Steroid Synthesis. Molecular and cellular biology 10 30348838
2015 Down regulation of Tim50 in Trypanosoma brucei increases tolerance to oxidative stress. Molecular and biochemical parasitology 10 25791316
2012 Interaction of presequence with human translocase of the inner membrane of mitochondria Tim50. The journal of physical chemistry. B 10 22335443
2024 eIF5A controls mitoprotein import by relieving ribosome stalling at TIM50 translocase mRNA. The Journal of cell biology 8 39509053
2011 Expression and structural characterization of human translocase of inner membrane of mitochondria Tim50. Protein expression and purification 8 21742040
2024 Reduced Protein Import via TIM23 SORT Drives Disease Pathology in TIMM50-Associated Mitochondrial Disease. Molecular and cellular biology 6 38828998
2023 Two domains of Tim50 coordinate translocation of proteins across the two mitochondrial membranes. Life science alliance 6 37748811
2022 A High-Throughput Search for SFXN1 Physical Partners Led to the Identification of ATAD3, HSD10 and TIM50. Biology 6 36138777
2012 Interaction of divalent metal ions with human translocase of inner membrane of mitochondria Tim50. Biochemical and biophysical research communications 6 23098911
2024 Biochemical and neurophysiological effects of deficiency of the mitochondrial import protein TIMM50. eLife 4 39680434
2021 Outer and inner mitochondrial membrane proteins TOMM40 and TIMM50 are intensively concentrated and localized at Purkinje and pyramidal neurons in the New Zealand white rabbit brain. Anatomical record (Hoboken, N.J. : 2007) 3 34041863
2021 Trypanosoma brucei Tim50 Possesses PAP Activity and Plays a Critical Role in Cell Cycle Regulation and Parasite Infectivity. mBio 3 34517757
2024 Biochemical and neurophysiological effects of deficiency of the mitochondrial import protein TIMM50. bioRxiv : the preprint server for biology 1 38826427
2023 eIF5A controls mitoprotein import by relieving ribosome stalling at the TIM50 translocase mRNA. bioRxiv : the preprint server for biology 1 38187585
2022 Correction: Chaudhuri et al. Diverse Functions of Tim50, a Component of the Mitochondrial Inner Membrane Protein Translocase. Int. J. Mol. Sci. 2021, 22, 7779. International journal of molecular sciences 1 35887402
2025 Ameliorating TIMM50 Loss Slows Senescence by Improving Mitochondrial Structure and Function. Advanced biology 0 40128440