Affinage

TIMM10

Mitochondrial import inner membrane translocase subunit Tim10 · UniProt P62072

Length
90 aa
Mass
10.3 kDa
Annotated
2026-06-10
14 papers in source corpus 14 papers cited in narrative 14 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

TIMM10 (Tim10) is a small intermembrane-space (IMS) chaperone of mitochondria that, together with Tim9, escorts hydrophobic carrier-family precursor proteins across the IMS toward Tim22-mediated insertion into the inner membrane (PMID:9495346, PMID:11483513). Tim10 and Tim9 assemble exclusively with one another into the functional chaperone; recombinant Tim9-Tim10 complex alone is sufficient to restore ADP/ATP carrier import and inner-membrane insertion in mitochondria lacking endogenous Tim10 (PMID:11483513). The crystal structure reveals a heterohexamer in which each subunit contributes a central loop flanked by disulfide bonds and N- and C-terminal tentacle-like helices, with buried salt bridges between conserved lysine and glutamate residues stapling alternating subunits; mutation of these salt-bridge residues, or of a conserved core glutamate, destabilizes the complex and impairs precursor import (PMID:17618651, PMID:19037098). Tim10's N-terminal substrate-binding tentacle is essential for cell viability and substrate recognition (PMID:17618651). Biogenesis of Tim10 is redox- and metal-regulated: it is held in a reduced, zinc-stabilized, import-competent state that resists oxidative folding, and is delivered across the outer membrane through a nine-residue IMS sorting signal recognized by the Mia40 receptor, which engages specifically the most N-terminal cysteine (PMID:16199054, PMID:17553782, PMID:19297525). Following import, zinc is released and intramolecular disulfide bonds form between the four conserved cysteines to generate the assembly-competent, oxidatively folded subunit (PMID:14973127, PMID:16199054). Within the complex, Tim9 protects Tim10 from degradation by the i-AAA protease Yme1 (PMID:26182355).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 1998 High

    Established that Tim10 functions in the IMS to chaperone carrier precursors across the outer membrane and hand them to the Tim22 inner-membrane insertion machinery, defining the carrier import pathway.

    Evidence Biochemical fractionation, co-immunoprecipitation, and reconstitution of import in yeast mitochondria

    PMID:9495346

    Open questions at the time
    • Did not define the minimal complement of subunits forming the functional chaperone
    • Structural basis of substrate recognition unknown
  2. 2001 High

    Resolved which subunits constitute the functional chaperone by showing the complex is composed exclusively of Tim9 and Tim10 and that recombinant Tim9-Tim10 alone restores carrier import.

    Evidence Functional reconstitution with E. coli-expressed proteins and import assay in tim10-ts mitochondria

    PMID:11483513

    Open questions at the time
    • Stoichiometry and architecture of the complex not yet determined
    • Mechanism of substrate engagement not addressed
  3. 2004 High

    Showed Tim10 assembly is redox-controlled, proceeding from a reduced unfolded import substrate to an intramolecularly disulfide-bonded, zinc-devoid, assembly-competent form, distinguishing productive from abortive disulfide states.

    Evidence Redox trapping, import assays, cysteine mutagenesis, and in vivo labeling

    PMID:14973127

    Open questions at the time
    • Catalyst driving in vivo oxidation not identified
    • Role of zinc in maintaining import competence not yet quantified
  4. 2005 High

    Defined the role of zinc as a kinetic brake that stabilizes reduced Tim10 and slows oxidative folding, coupling cytosolic metal binding to import competence and post-import folding upon zinc release.

    Evidence In vitro redox potential measurement, CD, fluorescence, and glutathione redox assays

    PMID:16199054

    Open questions at the time
    • In vivo timing of zinc release not directly observed
    • Identity of the cellular zinc donor/acceptor not established
  5. 2007 High

    Identified Mia40 as a site-specific receptor that recognizes only the most N-terminal cysteine of Tim10 for outer-membrane translocation, separating the import-recognition determinant from the four-cysteine assembly requirement.

    Evidence Systematic cysteine mutagenesis with in organello and in vitro import and Mia40 co-immunoprecipitation

    PMID:17553782

    Open questions at the time
    • Structure of the Mia40-Tim10 import intermediate not resolved
    • Sequence context beyond the cysteine not yet defined
  6. 2007 High

    Mapped functional determinants by showing a conserved core glutamate is required for hexamer assembly while the N-terminal substrate-binding region is essential for viability under all conditions.

    Evidence Site-directed mutagenesis, in vitro assembly assays, and in vivo complementation in a MET3-TIM10 strain

    PMID:17618651

    Open questions at the time
    • Atomic basis of the glutamate contact not yet visualized
    • How the N-terminal region contacts substrate not defined
  7. 2007 Medium

    Dissected the assembly pathway, showing Tim9 dimerizes while Tim10 is monomeric and that the hexamer forms through tetrameric intermediates with N-terminal helices assembling before C-terminal helices.

    Evidence Stopped-flow fluorescence/light scattering with tryptophan mutagenesis and analytical ultracentrifugation

    PMID:18022191

    Open questions at the time
    • Intermediates inferred in vitro and not validated in vivo
    • Single-lab biophysical observation
  8. 2008 High

    Provided the atomic architecture of the Tim9-Tim10 hexamer, revealing tentacle helices, disulfide-flanked central loops, and inter-subunit salt bridges whose mutation destabilizes the complex and blocks import.

    Evidence X-ray crystallography at 2.5 Å with mutagenesis and functional import assays

    PMID:19037098

    Open questions at the time
    • No structure of a substrate-bound complex
    • Dynamics of the tentacles not captured by the static structure
  9. 2008 Medium

    Characterized the metal-binding mechanism, defining two-step zinc binding with sub-nanomolar affinity and showing oxidized Tim10 cannot bind zinc, linking redox state to metal occupancy.

    Evidence CD, fluorescence and stopped-flow spectrometry with chelator competition (idx 8); stopped-flow kinetics under varied pH and salt (idx 9)

    PMID:17963238 PMID:18462749

    Open questions at the time
    • Cooperativity and allostery inferred from in vitro kinetics only
    • Physiological relevance of measured affinities not tested in vivo
  10. 2011 Low

    Linked hydrophobic-residue dynamics within the complex to its chaperone function, suggesting multiple functional conformational states exist at equilibrium for substrate binding.

    Evidence Temperature-dependent biochemical assays and molecular dynamics simulation with energy decomposition

    PMID:22095685

    Open questions at the time
    • Computationally driven with no mutagenesis validation
    • Proposed conformational states not directly observed
  11. 2009 High

    Identified a transferable nine-residue IMS sorting signal in the Tim10 precursor sufficient for Mia40 engagement and outer-membrane transfer, defining the import targeting element.

    Evidence Deletion and chimeric-construct analysis with in organello and in vitro import assays

    PMID:19297525

    Open questions at the time
    • Structural details of signal-Mia40 recognition not resolved
    • Generality of the signal across small Tim proteins not established
  12. 2015 Medium

    Revealed a quality-control axis in which Tim9 protects Tim10 from Yme1-mediated degradation through complex assembly, with loss of Tim9's inner disulfide triggering Yme1-dependent turnover of both subunits.

    Evidence Yeast genetics (ts mutants, YME1 deletion epistasis) with biochemical stability and abundance assays

    PMID:26182355

    Open questions at the time
    • Molecular basis of Yme1 recognition not defined
    • Single-lab observation of degradation suppression
  13. 2025 Medium

    Defined the determinants of Tim10 turnover by showing Yme1 preferentially binds Tim10 via its N-terminal tentacle independent of disulfide status, with unfolding exposing additional contacts that commit it to degradation.

    Evidence Binding and degradation assays with disulfide-bond variants (preprint)

    PMID:bio_10.1101_2025.07.23.666395

    Open questions at the time
    • Not yet peer-reviewed
    • In vivo relevance of tentacle-mediated Yme1 engagement not confirmed

Open questions

Synthesis pass · forward-looking unresolved questions
  • How the Tim9-Tim10 chaperone physically engages and releases diverse hydrophobic carrier substrates during transit, and how this is coordinated with handoff to Tim22, remains structurally undefined.
  • No substrate-bound structure of the complex
  • Mechanism of substrate handoff to Tim22 not resolved
  • Human TIMM10 in vivo studies absent from this corpus

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0044183 protein folding chaperone 4
Pathway
R-HSA-392499 Metabolism of proteins 3 R-HSA-9609507 Protein localization 2
Partners
MIA40TIMM12TIMM22TIMM9YME1
Complex memberships
TIM22 carrier translocaseTIM9-TIM10 hexameric chaperone complex

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1998 Tim10 (with Tim12) localizes to the mitochondrial intermembrane space and interacts sequentially with carrier family precursor proteins to facilitate their translocation across the outer membrane in a membrane-potential-independent manner; Tim10 and Tim12 are found in a complex with Tim22, which mediates membrane-potential-dependent insertion into the inner membrane. Both Tim10 and Tim12 contain a zinc-finger-like motif with four cysteines and bind equimolar zinc ions; interaction with precursors depends on divalent metal ions. Biochemical fractionation, co-immunoprecipitation, reconstitution of import in yeast Nature High 9495346
2001 The TIM10 complex is composed exclusively of Tim9 and Tim10 (no other mitochondrial protein is required for complex formation); reconstituted recombinant Tim9-Tim10 complex restores ADP/ATP carrier import across the outer membrane and accurate inner membrane insertion in tim10-ts mitochondria lacking endogenous Tim10. Functional reconstitution using E. coli-expressed recombinant proteins, import assay in tim10-ts mitochondria The EMBO journal High 11483513
2004 TIM10 assembly is redox-regulated: subunits are imported in a cysteine-reduced, unfolded state, then undergo intramolecular disulfide bonding (between their four conserved cysteines) to a zinc-devoid, assembly-competent structure, and finally assemble into the functional hexameric complex. Intramolecular disulfides form in vivo; intermolecular disulfides observed in vitro are abortive intermediates. Biochemical assays (redox trapping, import assays, mutagenesis of cysteines), in vivo labeling The Journal of biological chemistry High 14973127
2005 Zinc binding stabilizes reduced Tim10 and slows oxidative folding by more than tenfold, maintaining it in an import-competent state in the cytosol; once imported, zinc must be released to permit oxidative folding and assembly. Oxidized (disulfide-bonded) Tim10 cannot be further reduced by glutathione, while reduced Tim10 is rapidly oxidized by oxidized glutathione at physiological concentrations. Protein disulfide isomerase can catalyze oxidative folding of Tim10 only after zinc removal. In vitro biochemical and biophysical assays (redox potential measurement, CD spectroscopy, fluorescence, glutathione redox assays) Journal of molecular biology High 16199054
2007 Mia40 acts as a site-specific receptor for Tim10 biogenesis: only the most amino-terminal cysteine residue of Tim10 is critical for translocation across the outer membrane and interaction with Mia40, whereas all four cysteines are required for assembly of the Tim9-Tim10 chaperone complex. Systematic cysteine mutagenesis, in organello and in vitro import assays, co-immunoprecipitation with Mia40 The Journal of biological chemistry High 17553782
2007 A conserved glutamate residue in the central core domain of Tim10 (within the CX3C motif region) is required for assembly of the hexameric TIM10 complex; mutations abolishing complex assembly are lethal on non-fermentable carbon sources but allow growth on glucose. The N-terminal substrate-binding region of Tim10 is essential for cell viability under all conditions. Site-directed mutagenesis, in vitro complex assembly assays, in vivo complementation using MET3-TIM10 strain Journal of molecular biology High 17618651
2008 The crystal structure of the yeast Tim9-Tim10 hexameric complex was determined to 2.5 Å; each subunit contains a central loop flanked by disulfide bonds with N- and C-terminal tentacle-like helices. Buried salt bridges between conserved lysine and glutamate residues connect alternating subunits; mutation of these residues destabilizes the complex and causes defective precursor import. The N-terminal region of Tim9 is required for efficient trapping of incoming substrates into the IMS. X-ray crystallography (2.5 Å), site-directed mutagenesis, yeast growth assays, in vitro import assays Molecular biology of the cell High 19037098
2007 The Tim9-Tim10 complex assembles via a multi-step pathway with transient tetrameric intermediates before the final hexamer is formed; Tim9 forms a homodimer while Tim10 is a monomer. The N-terminal helices of subunits are assembled before the C-terminal helices during complex formation. Stopped-flow fluorescence with tryptophan mutagenesis, stopped-flow light scattering, analytical ultracentrifugation Journal of molecular biology Medium 18022191
2008 Tim10 zinc binding proceeds via a two-step mechanism: an initial selective binding of Zn2+ to cysteine residues forming a structurally unfolded intermediate, followed by folding upon higher zinc concentrations. Zinc-binding affinity of Tim10 is ~8×10^-10 M. Oxidized (disulfide-bonded) Tim10 cannot bind zinc. Circular dichroism, fluorescence spectrometry, stopped-flow fluorescence, metal chelator competition assays Proteins Medium 17963238
2008 Assembly of the Tim9-Tim10 complex is driven by electrostatic interactions (initial driving force, salt and pH dependence matching subunit isoelectric points) and is also regulated allosterically; Tim10 displays sigmoidal concentration dependence suggesting cooperativity, while Tim9 shows linear dependence. Stopped-flow kinetics with mutagenesis, pH and salt concentration variation Journal of molecular biology Medium 18462749
2009 A nine-amino-acid region within the Tim10 precursor (the IMS sorting signal) is sufficient for engagement with the Mia40 receptor and for transfer of proteins across the outer membrane to the IMS. Mutagenesis/deletion analysis, in organello and in vitro import assays, chimeric protein constructs Molecular biology of the cell High 19297525
2011 Dynamics of hydrophobic residues in the Tim9-Tim10 complex regulates its chaperone function; temperature-dependent conformational changes mimicking biological substrate-binding activity correspond to disruption of hydrophobic interactions, suggesting different functional conformational states exist at equilibrium. Temperature-dependent biochemical assays, substrate binding measurements, molecular dynamics simulation with energy decomposition analysis Proteins Low 22095685
2015 Tim9 protects Tim10 from degradation by the mitochondrial i-AAA protease Yme1 by assembling into the Tim9-Tim10 complex; loss of Tim9's inner disulfide bond leads to degradation of both Tim9 and Tim10, and this is suppressed by deletion of YME1. Tim10 (rather than the hexameric complex) is proposed as the primary functional unit. Yeast genetics (temperature-sensitive mutants, YME1 deletion), biochemical and biophysical methods (complex stability, protein levels) Bioscience reports Medium 26182355
2025 Yme1 protease preferentially binds Tim10 over other small Tim proteins via a high-affinity interaction mediated primarily by Tim10's flexible N-terminal tentacle region, irrespective of disulfide bond status; substrate unfolding (disruption of disulfide bonds) exposes additional contact sites that enhance engagement and commit Tim10 to degradation. Yme1 also binds assembled Tim9-Tim10 complex independently of the Tim10 N-terminal tentacle. Biochemical and biophysical approaches (binding assays, degradation assays), analysis of disulfide bond variants bioRxivpreprint Medium bio_10.1101_2025.07.23.666395

Source papers

Stage 0 corpus · 14 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1998 Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5. Nature 250 9495346
2009 Identification of the signal directing Tim9 and Tim10 into the intermembrane space of mitochondria. Molecular biology of the cell 146 19297525
2004 Functional TIM10 chaperone assembly is redox-regulated in vivo. The Journal of biological chemistry 105 14973127
2007 Biogenesis of the essential Tim9-Tim10 chaperone complex of mitochondria: site-specific recognition of cysteine residues by the intermembrane space receptor Mia40. The Journal of biological chemistry 74 17553782
2008 Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import. Molecular biology of the cell 64 19037098
2005 Zinc binding stabilizes mitochondrial Tim10 in a reduced and import-competent state kinetically. Journal of molecular biology 50 16199054
2001 Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex. The EMBO journal 50 11483513
2015 Mitochondrial Tim9 protects Tim10 from degradation by the protease Yme1. Bioscience reports 23 26182355
2007 Assembly of the mitochondrial Tim9-Tim10 complex: a multi-step reaction with novel intermediates. Journal of molecular biology 22 18022191
2007 Mutation of conserved charged residues in mitochondrial TIM10 subunits precludes TIM10 complex assembly, but does not abolish growth of yeast cells. Journal of molecular biology 16 17618651
2008 Zinc binding of Tim10: evidence for existence of an unstructured binding intermediate for a zinc finger protein. Proteins 15 17963238
2008 Allosteric and electrostatic protein-protein interactions regulate the assembly of the heterohexameric Tim9-Tim10 complex. Journal of molecular biology 10 18462749
2011 Temperature-dependent study reveals that dynamics of hydrophobic residues plays an important functional role in the mitochondrial Tim9-Tim10 complex. Proteins 4 22095685
2000 Isolation and characterization of the TIM10 homologue from the yeast Pichia sorbitophila: a putative component of the mitochondrial protein import system. Yeast (Chichester, England) 3 10806421

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