TIM9

id: O74700
gene_symbol: TIM9
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:559292
  label: Saccharomyces cerevisiae
description: >-
  TIM9 is a small mitochondrial intermembrane space (IMS) chaperone that forms a hexameric
  complex with TIM10 (the Tim9-Tim10 or TIM10 complex, composed of 3 copies of each subunit).
  This soluble 70 kDa complex functions as a carrier-holdase that escorts hydrophobic
  transmembrane protein precursors (carrier proteins, beta-barrel precursors) from the TOM
  complex across the aqueous IMS to the TIM22 complex for insertion into the inner membrane,
  or to the SAM complex for outer membrane beta-barrel assembly. TIM9 contains a twin CX3C
  motif that forms two intramolecular disulfide bonds in the IMS; during cytoplasmic transit
  these cysteines may coordinate zinc. TIM9 is essential for viability and plays both a
  structural role in complex assembly and a functional role in substrate recognition, with its
  N-terminal region required for efficient trapping of incoming substrates.
tags:
  - UPB
existing_annotations:
# --- IBA annotations ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      TIM9 is a peripheral membrane protein on the IMS face of the mitochondrial inner membrane
      (PMID:9822593, PMID:9889188). The IBA annotation to mitochondrial inner membrane is
      phylogenetically consistent and supported by experimental evidence in yeast. UniProt
      records the subcellular location as "Mitochondrion inner membrane; Peripheral membrane
      protein; Intermembrane side."
    action: ACCEPT
    reason: >-
      TIM9 is well-established as associated with the mitochondrial inner membrane, where
      a fraction of Tim9 is part of the membrane-associated 300 kDa TIM22 complex (PMID:9822593).
      The IBA annotation is phylogenetically sound and consistent with the IDA annotations.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa
        complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p.
    - reference_id: PMID:9889188
      supporting_text: >-
        Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct
        hetero-oligomeric assemblies with Tim10 and Tim12.

- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      The Tim9-Tim10 complex escorts carrier protein precursors across the IMS to the TIM22
      complex for insertion into the inner membrane. This is a core function of TIM9, supported
      by multiple experimental studies (PMID:9822593, PMID:9889188, PMID:10469659, PMID:11483513).
      The IBA annotation is phylogenetically consistent.
    action: ACCEPT
    reason: >-
      This is the core biological process that TIM9 participates in. The Tim9-Tim10 complex
      mediates partial translocation of mitochondrial carrier proteins across the outer membrane
      and their subsequent insertion into the inner membrane via TIM22 (PMID:9889188). Multiple
      experimental evidence codes support this for yeast TIM9 directly.
    supported_by:
    - reference_id: PMID:9889188
      supporting_text: >-
        The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates partial
        translocation of mitochondrial carriers proteins across the outer membrane.
    - reference_id: PMID:9822593
      supporting_text: >-
        Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane
        proteins across the aqueous intermembrane space.

- term:
    id: GO:0140318
    label: protein transporter activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      TIM9 functions as part of the Tim9-Tim10 carrier complex that escorts hydrophobic
      precursors across the IMS. However, TIM9 is more precisely a carrier-holdase (it moves
      with its cargo) than a transporter (which facilitates movement without moving itself).
      GO:0140318 "protein transporter activity" may not be the optimal term; GO:0140309
      "unfolded protein carrier activity" is more specific and accurate for TIM9 function.
      However, this IBA is not incorrect and the IDA annotations also use this term, so
      it is acceptable to keep.
    action: ACCEPT
    reason: >-
      While GO:0140309 "unfolded protein carrier activity" would be more precise for TIM9
      (as a carrier-holdase rather than a transporter), GO:0140318 is not incorrect -- TIM9
      does facilitate protein delivery between compartments. The IBA annotation is phylogenetically
      sound. The more specific annotation to GO:0140309 is proposed as a NEW annotation below.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane
        proteins across the aqueous intermembrane space.

# --- IEA annotations ---
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: >-
      IEA annotation mapping from UniProtKB subcellular location. TIM9 is annotated in UniProt
      as "Mitochondrion inner membrane; Peripheral membrane protein; Intermembrane side."
      This is consistent with experimental evidence.
    action: ACCEPT
    reason: >-
      This IEA is broader than the IBA and IDA annotations for the same term and is consistent
      with the known localization of TIM9 at the mitochondrial inner membrane (PMID:9822593).
    supported_by:
    - reference_id: UniProt:O74700
      supporting_text: >-
        SUBCELLULAR LOCATION: Mitochondrion inner membrane

- term:
    id: GO:0005758
    label: mitochondrial intermembrane space
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: >-
      IEA annotation mapping from UniProtKB subcellular location. TIM9 is localized to the
      mitochondrial intermembrane space where it forms the soluble Tim9-Tim10 70 kDa complex.
      Well supported by experimental evidence.
    action: ACCEPT
    reason: >-
      The IMS localization is well-established experimentally (PMID:9822593, PMID:9889188)
      and confirmed by proteomics (PMID:22984289, cited in UniProt). The IEA is consistent
      with direct evidence.
    supported_by:
    - reference_id: PMID:9889188
      supporting_text: >-
        Tim9 is located in the mitochondrial intermembrane space and is organized into two
        distinct hetero-oligomeric assemblies with Tim10 and Tim12.

- term:
    id: GO:0015031
    label: protein transport
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: >-
      IEA annotation from UniProtKB keyword mapping (KW-0653 Protein transport, KW-0811
      Translocation). TIM9 participates in protein transport across the IMS. This is a
      general parent term; the more specific GO:0045039 (protein insertion into mitochondrial
      inner membrane) is also annotated.
    action: ACCEPT
    reason: >-
      While broad, this IEA is not incorrect. TIM9 does participate in protein transport.
      The more specific annotations to GO:0045039 provide the necessary precision. Keeping
      this general IEA is fine alongside the specific experimental annotations.
    supported_by:
    - reference_id: UniProt:O74700
      supporting_text: >-
        Mitochondrial intermembrane chaperone that participates in the import and insertion
        of multi-pass transmembrane proteins into the mitochondrial inner membrane.

- term:
    id: GO:0042719
    label: mitochondrial intermembrane space chaperone complex
  evidence_type: IEA
  original_reference_id: GO_REF:0000117
  review:
    summary: >-
      IEA annotation from ARBA machine learning. TIM9 is a component of the mitochondrial
      intermembrane space chaperone complex (the Tim9-Tim10 hexameric complex). Well supported
      by experimental evidence.
    action: ACCEPT
    reason: >-
      This IEA is consistent with the IDA annotations for the same term (PMID:9822593,
      PMID:9889188). TIM9 is a canonical subunit of the IMS chaperone complex.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex.

- term:
    id: GO:0046872
    label: metal ion binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: >-
      IEA annotation from UniProtKB keyword mapping (KW-0479 Metal-binding). TIM9 has a
      twin CX3C motif with four conserved cysteines. However, in the mature IMS-localized
      form, these cysteines form disulfide bonds rather than coordinating metal ions.
      Zinc coordination is thought to occur only transiently during cytoplasmic transit,
      and the mature functional form does not bind zinc (PMID:11867522). This IEA is
      misleading for the functional state of TIM9.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      While TIM9 may transiently coordinate zinc during its own import through the TOM
      complex, the mature functional form in the IMS contains disulfide bonds rather than
      zinc-coordinated cysteines (PMID:11867522). The UniProt entry itself states that the
      twin CX3C motif "contains 4 conserved Cys residues that form 2 disulfide bonds in
      the mitochondrial intermembrane space" and zinc coordination is only probable during
      transit. Annotating TIM9 as a metal ion binding protein is misleading for its
      functional state.
    supported_by:
    - reference_id: UniProt:O74700
      supporting_text: >-
        The twin CX3C motif contains 4 conserved Cys residues that form 2 disulfide bonds
        in the mitochondrial intermembrane space. However, during the transit of TIM9 from
        cytoplasm into mitochondrion, the Cys residues probably coordinate zinc, thereby
        preventing folding and allowing its transfer across mitochondrial outer membrane

# --- IPI annotation ---
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:11483513
  review:
    summary: >-
      IPI annotation based on physical interaction with TIM10 (PMID:11483513). Tim9 and
      Tim10 purified from E. coli can form a complex of the same size as the endogenous
      complex. This demonstrates direct protein-protein interaction. However, "protein
      binding" is uninformative -- TIM9 binding to TIM10 reflects its role in forming
      the functional hexameric chaperone complex.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      GO:0005515 "protein binding" is too vague and does not capture the specific functional
      interaction. TIM9 interacts with TIM10 to form the functional hexameric chaperone
      complex, and also binds to substrate carrier proteins during transit. The specific
      functions (unfolded protein carrier activity, complex membership) are captured by
      other annotations. Per curation guidelines, "protein binding" should be avoided in
      favor of more informative MF terms.
    supported_by:
    - reference_id: PMID:11483513
      supporting_text: >-
        Tim9 and Tim10 purified from Escherichia coli can form a complex of the same size
        as the endogenous complex from yeast mitochondria. This shows that no other
        mitochondrial protein is required for the formation of the TIM10 complex.

# --- IDA annotations: localization ---
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: IDA
  original_reference_id: PMID:9889188
  review:
    summary: >-
      IDA annotation for mitochondrial localization of TIM9 based on Adam et al. (1999),
      who identified Tim9 as a new component of the TIM22.54 translocase and demonstrated
      its mitochondrial localization.
    action: ACCEPT
    reason: >-
      TIM9 is a well-established mitochondrial protein. This IDA is correct and supported
      by direct experimental evidence.
    supported_by:
    - reference_id: PMID:9889188
      supporting_text: >-
        Tim9 is located in the mitochondrial intermembrane space and is organized into two
        distinct hetero-oligomeric assemblies with Tim10 and Tim12.

- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: IDA
  original_reference_id: PMID:10648604
  review:
    summary: >-
      IDA annotation for mitochondrial inner membrane localization based on Koehler et al.
      (2000), who characterized the TIM22 complex in the inner membrane containing Tim9p
      among its peripheral subunits.
    action: ACCEPT
    reason: >-
      A fraction of TIM9 is associated with the inner membrane as part of the 300 kDa
      TIM22 complex. This is consistent with the UniProt annotation and the known biology
      of TIM9.
    supported_by:
    - reference_id: PMID:10648604
      supporting_text: >-
        The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and
        the integral membrane subunits Tim22p and Tim54p.

- term:
    id: GO:0005758
    label: mitochondrial intermembrane space
  evidence_type: IDA
  original_reference_id: PMID:19037698
  review:
    summary: >-
      IDA annotation for IMS localization. However, PMID:19037698 is about "The Dutch
      multicenter experience of the endo-sponge treatment for anastomotic leakage after
      colorectal surgery" -- this is clearly an incorrect PMID. The intended reference
      is likely PMID:19037098 (Baker et al. 2009, about Tim9-Tim10 complex structure
      and function). The annotation itself is correct -- TIM9 is localized to the IMS --
      but the reference is wrong.
    action: ACCEPT
    reason: >-
      The localization of TIM9 to the mitochondrial IMS is well-established by multiple
      independent studies (PMID:9822593, PMID:9889188). Although the cited PMID:19037698
      appears to be a data entry error (likely meant PMID:19037098), the annotation itself
      is correct. Note: the PMID should be corrected from 19037698 to 19037098.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex.
    - reference_id: PMID:19037098
      supporting_text: >-
        The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by
        chaperoning select hydrophobic precursor proteins across the intermembrane space.

# --- IDA annotations: biological process ---
- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IDA
  original_reference_id: PMID:10648604
  review:
    summary: >-
      IDA annotation based on Koehler et al. (2000), who showed that Tim9p is part of the
      TIM22 complex mediating insertion of carrier proteins into the inner membrane.
      Deletion of TIM18 (a TIM22 complex subunit) is synthetically lethal with
      temperature-sensitive mutations in Tim9p.
    action: ACCEPT
    reason: >-
      This is a core function of TIM9. The Tim9-Tim10 complex escorts carrier proteins
      to the TIM22 complex for insertion into the inner membrane.
    supported_by:
    - reference_id: PMID:10648604
      supporting_text: >-
        Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two
        and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p.

- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IDA
  original_reference_id: PMID:19037098
  review:
    summary: >-
      IDA annotation based on Baker et al. (2009), who solved the Tim9-Tim10 crystal structure
      at 2.5 A resolution and performed mutational analysis showing that Tim9 plays an important
      functional role in facilitating the initial steps of translocating precursor substrates
      into the IMS.
    action: ACCEPT
    reason: >-
      Baker et al. provide structural and functional evidence that Tim9 is directly involved
      in substrate recognition and translocation, not just complex stabilization.
    supported_by:
    - reference_id: PMID:19037098
      supporting_text: >-
        We conclude that Tim9 plays an important functional role that includes facilitating
        the initial steps in translocating precursor substrates into the intermembrane space.

# --- RCA annotation: zinc ion binding ---
- term:
    id: GO:0008270
    label: zinc ion binding
  evidence_type: RCA
  original_reference_id: PMID:30358795
  review:
    summary: >-
      RCA annotation for zinc ion binding from Wang et al. (2018), a study characterizing
      the yeast zinc proteome by bioinformatic domain and motif searches. TIM9 was identified
      as a potential zinc-binding protein based on its CX3C motif. However, experimental
      evidence from Curran et al. (2002, PMID:11867522) demonstrated that TIM9 in the mature
      IMS form contains disulfide bonds, not zinc-coordinated cysteines.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      The RCA annotation is based on computational prediction from the CX3C motif. While
      TIM9 may transiently bind zinc during its cytoplasmic import phase, the mature
      functional form in the IMS uses disulfide bonds rather than zinc coordination
      (PMID:11867522, cited in UniProt). The annotation to GO:0008270 is misleading for
      the steady-state functional protein.
    supported_by:
    - reference_id: UniProt:O74700
      supporting_text: >-
        The twin CX3C motif contains 4 conserved Cys residues that form 2 disulfide bonds
        in the mitochondrial intermembrane space. However, during the transit of TIM9 from
        cytoplasm into mitochondrion, the Cys residues probably coordinate zinc
    - reference_id: PMID:30358795
      supporting_text: >-
        The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified
        using a bioinformatics analysis that combined global domain searches with local motif
        searches.

# --- TAS annotations: IMS localization ---
- term:
    id: GO:0005758
    label: mitochondrial intermembrane space
  evidence_type: TAS
  original_reference_id: Reactome:R-SCE-1252259
  review:
    summary: >-
      TAS annotation from Reactome pathway "TIM9:TIM10 binds hydrophobic proteins." The
      IMS localization is consistent with all experimental evidence.
    action: ACCEPT
    reason: >-
      Correctly reflects the localization of TIM9 in the IMS as part of the Tim9-Tim10
      complex. Redundant with IDA annotations but not incorrect.
    supported_by:
    - reference_id: Reactome:R-SCE-1252259
      supporting_text: TIM9:TIM10 binds hydrophobic proteins

- term:
    id: GO:0005758
    label: mitochondrial intermembrane space
  evidence_type: TAS
  original_reference_id: Reactome:R-SCE-1252260
  review:
    summary: >-
      TAS annotation from Reactome pathway "MIA40:ERV1 oxidizes cysteine residues to cystine
      disulfide bonds." This Reactome entry relates to the MIA pathway by which TIM9 is
      imported and oxidized in the IMS. The IMS localization is correct.
    action: ACCEPT
    reason: >-
      The MIA pathway (Mia40/Erv1) is responsible for the oxidative folding of TIM9 in the
      IMS, forming the disulfide bonds in its twin CX3C motif. This correctly places TIM9
      in the IMS. Redundant with other IMS localization annotations but not incorrect.
    supported_by:
    - reference_id: Reactome:R-SCE-1252260
      supporting_text: MIA40:ERV1 oxidizes cysteine residues to cystine disulfide bonds

# --- IDA annotations: protein transporter activity ---
- term:
    id: GO:0140318
    label: protein transporter activity
  evidence_type: IDA
  original_reference_id: PMID:9822593
  review:
    summary: >-
      IDA annotation for protein transporter activity based on Koehler et al. (1998), who
      demonstrated that Tim9p is an essential partner of Tim10p for the import of mitochondrial
      carrier proteins and can be cross-linked to partly translocated carrier proteins.
    action: ACCEPT
    reason: >-
      TIM9 does facilitate protein delivery across the IMS. The term GO:0140318 captures
      the transporter aspect. While GO:0140309 "unfolded protein carrier activity" would be
      more precise (as TIM9 moves with its cargo rather than facilitating movement without
      moving), this annotation is not incorrect. The more specific GO:0140309 is proposed
      as a NEW annotation.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        Tim9p can be cross-linked to a partly translocated carrier protein.
    - reference_id: PMID:9822593
      supporting_text: >-
        Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane
        proteins across the aqueous intermembrane space.

- term:
    id: GO:0140318
    label: protein transporter activity
  evidence_type: IGI
  original_reference_id: PMID:9822593
  review:
    summary: >-
      IGI annotation for protein transporter activity from the same study. Tim9p function
      was shown through genetic interaction with Tim10p (Ser67Cys mutation in Tim9p
      suppresses temperature-sensitive growth defects of tim10-1 and tim12-1 mutants).
    action: ACCEPT
    reason: >-
      The genetic interaction data (suppression of tim10-1 and tim12-1) support the functional
      involvement of TIM9 in protein transport, consistent with its role in the carrier
      import pathway.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect
        of tim10-1 and tim12-1 mutants.

# --- HDA annotations: mitochondrion ---
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: HDA
  original_reference_id: PMID:24769239
  review:
    summary: >-
      HDA (high-throughput direct assay) annotation for mitochondrial localization based on
      Renvoise et al. (2014), a quantitative proteomic study of isolated yeast mitochondria.
      TIM9 was identified by mass spectrometry in mitochondrial fractions.
    action: ACCEPT
    reason: >-
      Proteomic identification of TIM9 in mitochondrial fractions is consistent with all
      other evidence for its mitochondrial localization.
    supported_by:
    - reference_id: PMID:24769239
      supporting_text: >-
        Label free quantitative analysis of protein accumulation revealed significant variation
        of 176 mitochondrial proteins

- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: HDA
  original_reference_id: PMID:16823961
  review:
    summary: >-
      HDA annotation for mitochondrial localization based on Reinders et al. (2006), a
      comprehensive mitochondrial proteomics study that identified 851 proteins in yeast
      mitochondria using multidimensional LC-MS/MS approaches.
    action: ACCEPT
    reason: >-
      Mass spectrometry identification of TIM9 in purified mitochondria confirms its
      mitochondrial localization. The UniProt entry cites this study for subcellular location.
    supported_by:
    - reference_id: PMID:16823961
      supporting_text: >-
        A total of 851 different proteins (PROMITO dataset) were identified by use of
        multidimensional LC-MS/MS, 1D-SDS-PAGE combined with nano-LC-MS/MS and 2D-PAGE
        with subsequent MALDI-mass fingerprinting.

# --- IDA annotations: complex membership ---
- term:
    id: GO:0042719
    label: mitochondrial intermembrane space chaperone complex
  evidence_type: IDA
  original_reference_id: PMID:9822593
  review:
    summary: >-
      IDA annotation for membership in the mitochondrial IMS chaperone complex (the soluble
      Tim9-Tim10 70 kDa complex) based on Koehler et al. (1998), who co-purified Tim9p
      and Tim10p and showed co-immunoprecipitation.
    action: ACCEPT
    reason: >-
      TIM9 is a canonical and essential subunit of the Tim9-Tim10 IMS chaperone complex.
      This is a core annotation.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and
        Tim10p co-purify in successive chromatographic fractionations and
        co-immunoprecipitated with each other.

- term:
    id: GO:0042719
    label: mitochondrial intermembrane space chaperone complex
  evidence_type: IDA
  original_reference_id: PMID:9889188
  review:
    summary: >-
      IDA annotation for IMS chaperone complex membership based on Adam et al. (1999), who
      independently identified Tim9 and showed it forms distinct complexes with Tim10 and
      Tim12.
    action: ACCEPT
    reason: >-
      Independent confirmation of TIM9 as a component of the IMS chaperone complex.
    supported_by:
    - reference_id: PMID:9889188
      supporting_text: >-
        Tim9 is located in the mitochondrial intermembrane space and is organized into two
        distinct hetero-oligomeric assemblies with Tim10 and Tim12. One complex contains
        Tim9 and Tim10.

- term:
    id: GO:0042721
    label: TIM22 mitochondrial import inner membrane insertion complex
  evidence_type: IDA
  original_reference_id: PMID:9495346
  review:
    summary: >-
      IDA annotation for TIM22 complex membership based on Sirrenberg et al. (1998), who
      showed that Tim10 and Tim12 (and by extension Tim9) interact with precursors and
      are part of a complex with Tim22 that mediates inner membrane insertion. While Tim9
      was not directly characterized in this specific paper (it was not yet discovered),
      Tim9 was subsequently shown to be part of the TIM22 300 kDa complex (PMID:9822593,
      PMID:9889188).
    action: ACCEPT
    reason: >-
      TIM9 is a peripheral subunit of the TIM22 complex via its association with Tim10 and
      Tim12. The 300 kDa membrane complex containing Tim22, Tim54, Tim18, Tim12, Tim10,
      and Tim9 is well-established. ComplexPortal entry CPX-1629 lists TIM9 as a component.
    supported_by:
    - reference_id: PMID:9495346
      supporting_text: >-
        Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor
        and mediates its membrane-potential-dependent insertion into the inner membrane.
    - reference_id: PMID:9822593
      supporting_text: >-
        A small fraction of Tim9p is bound to the outer face of the inner membrane in a
        300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p.

# --- IDA/IGI/IMP annotations: protein insertion into mitochondrial inner membrane ---
- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IDA
  original_reference_id: PMID:10469659
  review:
    summary: >-
      IDA annotation based on Leuenberger et al. (1999), who showed that the Tim9p-Tim10p
      complex mediates the import of a specific subset of integral inner membrane proteins
      and can transfer these proteins to one of three different membrane insertion sites.
    action: ACCEPT
    reason: >-
      This study extends the known role of TIM9 beyond carrier proteins to additional
      inner membrane protein substrates, demonstrating the breadth of its involvement in
      protein insertion into the inner membrane.
    supported_by:
    - reference_id: PMID:10469659
      supporting_text: >-
        the two 70 kDa complexes each mediate the import of a different subset of integral
        inner membrane proteins and that they can transfer these proteins to one of three
        different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as
        yet uncharacterized insertion site.

- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IDA
  original_reference_id: PMID:9822593
  review:
    summary: >-
      IDA annotation based on Koehler et al. (1998), the original identification of Tim9p
      as an essential partner of Tim10p for import of mitochondrial carrier proteins.
    action: ACCEPT
    reason: >-
      Foundational study demonstrating TIM9 involvement in carrier protein import. Tim9p
      was shown to be cross-linkable to partly translocated carrier proteins.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        Tim9p can be cross-linked to a partly translocated carrier protein.

- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IGI
  original_reference_id: PMID:9822593
  review:
    summary: >-
      IGI annotation based on genetic interaction evidence from Koehler et al. (1998).
      The Ser67Cys mutation in Tim9p suppresses ts growth defects of tim10-1 and tim12-1,
      demonstrating genetic interaction within the carrier import pathway.
    action: ACCEPT
    reason: >-
      Genetic suppression data (Tim9p Ser67Cys suppresses tim10-1 and tim12-1) support
      the functional involvement of TIM9 in the carrier protein import pathway.
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect
        of tim10-1 and tim12-1 mutants.

- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IMP
  original_reference_id: PMID:9889188
  review:
    summary: >-
      IMP annotation based on Adam et al. (1999), who demonstrated that Tim9 is an essential
      protein and that the TIM9.10 complex mediates partial translocation of carrier proteins.
    action: ACCEPT
    reason: >-
      Mutant phenotype evidence (essentiality and translocation defects) supports the role
      of TIM9 in protein insertion into the mitochondrial inner membrane.
    supported_by:
    - reference_id: PMID:9889188
      supporting_text: >-
        We have identified Tim9, a new component of the TIM22.54 import machinery, which
        mediates transport of proteins into the inner membrane of mitochondria. Tim9, an
        essential protein of Saccharomyces cerevisiae

# --- THE KEY ANNOTATION: unfolded protein binding ---
- term:
    id: GO:0051082
    label: unfolded protein binding
  evidence_type: IDA
  original_reference_id: PMID:12138093
  review:
    summary: >-
      IDA annotation for unfolded protein binding based on Vial et al. (2002), who demonstrated
      that the reconstituted Tim9-Tim10 complex binds to the physiological substrate ADP/ATP
      carrier (AAC) and displays chaperone activity in refolding the model substrate firefly
      luciferase. TIM9 is the canonical carrier-holdase: it binds unfolded/hydrophobic protein
      precursors AND escorts them across the mitochondrial IMS from the TOM complex to the
      TIM22 complex. GO:0051082 "unfolded protein binding" captures only the binding aspect
      but misses the essential carrier/escort function. GO:0140309 "unfolded protein carrier
      activity" was created specifically for this class of proteins (go-ontology#30552) and
      is the correct replacement term.
    action: MODIFY
    reason: >-
      TIM9 (as part of the Tim9-Tim10 complex) is the textbook carrier-holdase: it binds
      unfolded hydrophobic precursor proteins AND escorts them between cellular compartments
      (from TOM to TIM22/SAM across the IMS). GO:0051082 "unfolded protein binding" captures
      only the binding aspect. GO:0140309 "unfolded protein carrier activity" was created in
      Nov 2025 specifically for TIM carrier-holdases (go-ontology#30552). Its definition --
      "A protein carrier activity that binds to a protein in an unfolded state and escorts it
      between two different cellular components. The unfolded protein carrier prevents
      aggregation of the target protein" -- precisely describes TIM9 function. This is a
      child of GO:0140597 "protein carrier chaperone" and GO:0140104 "molecular carrier
      activity." The Tim9-Tim10 complex acts as a "chaperone-like protein that protects the
      hydrophobic precursors from aggregation and guides them through the mitochondrial
      intermembrane space" (UniProt). PMID:12138093 shows the reconstituted complex is
      functional because it binds AAC and displays chaperone activity.
    proposed_replacement_terms:
    - id: GO:0140309
      label: unfolded protein carrier activity
    supported_by:
    - reference_id: PMID:12138093
      supporting_text: >-
        the reconstituted TIM10 complex is functional because it bound to the physiological
        substrate ADP/ATP carrier and displayed chaperone activity in refolding the model
        substrate firefly luciferase.
    - reference_id: PMID:9822593
      supporting_text: >-
        Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane
        proteins across the aqueous intermembrane space.
    - reference_id: UniProt:O74700
      supporting_text: >-
        Acts as a chaperone-like protein that protects the hydrophobic precursors from
        aggregation and guide them through the mitochondrial intermembrane space.

# --- NEW annotation: unfolded protein carrier activity ---
- term:
    id: GO:0140309
    label: unfolded protein carrier activity
  evidence_type: IDA
  original_reference_id: PMID:12138093
  review:
    summary: >-
      NEW annotation. TIM9 (as part of the Tim9-Tim10 hexameric complex) is the canonical
      carrier-holdase for which GO:0140309 was created (go-ontology#30552). The Tim9-Tim10
      complex binds unfolded hydrophobic transmembrane protein precursors and escorts them
      from the TOM complex across the IMS to the TIM22 complex (for inner membrane insertion)
      or to the SAM complex (for beta-barrel assembly). Vial et al. (2002, PMID:12138093)
      showed the reconstituted Tim9-Tim10 complex binds the ADP/ATP carrier substrate and
      displays chaperone activity. Multiple studies demonstrate the carrier/escort function:
      the complex guides hydrophobic proteins through the aqueous IMS (PMID:9822593), mediates
      partial translocation of carrier proteins (PMID:9889188), and can transfer substrates
      to multiple insertion sites (PMID:10469659).
    action: NEW
    reason: >-
      GO:0140309 "unfolded protein carrier activity" was created specifically for TIM
      carrier-holdases. TIM9 is the founding member of this functional class. This term
      is not currently annotated to TIM9 in GOA despite being the most precise MF term
      for its chaperone function. It should be added as a new annotation.
    supported_by:
    - reference_id: PMID:12138093
      supporting_text: >-
        the reconstituted TIM10 complex is functional because it bound to the physiological
        substrate ADP/ATP carrier and displayed chaperone activity in refolding the model
        substrate firefly luciferase.
    - reference_id: PMID:9822593
      supporting_text: >-
        Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane
        proteins across the aqueous intermembrane space.
    - reference_id: PMID:9889188
      supporting_text: >-
        The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates
        partial translocation of mitochondrial carriers proteins across the outer membrane.
    - reference_id: PMID:10469659
      supporting_text: >-
        the two 70 kDa complexes each mediate the import of a different subset of integral
        inner membrane proteins and that they can transfer these proteins to one of three
        different membrane insertion sites

references:
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  findings: []
- id: GO_REF:0000043
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
    vocabulary mapping, accompanied by conservative changes to GO terms applied by
    UniProt
  findings: []
- id: GO_REF:0000117
  title: Electronic Gene Ontology annotations created by ARBA machine learning models
  findings: []
- id: PMID:10469659
  title: Different import pathways through the mitochondrial intermembrane space for
    inner membrane proteins.
  findings:
  - statement: >-
      Tim9p-Tim10p and Tim8p-Tim13p complexes each mediate import of different subsets
      of inner membrane proteins, transferring them to TIM22, TIM23, or other sites.
- id: PMID:10648604
  title: Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported
    proteins into the yeast mitochondrial inner membrane.
  findings:
  - statement: >-
      Tim18p is an additional subunit of the TIM22 complex; its deletion is synthetically
      lethal with ts mutations in Tim9p or Tim10p.
- id: PMID:11483513
  title: Functional reconstitution of the import of the yeast ADP/ATP carrier mediated
    by the TIM10 complex.
  findings:
  - statement: >-
      Tim9 and Tim10 purified from E. coli form a complex identical in size to the
      endogenous complex. The reconstituted TIM10 complex restores AAC import in tim10-ts
      mitochondria.
- id: PMID:11867522
  title: The Tim9p-Tim10p complex binds to the transmembrane domains of the ADP/ATP
    carrier.
  findings:
  - statement: >-
      Mature Tim9-Tim10 complex in the IMS contains disulfide bonds, not zinc; zinc
      coordination occurs only during cytoplasmic transit.
- id: PMID:12138093
  title: Assembly of Tim9 and Tim10 into a functional chaperone.
  findings:
  - statement: >-
      The reconstituted Tim9-Tim10 hexameric complex binds the ADP/ATP carrier substrate
      and displays chaperone activity in refolding firefly luciferase.
- id: PMID:16823961
  title: 'Toward the complete yeast mitochondrial proteome: multidimensional separation
    techniques for mitochondrial proteomics.'
  findings:
  - statement: >-
      TIM9 identified by mass spectrometry in purified yeast mitochondria as part of
      comprehensive proteomics.
- id: PMID:19037098
  title: Structural and functional requirements for activity of the Tim9-Tim10 complex
    in mitochondrial protein import.
  findings:
  - statement: >-
      Crystal structure of yeast Tim9-Tim10 hexamer at 2.5A. Tim9 N-terminal region
      required for efficient substrate trapping; Tim9 plays an important functional
      (not just structural) role.
- id: PMID:19037698
  title: The Dutch multicenter experience of the endo-sponge treatment for anastomotic
    leakage after colorectal surgery.
  findings:
  - statement: >-
      NOTE: This PMID appears to be a data entry error in the GOA. It is about colorectal
      surgery, not mitochondrial biology. The intended reference is likely PMID:19037098.
- id: PMID:24769239
  title: Quantitative variations of the mitochondrial proteome and phosphoproteome
    during fermentative and respiratory growth in Saccharomyces cerevisiae.
  findings:
  - statement: >-
      TIM9 identified in quantitative proteomic analysis of yeast mitochondria under
      different growth conditions.
- id: PMID:30358795
  title: The cellular economy of the Saccharomyces cerevisiae zinc proteome.
  findings:
  - statement: >-
      TIM9 identified as a potential zinc-binding protein by bioinformatic analysis of
      the CX3C motif, but mature IMS form uses disulfide bonds.
- id: PMID:9495346
  title: Carrier protein import into mitochondria mediated by the intermembrane proteins
    Tim10/Mrs11 and Tim12/Mrs5.
  findings:
  - statement: >-
      Tim10 and Tim12 interact with carrier protein precursors and facilitate translocation
      across the outer membrane; they form a complex with Tim22 for inner membrane insertion.
- id: PMID:9822593
  title: Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial
    carrier proteins.
  findings:
  - statement: >-
      Tim9p identified as essential partner of Tim10p in a soluble 70 kDa IMS complex.
      Tim9p cross-links to partly translocated carrier proteins. A fraction associates
      with the inner membrane 300 kDa TIM22 complex.
- id: PMID:9889188
  title: Tim9, a new component of the TIM22.54 translocase in mitochondria.
  findings:
  - statement: >-
      Tim9 independently identified as essential component of TIM22.54 machinery. The
      TIM9.10 complex mediates partial translocation of carrier proteins; the TIM9.10.12
      complex assists further translocation via TIM22.54.
- id: Reactome:R-SCE-1252259
  title: TIM9:TIM10 binds hydrophobic proteins
  findings: []
- id: Reactome:R-SCE-1252260
  title: MIA40:ERV1 oxidizes cysteine residues to cystine disulfide bonds
  findings: []
- id: UniProt:O74700
  title: UniProtKB entry for TIM9_YEAST
  findings:
  - statement: >-
      Mitochondrial intermembrane chaperone; participates in import and insertion of
      multi-pass transmembrane proteins into the inner membrane and transfer of
      beta-barrel precursors to SAM complex.

core_functions:
- molecular_function:
    id: GO:0140309
    label: unfolded protein carrier activity
  description: >-
    TIM9 functions as part of the Tim9-Tim10 hexameric carrier-holdase complex that binds
    unfolded hydrophobic transmembrane protein precursors and escorts them from the TOM
    complex across the aqueous IMS to the TIM22 complex for inner membrane insertion, or
    to the SAM complex for outer membrane beta-barrel assembly. This carrier-holdase
    activity -- binding unfolded proteins AND transporting them between compartments --
    is the core molecular function of TIM9.
  directly_involved_in:
  - id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  locations:
  - id: GO:0005758
    label: mitochondrial intermembrane space
  in_complex:
    id: GO:0042719
    label: mitochondrial intermembrane space chaperone complex