TIM9

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0005743 mitochondrial inner membrane	IBA GO_REF:0000033	ACCEPT	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." 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. Supporting Evidence: PMID:9822593 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. PMID:9889188 Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12.
GO:0045039 protein insertion into mitochondrial inner membrane	IBA GO_REF:0000033	ACCEPT	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. 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. Supporting Evidence: PMID:9889188 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. PMID:9822593 Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space. file:yeast/TIM9/TIM9-deep-research-falcon.md Tim9 acts as a chaperone/escort factor that prevents aggregation of hydrophobic precursors in the aqueous IMS and transfers them from TOM to TIM22.
GO:0140318 protein transporter activity	IBA GO_REF:0000033	ACCEPT	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. 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. Supporting Evidence: PMID:9822593 Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.
GO:0005743 mitochondrial inner membrane	IEA GO_REF:0000044	ACCEPT	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. 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). Supporting Evidence: UniProt:O74700 SUBCELLULAR LOCATION: Mitochondrion inner membrane
GO:0005758 mitochondrial intermembrane space	IEA GO_REF:0000044	ACCEPT	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. 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. Supporting Evidence: PMID:9889188 Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12. file:yeast/TIM9/TIM9-deep-research-falcon.md Tim9 is located in the mitochondrial IMS
GO:0015031 protein transport	IEA GO_REF:0000043	ACCEPT	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. 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. Supporting Evidence: UniProt:O74700 Mitochondrial intermembrane chaperone that participates in the import and insertion of multi-pass transmembrane proteins into the mitochondrial inner membrane.
GO:0042719 mitochondrial intermembrane space chaperone complex	IEA GO_REF:0000117	ACCEPT	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. 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. Falcon deep research independently confirms the Tim9-Tim10 heterohexamer with 3:3 stoichiometry (~70 kDa soluble complex). Supporting Evidence: PMID:9822593 Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. file:yeast/TIM9/TIM9-deep-research-falcon.md Tim9 forms the canonical Tim9–Tim10 heterohexamer and also participates in a Tim9–Tim10–Tim12 docking complex linked to TIM22. file:yeast/TIM9/TIM9-deep-research-falcon.md Tim9:Tim10 complex stoichiometry is 3:3; the TIM22-associated complex is Tim9–Tim10–Tim12 = 3:2:1; the soluble Tim9/Tim10 complex is about ~70 kDa
GO:0046872 metal ion binding	IEA GO_REF:0000043	MARK AS OVER ANNOTATED	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. 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. Supporting Evidence: UniProt:O74700 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 file:yeast/TIM9/TIM9-deep-research-falcon.md Small TIM proteins (including Tim9) are imported through TOM and undergo oxidative folding in the IMS via the MIA (mitochondrial import and assembly) pathway, which introduces disulfides in the conserved CX3C motifs
GO:0005515 protein binding	IPI PMID:11483513 Functional reconstitution of the import of the yeast ADP/ATP...	MARK AS OVER ANNOTATED	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. 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. Falcon deep research notes that within the complex Tim10 provides the key substrate-binding elements (Tim9 alone does not bind the AAC carrier), and that the obligate Tim9-Tim10 partnership forms an essential heterohexamer -- the functionally meaningful interaction, not generic protein binding. Supporting Evidence: PMID:11483513 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. file:yeast/TIM9/TIM9-deep-research-falcon.md Tim9 and Tim10 assemble into an essential heterohexameric Tim9/10 complex that chaperones hydrophobic preproteins from TOM to TIM22 file:yeast/TIM9/TIM9-deep-research-falcon.md Biochemical substrate-binding evidence indicates that Tim10 (alone) can bind the ATP/ADP carrier (AAC) similarly to the Tim9–Tim10 complex, whereas Tim9 alone does not bind AAC**, supporting a model where Tim10 provides key substrate-binding elements within the complex
GO:0005739 mitochondrion	IDA PMID:9889188 Tim9, a new component of the TIM22.54 translocase in mitocho...	ACCEPT	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. Reason: TIM9 is a well-established mitochondrial protein. This IDA is correct and supported by direct experimental evidence. Supporting Evidence: PMID:9889188 Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12.
GO:0005743 mitochondrial inner membrane	IDA PMID:10648604 Tim18p, a new subunit of the TIM22 complex that mediates ins...	ACCEPT	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. 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. Supporting Evidence: PMID:10648604 The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p.
GO:0005758 mitochondrial intermembrane space	IDA PMID:19037698 The Dutch multicenter experience of the endo-sponge treatmen...	ACCEPT	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. 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. Supporting Evidence: PMID:9822593 Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. PMID:19037098 The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space.
GO:0045039 protein insertion into mitochondrial inner membrane	IDA PMID:10648604 Tim18p, a new subunit of the TIM22 complex that mediates ins...	ACCEPT	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. 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. Supporting Evidence: PMID:10648604 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.
GO:0045039 protein insertion into mitochondrial inner membrane	IDA PMID:19037098 Structural and functional requirements for activity of the T...	ACCEPT	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. Reason: Baker et al. provide structural and functional evidence that Tim9 is directly involved in substrate recognition and translocation, not just complex stabilization. Supporting Evidence: PMID:19037098 We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.
GO:0008270 zinc ion binding	RCA PMID:30358795 The cellular economy of the Saccharomyces cerevisiae zinc pr...	MARK AS OVER ANNOTATED	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. 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. Supporting Evidence: UniProt:O74700 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 PMID:30358795 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. file:yeast/TIM9/TIM9-deep-research-falcon.md the conserved CX3C cysteines form two intramolecular disulfide bonds important for structural integrity
GO:0005758 mitochondrial intermembrane space	TAS Reactome:R-SCE-1252259	ACCEPT	Summary: TAS annotation from Reactome pathway "TIM9:TIM10 binds hydrophobic proteins." The IMS localization is consistent with all experimental evidence. Reason: Correctly reflects the localization of TIM9 in the IMS as part of the Tim9-Tim10 complex. Redundant with IDA annotations but not incorrect. Supporting Evidence: Reactome:R-SCE-1252259 TIM9:TIM10 binds hydrophobic proteins
GO:0005758 mitochondrial intermembrane space	TAS Reactome:R-SCE-1252260	ACCEPT	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. 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. Supporting Evidence: Reactome:R-SCE-1252260 MIA40:ERV1 oxidizes cysteine residues to cystine disulfide bonds
GO:0140318 protein transporter activity	IDA PMID:9822593 Tim9p, an essential partner subunit of Tim10p for the import...	ACCEPT	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. 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. Supporting Evidence: PMID:9822593 Tim9p can be cross-linked to a partly translocated carrier protein. PMID:9822593 Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.
GO:0140318 protein transporter activity	IGI PMID:9822593 Tim9p, an essential partner subunit of Tim10p for the import...	ACCEPT	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). 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. Supporting Evidence: PMID:9822593 A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants.
GO:0005739 mitochondrion	HDA PMID:24769239 Quantitative variations of the mitochondrial proteome and ph...	ACCEPT	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. Reason: Proteomic identification of TIM9 in mitochondrial fractions is consistent with all other evidence for its mitochondrial localization. Supporting Evidence: PMID:24769239 Label free quantitative analysis of protein accumulation revealed significant variation of 176 mitochondrial proteins
GO:0005739 mitochondrion	HDA PMID:16823961 Toward the complete yeast mitochondrial proteome: multidimen...	ACCEPT	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. Reason: Mass spectrometry identification of TIM9 in purified mitochondria confirms its mitochondrial localization. The UniProt entry cites this study for subcellular location. Supporting Evidence: PMID:16823961 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.
GO:0042719 mitochondrial intermembrane space chaperone complex	IDA PMID:9822593 Tim9p, an essential partner subunit of Tim10p for the import...	ACCEPT	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. Reason: TIM9 is a canonical and essential subunit of the Tim9-Tim10 IMS chaperone complex. This is a core annotation. Supporting Evidence: PMID:9822593 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.
GO:0042719 mitochondrial intermembrane space chaperone complex	IDA PMID:9889188 Tim9, a new component of the TIM22.54 translocase in mitocho...	ACCEPT	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. Reason: Independent confirmation of TIM9 as a component of the IMS chaperone complex. Supporting Evidence: PMID:9889188 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.
GO:0042721 TIM22 mitochondrial import inner membrane insertion complex	IDA PMID:9495346 Carrier protein import into mitochondria mediated by the int...	ACCEPT	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). 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. Supporting Evidence: PMID:9495346 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. PMID:9822593 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.
GO:0045039 protein insertion into mitochondrial inner membrane	IDA PMID:10469659 Different import pathways through the mitochondrial intermem...	ACCEPT	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. 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. Supporting Evidence: PMID:10469659 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.
GO:0045039 protein insertion into mitochondrial inner membrane	IDA PMID:9822593 Tim9p, an essential partner subunit of Tim10p for the import...	ACCEPT	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. Reason: Foundational study demonstrating TIM9 involvement in carrier protein import. Tim9p was shown to be cross-linkable to partly translocated carrier proteins. Supporting Evidence: PMID:9822593 Tim9p can be cross-linked to a partly translocated carrier protein.
GO:0045039 protein insertion into mitochondrial inner membrane	IGI PMID:9822593 Tim9p, an essential partner subunit of Tim10p for the import...	ACCEPT	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. Reason: Genetic suppression data (Tim9p Ser67Cys suppresses tim10-1 and tim12-1) support the functional involvement of TIM9 in the carrier protein import pathway. Supporting Evidence: PMID:9822593 A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants.
GO:0045039 protein insertion into mitochondrial inner membrane	IMP PMID:9889188 Tim9, a new component of the TIM22.54 translocase in mitocho...	ACCEPT	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. Reason: Mutant phenotype evidence (essentiality and translocation defects) supports the role of TIM9 in protein insertion into the mitochondrial inner membrane. Supporting Evidence: PMID:9889188 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
GO:0051082 unfolded protein binding	IDA PMID:12138093 Assembly of Tim9 and Tim10 into a functional chaperone.	MODIFY	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. 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 replacements: unfolded protein carrier activity Supporting Evidence: PMID:12138093 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. PMID:9822593 Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space. UniProt:O74700 Acts as a chaperone-like protein that protects the hydrophobic precursors from aggregation and guide them through the mitochondrial intermembrane space. file:yeast/TIM9/TIM9-deep-research-falcon.md Tim9 acts as a chaperone/escort factor that prevents aggregation of hydrophobic precursors in the aqueous IMS and transfers them from TOM to TIM22. file:yeast/TIM9/TIM9-deep-research-falcon.md TIM9·10 recognizes hydrophobic clients primarily through a hydrophobic cleft formed by chaperone helices
GO:0140309 unfolded protein carrier activity	IDA PMID:12138093 Assembly of Tim9 and Tim10 into a functional chaperone.	NEW	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). 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. Supporting Evidence: PMID:12138093 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. PMID:9822593 Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space. PMID:9889188 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. PMID:10469659 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 file:yeast/TIM9/TIM9-deep-research-falcon.md Tim9 is best viewed as a specialized, soluble IMS holdase chaperone file:yeast/TIM9/TIM9-deep-research-falcon.md Small TIMs can also assist trafficking of some outer-membrane β-barrel proteins toward the SAM complex

Core Functions

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.

Molecular Function:

unfolded protein carrier activity

Directly Involved In:

protein insertion into mitochondrial inner membrane

Cellular Locations:

mitochondrial intermembrane space

In Complex:

mitochondrial intermembrane space chaperone complex

References

GO_REF:0000033

Annotation inferences using phylogenetic trees

GO_REF:0000043

Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping

GO_REF:0000044

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

GO_REF:0000117

Electronic Gene Ontology annotations created by ARBA machine learning models

PMID:10469659

Different import pathways through the mitochondrial intermembrane space for inner membrane proteins.

Tim9p-Tim10p and Tim8p-Tim13p complexes each mediate import of different subsets of inner membrane proteins, transferring them to TIM22, TIM23, or other sites.

PMID:10648604

Tim18p, a new subunit of the TIM22 complex that mediates insertion of imported proteins into the yeast mitochondrial inner membrane.

Tim18p is an additional subunit of the TIM22 complex; its deletion is synthetically lethal with ts mutations in Tim9p or Tim10p.

PMID:11483513

Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex.

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.

PMID:11867522

The Tim9p-Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier.

Mature Tim9-Tim10 complex in the IMS contains disulfide bonds, not zinc; zinc coordination occurs only during cytoplasmic transit.

PMID:12138093

Assembly of Tim9 and Tim10 into a functional chaperone.

The reconstituted Tim9-Tim10 hexameric complex binds the ADP/ATP carrier substrate and displays chaperone activity in refolding firefly luciferase.

PMID:16823961

Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics.

TIM9 identified by mass spectrometry in purified yeast mitochondria as part of comprehensive proteomics.

PMID:19037098

Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import.

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.

PMID:19037698

The Dutch multicenter experience of the endo-sponge treatment for anastomotic leakage after colorectal surgery.

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.

PMID:24769239

Quantitative variations of the mitochondrial proteome and phosphoproteome during fermentative and respiratory growth in Saccharomyces cerevisiae.

TIM9 identified in quantitative proteomic analysis of yeast mitochondria under different growth conditions.

PMID:30358795

The cellular economy of the Saccharomyces cerevisiae zinc proteome.

TIM9 identified as a potential zinc-binding protein by bioinformatic analysis of the CX3C motif, but mature IMS form uses disulfide bonds.

PMID:9495346

Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5.

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.

PMID:9822593

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins.

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.

PMID:9889188

Tim9, a new component of the TIM22.54 translocase in mitochondria.

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.

Reactome:R-SCE-1252259

TIM9:TIM10 binds hydrophobic proteins

Reactome:R-SCE-1252260

MIA40:ERV1 oxidizes cysteine residues to cystine disulfide bonds

UniProt:O74700

UniProtKB entry for TIM9_YEAST

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.

file:yeast/TIM9/TIM9-deep-research-falcon.md

Falcon deep research on TIM9 (Edison Scientific Literature)

Tim9 is a small TIM family soluble IMS chaperone (holdase) whose primary role is chaperone-like binding and transfer of hydrophobic precursors, not catalysis or transport.

"Tim9 is best viewed as a **specialized, soluble IMS holdase chaperone**"
Tim9 is located in the mitochondrial intermembrane space, between TOM-mediated entry and TIM22-mediated inner-membrane insertion.

"Tim9 is located in the **mitochondrial IMS**"
Tim9 forms the canonical Tim9-Tim10 heterohexamer (3:3 stoichiometry, ~70 kDa soluble complex) and a Tim9-Tim10-Tim12 (3:2:1) docking complex linked to TIM22.

"Tim9:Tim10 complex stoichiometry is **3:3**; the TIM22-associated complex is **Tim9–Tim10–Tim12 = 3:2:1**; the soluble Tim9/Tim10 complex is about **~70 kDa**"
Tim9 acts as a chaperone/escort factor that prevents aggregation of hydrophobic precursors in the aqueous IMS and transfers them from TOM to TIM22.

"Tim9 acts as a **chaperone/escort factor** that prevents aggregation of hydrophobic precursors in the aqueous IMS and transfers them from **TOM** to **TIM22**."
Within the complex, Tim10 provides the key substrate-binding elements; Tim9 alone does not bind the AAC carrier (consistent with UniProt noting Tim9 has a strong structural role relative to Tim10).

"Biochemical substrate-binding evidence indicates that **Tim10** (alone) can bind the **ATP/ADP carrier (AAC)** similarly to the Tim9–Tim10 complex, whereas **Tim9 alone does not bind AAC**, supporting a model where Tim10 provides key substrate-binding elements within the complex"
The Tim9-Tim10 complex recognizes hydrophobic clients through a hydrophobic cleft formed by chaperone helices.

"TIM9·10 recognizes hydrophobic clients primarily through a **hydrophobic cleft** formed by chaperone helices"
Small TIMs including Tim9 can also assist trafficking of some outer-membrane beta-barrel proteins toward the SAM complex.

"Small TIMs can also assist trafficking of some outer-membrane β-barrel proteins toward the SAM complex"
Tim9 is itself imported via TOM and oxidatively folded in the IMS by the MIA pathway, which introduces the two intramolecular disulfides in its conserved CX3C motifs (rather than stable metal coordination in the mature form).

"Small TIM proteins (including Tim9) are imported through TOM and undergo oxidative folding in the IMS via the **MIA (mitochondrial import and assembly) pathway**, which introduces disulfides in the conserved CX3C motifs"
Tim9 and Tim10 assemble into an essential heterohexameric Tim9/10 complex; unassembled small TIMs can be degraded by Yme1, with assembly being protective.

"Tim9 and Tim10 assemble into an essential heterohexameric Tim9/10 complex** that chaperones hydrophobic preproteins from TOM to TIM22"
Conserved-residue mutants tim9-19 (E52G) and tim9-3 (V40A + S60P) abolish a detectable Tim9-Tim10 heterohexamer, linking these residues to complex integrity and import function.

"tim9-19 (E52G)** and **tim9-3 (V40A + S60P)** show **no detectable Tim9–Tim10 heterohexamer** in detergent-solubilized mitochondria"

Deep Research

Falcon

(TIM9-deep-research-falcon.md)

this with annotations you find in gene/protein databases, but these can be outdated or inaccurate. Falcon Edison Scientific Literature 32 citations 2 artifacts 2026-05-31T17:46:08.534441

Edison artifact artifact-00 (text/markdown)

## Context ID: pqac-00000008 The document provides several structural representations of the TIM9·10 and TIM23 complexes. Figure 1F (page 3) shows a top-view st (image/png)

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Research Report: Functional Annotation of S. cerevisiae TIM9 (UniProt O74700)

0) Mandatory identity verification (disambiguation)

The target protein is Tim9 from Saccharomyces cerevisiae S288c, encoded by TIM9 (YEL020W-A / YEL020BW) and annotated in UniProt as “Mitochondrial import inner membrane translocase subunit TIM9” (UniProt O74700; user-supplied context). The literature evidence gathered here consistently describes yeast Tim9 as a small TIM family protein localized to the mitochondrial intermembrane space (IMS), forming hetero-oligomeric chaperone complexes with Tim10 (and Tim12) that facilitate the TIM22 carrier pathway. This matches the UniProt description and the expected small Tim family/domain architecture (twin CX3C motifs; Tim10-like family) (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4).

1) Key concepts, definitions, and current understanding

1.1 Small TIM chaperones (definition)

Small TIMs are a set of small (∼10 kDa class) soluble IMS chaperones that escort hydrophobic mitochondrial membrane-protein precursors after they pass through the TOM complex, preventing aggregation in the aqueous IMS and enabling transfer to downstream insertases/translocases (gentle2007conservedmotifsreveal pages 1-1, chaudhuri2020tim17updatesa pages 2-5).

1.2 TIM22 “carrier pathway” and Tim9’s central role

Hydrophobic multi-pass inner-membrane proteins lacking N-terminal presequences (classically metabolite carrier proteins) are inserted via the TIM22 translocase, and small TIM complexes are required for their transit across the IMS from TOM → TIM22 (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4).

Tim9’s primary functional role is therefore chaperone-like binding and transfer of hydrophobic internal-signal precursors rather than catalysis or transport. In practice, Tim9 is a core component of the peripheral IMS module of the TIM22 pathway, physically and functionally coupled to TIM22 via Tim12 and membrane subunits (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4).

1.3 Localization and biogenesis (MIA pathway)

Tim9 is located in the mitochondrial IMS (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4). Small TIM proteins (including Tim9) are imported through TOM and undergo oxidative folding in the IMS via the MIA (mitochondrial import and assembly) pathway, which introduces disulfides in the conserved CX3C motifs (chaudhuri2020tim17updatesa pages 2-5, gentle2007conservedmotifsreveal pages 1-1).

2) Molecular function, complexes, substrates, and mechanism

2.1 Complex composition and stoichiometry

Multiple sources converge on two principal assemblies:

Soluble Tim9–Tim10 heterohexamer in the IMS
Stoichiometry: Tim9:Tim10 = 3:3 (chaudhuri2020tim17updatesa pages 2-5)
Approximate complex mass: ~70 kDa (kumar2020conservedregionsof pages 1-4)
TIM22-associated docking complex
Stoichiometry: Tim9–Tim10–Tim12 = 3:2:1, localized on the TIM22 translocase (chaudhuri2020tim17updatesa pages 2-5)
Tim12 is described as stably associated with Tim22 and facilitates docking/hand-off to the TIM22 insertase (chaudhuri2020tim17updatesa pages 2-5).

At the translocase scale, the yeast TIM22 complex is described as ~300 kDa, composed of Tim22/Tim54/Tim18/Sdh3 plus the small Tim module (Tim9/Tim10/Tim12) (kumar2023functionalcrosstalkbetween pages 1-2, kumar2020conservedregionsof pages 1-4).

2.2 Substrate specificity: carrier proteins and other hydrophobic clients

The canonical TIM22 substrate class is mitochondrial carrier proteins (MCPs), including exemplars such as AAC2/Pet9 (ADP/ATP carrier), Pic/Pic2, and Dic1, which are cited as model TIM22 cargos (kumar2023functionalcrosstalkbetween pages 1-2). Biochemical substrate-binding evidence indicates that Tim10 (alone) can bind the ATP/ADP carrier (AAC) similarly to the Tim9–Tim10 complex, whereas Tim9 alone does not bind AAC, supporting a model where Tim10 provides key substrate-binding elements within the complex (gentle2007conservedmotifsreveal pages 6-7).

Small TIMs can also assist trafficking of some outer-membrane β-barrel proteins toward the SAM complex, indicating broader IMS “holdase/escort” functions beyond only inner-membrane carriers (chaudhuri2020tim17updatesa pages 2-5, gentle2007conservedmotifsreveal pages 9-9).

2.3 Mechanism (handoff from TOM to TIM22)

In the prevailing model synthesized in authoritative reviews and pathway studies, Tim9-containing small TIM complexes:
1) capture/release hydrophobic precursors emerging from TOM into the IMS,
2) maintain them in an insertion-competent state,
3) dock onto the TIM22 translocase (via Tim12 and membrane adaptors such as Tim54), and
4) deliver precursors for membrane insertion through Tim22 (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4).

3) Structural basis of function (client binding and complex stability)

3.1 Core fold and conserved motifs

Small TIM proteins (including Tim9) contain conserved twin CX3C motifs that form two intramolecular disulfide bonds, stabilizing a compact helical bundle suitable for IMS chaperone activity (gentle2007conservedmotifsreveal pages 1-1, chaudhuri2020tim17updatesa pages 2-5).

3.2 Hexamer architecture and interfaces

A detailed comparative structural analysis identifies conserved intersubunit contacts (aromatic packing and electrostatic ion pairs) that stabilize the Tim9–Tim10 hexamer and rationalize why specific point mutations disrupt assembly (gentle2007conservedmotifsreveal pages 5-6). Conserved contacts include aromatic residues in Tim9 (e.g., F29/F36) and a network of intersubunit ion pairs (gentle2007conservedmotifsreveal pages 5-6).

3.3 Genetic evidence linking structure to function

Mutations in conserved residues of yeast Tim9 destabilize the Tim9–Tim10 complex:
- tim9-19 (E52G) and tim9-3 (V40A + S60P) show no detectable Tim9–Tim10 heterohexamer in detergent-solubilized mitochondria, linking those residues to complex integrity and thus function in import (gentle2007conservedmotifsreveal pages 5-6).

3.4 Client-binding clefts and specificity (molecular biophysics)

High-resolution biophysical work on small TIM chaperones demonstrates that TIM9·10 recognizes hydrophobic clients primarily through a hydrophobic cleft formed by chaperone helices, whereas TIM8·13 is tuned toward clients with substantial hydrophilic/disordered segments via electrostatic interactions (sucec2020structuralbasisof pages 1-2, sucec2020structuralbasisof pages 8-9). In this framework:
- The carrier-like client Ggc1 shows a ~5–10-fold preference for TIM9·10 (relative to TIM8·13), while Tim23 shows a modest preference for TIM8·13 (∼1.5-fold), capturing the client-specific division of labor between chaperone complexes (sucec2020structuralbasisof pages 2-3).
- TIM9·10 can form “fuzzy” complexes driven by many weak, rapidly interconverting (<1 ms) contacts (sucec2020structuralbasisof pages 2-3).

Quantitative binding example (Tim23 IMS segment): TIM8·13 binds the Tim23 IMS fragment with Kd = 66 ± 8 μM, whereas TIM9·10 binding to Tim23IMS is undetectable by ITC in the reported assays, consistent with a primarily hydrophobic-client specialization for TIM9·10 (sucec2020structuralbasisof pages 3-4, sucec2020structuralbasisof pages 8-9).

Stoichiometry (chaperone:precursor): Biophysical measurements converge on 1:1 chaperone:Tim23 stoichiometry for TIM9·10–Tim23 and TIM8·13–Tim23, while an exception is noted for a carrier (Ggc1) where two TIM9·10 complexes hold one precursor (2:1) (sucec2020structuralbasisof pages 7-8, sucec2020structuralbasisof pages 2-3).

4) Recent developments (prioritizing 2023–2024)

4.1 2023: TIM22 pathway is integrated with mitochondrial quality control via Yme1

A 2023 Journal of Cell Science study identified functional crosstalk between TIM22 and the i-AAA protease Yme1 in S. cerevisiae, providing a modern view in which carrier import capacity must be balanced with proteostasis:
- The authors state that excess TIM22 pathway cargos can cause proteostatic stress and cell death, and that Yme1 contributes to TIM22 substrate proteostasis and TIM22 complex stability (kumar2023functionalcrosstalkbetween pages 1-2).
- Phenotypically, yme1Δ cells show severe respiratory growth defects at 37°C on non-fermentable medium (YPG), and partial impairment of TIM22 (e.g., tim18Δ or tim22K127A) rescues the yme1Δ respiratory growth defect under these conditions (kumar2023functionalcrosstalkbetween pages 2-3).
- Conversely, Tim22 overexpression worsens growth of yme1Δ under tested conditions, consistent with a model where elevated carrier-pathway throughput can be deleterious when quality control is compromised (kumar2023functionalcrosstalkbetween pages 2-3).

Although this study focuses on TIM22 and Yme1, Tim9’s relevance is direct because Tim9 is part of the TIM22 peripheral module and thereby contributes to the flux of hydrophobic substrates that drive the proteostatic phenotype (kumar2023functionalcrosstalkbetween pages 1-2).

4.2 2024: Review synthesis—small TIM assembly state is surveilled by Yme1

A 2024 mini-review in Biochemical Society Transactions consolidates evidence that:
- Tim9 and Tim10 assemble into an essential heterohexameric Tim9/10 complex that chaperones hydrophobic preproteins from TOM to TIM22 (kan2024roleofyme1 pages 3-4).
- Small TIM proteins are imported via the redox-sensitive Mia40/MIA pathway, and unassembled small TIM components can be targeted for proteolytic degradation by Yme1, with assembly into the Tim9/10 complex being protective (kan2024roleofyme1 pages 3-4).
- TIM22 complex stability is decreased in Δyme1 cells and import efficiency is particularly affected under heat stress conditions, linking TIM22 pathway competence (and therefore Tim9/Tim10 function) to protease-mediated quality control (kan2024roleofyme1 pages 3-4).

4.3 2024: Updated structural interpretations of small TIM docking to TIM22

A 2024 structural thesis on TIM22 proposes mechanistic features relevant to Tim9/Tim10 delivery:
- Yeast Tim9/Tim10 termini are longer and flexible, with “tentacle” mobility hypothesized to help engage precursors, consistent with a stable hexamer core and flexible substrate-interaction periphery (valpadashi2024structuralandfunctional pages 51-51).
- The IMS-facing helix of TIM22 near its N-terminus is interpreted as a candidate small TIM docking site, positioned close to the membrane to facilitate insertion (valpadashi2024structuralandfunctional pages 51-51).

5) Current applications and real-world implementations

Model system for mitochondrial membrane protein biogenesis: TIM9 is routinely used in yeast as part of the canonical TIM22 carrier-pathway framework, enabling mechanistic dissection of how hydrophobic membrane proteins are escorted through aqueous compartments to their insertase (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4).
Structural/biophysical platforms for chaperone specificity: Small TIM chaperones (including TIM9·10) are used as experimentally tractable systems to study “fuzzy” chaperone-client binding, competition, and specificity tuning via hydrophobic versus electrostatic interactions (sucec2020structuralbasisof pages 2-3, sucec2020structuralbasisof pages 8-9).
Proteostasis and quality control studies: Tim9’s pathway role places it at the intersection of protein import and mitochondrial quality control. The demonstrated genetic interactions between TIM22 and Yme1 provide a functional context for studying how import flux, chaperone availability, and protease capacity jointly shape mitochondrial health (kumar2023functionalcrosstalkbetween pages 2-3, kan2024roleofyme1 pages 3-4).

6) Expert opinions and authoritative synthesis

Across reviews and primary studies, a consistent expert-level interpretation emerges:
- Tim9 is best viewed as a specialized, soluble IMS holdase chaperone, whose biological necessity stems from the requirement to traffic highly hydrophobic, multi-pass clients across the IMS without aggregation and with correct delivery to TIM22 (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4).
- Modern mechanistic work emphasizes that specificity is not purely “one chaperone per client,” but rather emerges from the physicochemical matching between client features (hydrophobic TM segments vs. hydrophilic disordered regions) and chaperone cleft properties, enabling competition and transfer between small TIM complexes (sucec2020structuralbasisof pages 2-3, sucec2020structuralbasisof pages 8-9).

7) Evidence map (summary table)

The following table consolidates the main functional annotation points, quantitative values, and citations.

Category	Key points	Evidence details (specific stoichiometries, sizes, mutations, residue pairs)	Key sources (author year journal)	URL
identity	TIM9 in this report matches Saccharomyces cerevisiae Tim9, a small TIM family mitochondrial protein corresponding to UniProt O74700; it is an intermembrane-space chaperone, not an enzyme or transporter.	Small TIM proteins are typically ~8–12 kDa; in yeast, Tim9 is one of five small TIMs (Tim8, Tim9, Tim10, Tim12, Tim13). Tim9 belongs to the zf-Tim10/DDP family with conserved twin CX3C motifs. (guillen2023uniqueinteractionsand pages 1-5, gentle2007conservedmotifsreveal pages 1-1)	Gentle et al. 2007 Mol Biol Evol; Guillén et al. 2023 bioRxiv	https://doi.org/10.1093/molbev/msm031 ; https://doi.org/10.1101/2023.05.29.542777
localization	Tim9 localizes to the mitochondrial intermembrane space (IMS), where it acts between TOM-mediated entry and TIM22-mediated inner-membrane insertion.	Multiple sources place Tim9 in the IMS as a soluble chaperone module that escorts hydrophobic precursors after TOM transit. (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4)	Chaudhuri et al. 2020 Biomolecules; Kumar et al. 2020 J Cell Sci	https://doi.org/10.3390/biom10121643 ; https://doi.org/10.1242/jcs.244632
complexes	Tim9 forms the canonical Tim9–Tim10 heterohexamer and also participates in a Tim9–Tim10–Tim12 docking complex linked to TIM22.	Tim9:Tim10 complex stoichiometry is 3:3; the TIM22-associated complex is Tim9–Tim10–Tim12 = 3:2:1; the soluble Tim9/Tim10 complex is about ~70 kDa; TIM22 machinery is about ~300 kDa. Tim12 is stably associated with Tim22 and helps dock the chaperone complex to the translocase. (chaudhuri2020tim17updatesa pages 2-5, kumar2020conservedregionsof pages 1-4)	Chaudhuri et al. 2020 Biomolecules; Kumar et al. 2020 J Cell Sci	https://doi.org/10.3390/biom10121643 ; https://doi.org/10.1242/jcs.244632
substrates	Tim9’s primary substrate class is hydrophobic multi-pass inner-membrane proteins, especially mitochondrial carrier proteins imported via the TIM22 pathway.	Substrates include metabolite carriers such as the ATP/ADP carrier (AAC) and other carrier-type proteins; sources also note involvement with some internal-signal translocase subunits (e.g., Tim22, Tim23, Tim17 in pathway context). Small TIMs can also contribute to transfer/assembly of some outer-membrane β-barrel proteins to SAM. (chaudhuri2020tim17updatesa pages 2-5, gentle2007conservedmotifsreveal pages 6-7, gentle2007conservedmotifsreveal pages 9-9, kumar2020conservedregionsof pages 1-4)	Chaudhuri et al. 2020 Biomolecules; Gentle et al. 2007 Mol Biol Evol; Kumar et al. 2020 J Cell Sci	https://doi.org/10.3390/biom10121643 ; https://doi.org/10.1093/molbev/msm031 ; https://doi.org/10.1242/jcs.244632
mechanism	Tim9 acts as a chaperone/escort factor that prevents aggregation of hydrophobic precursors in the aqueous IMS and transfers them from TOM to TIM22.	Tim9/Tim10 releases hydrophobic clients from TOM and hands them to TIM22; Tim12-containing complex mediates docking to the TIM22 translocase. Structural work supports hydrophobic client-binding clefts in the Tim9/Tim10 hexamer and flexible terminal “tentacles” involved in precursor engagement. (chaudhuri2020tim17updatesa pages 2-5, sucec2020structuralbasisof pages 3-3, sucec2020structuralbasisof media 57627111, valpadashi2024structuralandfunctional pages 51-51)	Chaudhuri et al. 2020 Biomolecules; Sučec et al. 2020 Sci Adv; Valpadashi 2024 PhD thesis	https://doi.org/10.3390/biom10121643 ; https://doi.org/10.1126/sciadv.abd0263 ; https://doi.org/10.53846/goediss-10678
structure & motifs	Tim9 is a small TIM chaperone with twin CX3C motifs, disulfide-stabilized fold, and conserved intersubunit contacts required for hexamer stability.	Small TIM hexamers are described as donut/propeller-like with a relatively flat face and terminal tentacle-like extensions. Conserved contacts include aromatic interactions involving Tim9-F29, Tim9-F36 and ion-pairing involving the conserved Tim9 glutamate. In mutant analysis, tim9-19 = E52G and tim9-3 = V40A + S60P caused loss of detectable Tim9–Tim10 heterohexamer in detergent-solubilized mitochondria. Crosslinks reported for yeast Tim9/Tim10 include K58–K81, K58–K45, and K81–K68. (gentle2007conservedmotifsreveal pages 5-6, gentle2007conservedmotifsreveal pages 6-7, valpadashi2024structuralandfunctional pages 51-51)	Gentle et al. 2007 Mol Biol Evol; Valpadashi 2024 PhD thesis	https://doi.org/10.1093/molbev/msm031 ; https://doi.org/10.53846/goediss-10678
biogenesis (MIA)	Tim9 itself is imported into the IMS and undergoes oxidative folding via the MIA pathway.	After TOM passage, small TIMs are oxidatively folded by the MIA (mitochondrial import and assembly) machinery; the conserved CX3C cysteines form two intramolecular disulfide bonds important for structural integrity. (chaudhuri2020tim17updatesa pages 2-5, gentle2007conservedmotifsreveal pages 6-7, gentle2007conservedmotifsreveal pages 1-1)	Chaudhuri et al. 2020 Biomolecules; Gentle et al. 2007 Mol Biol Evol	https://doi.org/10.3390/biom10121643 ; https://doi.org/10.1093/molbev/msm031
phenotypes & genetics	Tim9 function is genetically important for mitochondrial protein import and cell viability; disruption of conserved residues destabilizes the chaperone complex.	The 2023 comparative source states Tim9 (with Tim10 and Tim12) is essential in yeast. Older yeast mutant data show tim9-19 (E52G) and tim9-3 (V40A, S60P) abolish detectable Tim9–Tim10 hexamer. Related tim10ts mutants reduce Tim10 and Tim9 expression at 37°C, causing death unless functionally rescued, illustrating the essentiality of the Tim9/Tim10 chaperone function for at least one indispensable substrate. (guillen2023uniqueinteractionsand pages 1-5, gentle2007conservedmotifsreveal pages 5-6, gentle2007conservedmotifsreveal pages 7-7)	Guillén et al. 2023 bioRxiv; Gentle et al. 2007 Mol Biol Evol	https://doi.org/10.1101/2023.05.29.542777 ; https://doi.org/10.1093/molbev/msm031
recent developments 2023-2024	Recent work emphasizes the broader proteostasis integration of the TIM22 pathway and updated structural interpretation of small TIM–TIM22 interactions, rather than discovering a new primary function for Tim9.	2023 work linked TIM22 pathway activity to Yme1-dependent mitochondrial proteostasis, indicating that excess TIM22 substrates can drive stress and that Yme1 supports TIM22 complex stability. A 2024 structural analysis/thesis proposed an IMS-facing TIM22 helix as a small TIM docking site near the membrane; a nearby human disease mutation (TIM22 V33L) may impair chaperone binding. 2024 review literature also reiterates that Tim9/Tim10 is an essential IMS chaperone module in the TIM22 pathway. (valpadashi2024structuralandfunctional pages 51-51)	Valpadashi 2024 PhD thesis; Kumar et al. 2023 J Cell Sci	https://doi.org/10.53846/goediss-10678 ; https://doi.org/10.1242/jcs.260060

Table: This table summarizes the functional annotation of Saccharomyces cerevisiae Tim9 (UniProt O74700), including identity, localization, complexes, substrates, mechanism, structural features, MIA-dependent biogenesis, genetic evidence, and recent 2023–2024 developments. It is useful as a concise evidence map for curated gene-function annotation.

8) Key references (URLs and publication dates)

Chaudhuri M. et al. “Tim17 Updates: A Comprehensive Review…” Biomolecules (Dec 2020). https://doi.org/10.3390/biom10121643 (chaudhuri2020tim17updatesa pages 2-5)
Kumar A. et al. “Functional crosstalk between the TIM22 complex and YME1…” Journal of Cell Science (Jan 2023). https://doi.org/10.1242/jcs.260060 (kumar2023functionalcrosstalkbetween pages 2-3, kumar2023functionalcrosstalkbetween pages 1-2)
Kan K.T. et al. “Role of Yme1 in mitochondrial protein homeostasis…” Biochemical Society Transactions (Jun 2024). https://doi.org/10.1042/bst20240450 (kan2024roleofyme1 pages 3-4)
Sučec I. et al. “Structural basis of client specificity…” Science Advances (Dec 2020). https://doi.org/10.1126/sciadv.abd0263 (sucec2020structuralbasisof pages 2-3, sucec2020structuralbasisof pages 8-9)
Gentle I. et al. “Conserved motifs reveal details of ancestry…” Molecular Biology and Evolution (May 2007). https://doi.org/10.1093/molbev/msm031 (gentle2007conservedmotifsreveal pages 5-6)

9) Limitations and gaps

The gathered corpus contains strong mechanistic and pathway-level evidence for Tim9’s role but limited direct 2023–2024 primary data specifically manipulating TIM9 itself (as opposed to TIM22 or quality-control components). Recent work largely reframes Tim9’s function within updated proteostasis/quality-control models and TIM22 docking interpretations rather than redefining its core molecular role (kumar2023functionalcrosstalkbetween pages 2-3, kan2024roleofyme1 pages 3-4, valpadashi2024structuralandfunctional pages 51-51).

References

(chaudhuri2020tim17updatesa pages 2-5): Minu Chaudhuri, Chauncey Darden, Fidel Soto Gonzalez, Ujjal K. Singha, Linda Quinones, and Anuj Tripathi. Tim17 updates: a comprehensive review of an ancient mitochondrial protein translocator. Biomolecules, 10:1643, Dec 2020. URL: https://doi.org/10.3390/biom10121643, doi:10.3390/biom10121643. This article has 25 citations.
(kumar2020conservedregionsof pages 1-4): Abhishek Kumar, Srujan Kumar Matta, and Patrick D'Silva. Conserved regions of budding yeast tim22 have a role in structural organization of the carrier translocase. Journal of Cell Science, Jul 2020. URL: https://doi.org/10.1242/jcs.244632, doi:10.1242/jcs.244632. This article has 8 citations and is from a domain leading peer-reviewed journal.
(gentle2007conservedmotifsreveal pages 1-1): I. Gentle, A. Perry, F. Alcock, V. Likic, P. Doležal, E. T. Ng, A. Purcell, Malcolm McConnville, Thomas Naderer, Anne-Laure Chanez, Fabien Charrière, Caroline Aschinger, A. Schneider, K. Tokatlidis, and T. Lithgow. Conserved motifs reveal details of ancestry and structure in the small tim chaperones of the mitochondrial intermembrane space. Molecular biology and evolution, 24 5:1149-60, May 2007. URL: https://doi.org/10.1093/molbev/msm031, doi:10.1093/molbev/msm031. This article has 118 citations and is from a highest quality peer-reviewed journal.
(kumar2023functionalcrosstalkbetween pages 1-2): Abhishek Kumar, Tejashree Pradip Waingankar, and Patrick D'Silva. Functional crosstalk between the tim22 complex and yme1 machinery maintains mitochondrial proteostasis and integrity. Journal of Cell Science, Jan 2023. URL: https://doi.org/10.1242/jcs.260060, doi:10.1242/jcs.260060. This article has 9 citations and is from a domain leading peer-reviewed journal.
(gentle2007conservedmotifsreveal pages 6-7): I. Gentle, A. Perry, F. Alcock, V. Likic, P. Doležal, E. T. Ng, A. Purcell, Malcolm McConnville, Thomas Naderer, Anne-Laure Chanez, Fabien Charrière, Caroline Aschinger, A. Schneider, K. Tokatlidis, and T. Lithgow. Conserved motifs reveal details of ancestry and structure in the small tim chaperones of the mitochondrial intermembrane space. Molecular biology and evolution, 24 5:1149-60, May 2007. URL: https://doi.org/10.1093/molbev/msm031, doi:10.1093/molbev/msm031. This article has 118 citations and is from a highest quality peer-reviewed journal.
(gentle2007conservedmotifsreveal pages 9-9): I. Gentle, A. Perry, F. Alcock, V. Likic, P. Doležal, E. T. Ng, A. Purcell, Malcolm McConnville, Thomas Naderer, Anne-Laure Chanez, Fabien Charrière, Caroline Aschinger, A. Schneider, K. Tokatlidis, and T. Lithgow. Conserved motifs reveal details of ancestry and structure in the small tim chaperones of the mitochondrial intermembrane space. Molecular biology and evolution, 24 5:1149-60, May 2007. URL: https://doi.org/10.1093/molbev/msm031, doi:10.1093/molbev/msm031. This article has 118 citations and is from a highest quality peer-reviewed journal.
(gentle2007conservedmotifsreveal pages 5-6): I. Gentle, A. Perry, F. Alcock, V. Likic, P. Doležal, E. T. Ng, A. Purcell, Malcolm McConnville, Thomas Naderer, Anne-Laure Chanez, Fabien Charrière, Caroline Aschinger, A. Schneider, K. Tokatlidis, and T. Lithgow. Conserved motifs reveal details of ancestry and structure in the small tim chaperones of the mitochondrial intermembrane space. Molecular biology and evolution, 24 5:1149-60, May 2007. URL: https://doi.org/10.1093/molbev/msm031, doi:10.1093/molbev/msm031. This article has 118 citations and is from a highest quality peer-reviewed journal.
(sucec2020structuralbasisof pages 1-2): Iva Sučec, Yong Wang, Ons Dakhlaoui, Katharina Weinhäupl, Tobias Jores, Doriane Costa, Audrey Hessel, Martha Brennich, Doron Rapaport, Kresten Lindorff-Larsen, Beate Bersch, and Paul Schanda. Structural basis of client specificity in mitochondrial membrane-protein chaperones. Science Advances, Dec 2020. URL: https://doi.org/10.1126/sciadv.abd0263, doi:10.1126/sciadv.abd0263. This article has 38 citations and is from a highest quality peer-reviewed journal.
(sucec2020structuralbasisof pages 8-9): Iva Sučec, Yong Wang, Ons Dakhlaoui, Katharina Weinhäupl, Tobias Jores, Doriane Costa, Audrey Hessel, Martha Brennich, Doron Rapaport, Kresten Lindorff-Larsen, Beate Bersch, and Paul Schanda. Structural basis of client specificity in mitochondrial membrane-protein chaperones. Science Advances, Dec 2020. URL: https://doi.org/10.1126/sciadv.abd0263, doi:10.1126/sciadv.abd0263. This article has 38 citations and is from a highest quality peer-reviewed journal.
(sucec2020structuralbasisof pages 2-3): Iva Sučec, Yong Wang, Ons Dakhlaoui, Katharina Weinhäupl, Tobias Jores, Doriane Costa, Audrey Hessel, Martha Brennich, Doron Rapaport, Kresten Lindorff-Larsen, Beate Bersch, and Paul Schanda. Structural basis of client specificity in mitochondrial membrane-protein chaperones. Science Advances, Dec 2020. URL: https://doi.org/10.1126/sciadv.abd0263, doi:10.1126/sciadv.abd0263. This article has 38 citations and is from a highest quality peer-reviewed journal.
(sucec2020structuralbasisof pages 3-4): Iva Sučec, Yong Wang, Ons Dakhlaoui, Katharina Weinhäupl, Tobias Jores, Doriane Costa, Audrey Hessel, Martha Brennich, Doron Rapaport, Kresten Lindorff-Larsen, Beate Bersch, and Paul Schanda. Structural basis of client specificity in mitochondrial membrane-protein chaperones. Science Advances, Dec 2020. URL: https://doi.org/10.1126/sciadv.abd0263, doi:10.1126/sciadv.abd0263. This article has 38 citations and is from a highest quality peer-reviewed journal.
(sucec2020structuralbasisof pages 7-8): Iva Sučec, Yong Wang, Ons Dakhlaoui, Katharina Weinhäupl, Tobias Jores, Doriane Costa, Audrey Hessel, Martha Brennich, Doron Rapaport, Kresten Lindorff-Larsen, Beate Bersch, and Paul Schanda. Structural basis of client specificity in mitochondrial membrane-protein chaperones. Science Advances, Dec 2020. URL: https://doi.org/10.1126/sciadv.abd0263, doi:10.1126/sciadv.abd0263. This article has 38 citations and is from a highest quality peer-reviewed journal.
(kumar2023functionalcrosstalkbetween pages 2-3): Abhishek Kumar, Tejashree Pradip Waingankar, and Patrick D'Silva. Functional crosstalk between the tim22 complex and yme1 machinery maintains mitochondrial proteostasis and integrity. Journal of Cell Science, Jan 2023. URL: https://doi.org/10.1242/jcs.260060, doi:10.1242/jcs.260060. This article has 9 citations and is from a domain leading peer-reviewed journal.
(kan2024roleofyme1 pages 3-4): Kwan Ting Kan, Joel Wilcock, and Hui Lu. Role of yme1 in mitochondrial protein homeostasis: from regulation of protein import, oxphos function to lipid synthesis and mitochondrial dynamics. Biochemical Society Transactions, 52:1539-1548, Jun 2024. URL: https://doi.org/10.1042/bst20240450, doi:10.1042/bst20240450. This article has 9 citations and is from a peer-reviewed journal.
(valpadashi2024structuralandfunctional pages 51-51): Anusha Valpadashi. Structural and functional characterization of TIM22 complex in the inner mitochondrial membarne. PhD thesis, University Goettingen Repository, 2024. URL: https://doi.org/10.53846/goediss-10678, doi:10.53846/goediss-10678.
(guillen2023uniqueinteractionsand pages 1-5): Linda S. Quiñones Guillén, Fidel Soto Gonzalez, Chauncey Darden, Muhammad Bismillah Khan, A. Tripathi, Joseph T. Smith, Ayorinde Cooley, Victor Paromov, Jamaine Davis, Smitā Miśra, and Minu Chaudhuri. Unique interactions and functions of the mitochondrial small tims in trypanosoma brucei. bioRxiv, May 2023. URL: https://doi.org/10.1101/2023.05.29.542777, doi:10.1101/2023.05.29.542777. This article has 2 citations.
(sucec2020structuralbasisof pages 3-3): Iva Sučec, Yong Wang, Ons Dakhlaoui, Katharina Weinhäupl, Tobias Jores, Doriane Costa, Audrey Hessel, Martha Brennich, Doron Rapaport, Kresten Lindorff-Larsen, Beate Bersch, and Paul Schanda. Structural basis of client specificity in mitochondrial membrane-protein chaperones. Science Advances, Dec 2020. URL: https://doi.org/10.1126/sciadv.abd0263, doi:10.1126/sciadv.abd0263. This article has 38 citations and is from a highest quality peer-reviewed journal.
(sucec2020structuralbasisof media 57627111): Iva Sučec, Yong Wang, Ons Dakhlaoui, Katharina Weinhäupl, Tobias Jores, Doriane Costa, Audrey Hessel, Martha Brennich, Doron Rapaport, Kresten Lindorff-Larsen, Beate Bersch, and Paul Schanda. Structural basis of client specificity in mitochondrial membrane-protein chaperones. Science Advances, Dec 2020. URL: https://doi.org/10.1126/sciadv.abd0263, doi:10.1126/sciadv.abd0263. This article has 38 citations and is from a highest quality peer-reviewed journal.
(gentle2007conservedmotifsreveal pages 7-7): I. Gentle, A. Perry, F. Alcock, V. Likic, P. Doležal, E. T. Ng, A. Purcell, Malcolm McConnville, Thomas Naderer, Anne-Laure Chanez, Fabien Charrière, Caroline Aschinger, A. Schneider, K. Tokatlidis, and T. Lithgow. Conserved motifs reveal details of ancestry and structure in the small tim chaperones of the mitochondrial intermembrane space. Molecular biology and evolution, 24 5:1149-60, May 2007. URL: https://doi.org/10.1093/molbev/msm031, doi:10.1093/molbev/msm031. This article has 118 citations and is from a highest quality peer-reviewed journal.

Artifacts

Edison artifact artifact-00

Citations

kumar2020conservedregionsof pages 1-4
kumar2023functionalcrosstalkbetween pages 1-2
gentle2007conservedmotifsreveal pages 6-7
gentle2007conservedmotifsreveal pages 5-6
sucec2020structuralbasisof pages 2-3
kumar2023functionalcrosstalkbetween pages 2-3
valpadashi2024structuralandfunctional pages 51-51
gentle2007conservedmotifsreveal pages 1-1
gentle2007conservedmotifsreveal pages 9-9
sucec2020structuralbasisof pages 1-2
sucec2020structuralbasisof pages 8-9
sucec2020structuralbasisof pages 3-4
sucec2020structuralbasisof pages 7-8
guillen2023uniqueinteractionsand pages 1-5
sucec2020structuralbasisof pages 3-3
gentle2007conservedmotifsreveal pages 7-7
https://doi.org/10.1093/molbev/msm031
https://doi.org/10.1101/2023.05.29.542777
https://doi.org/10.3390/biom10121643
https://doi.org/10.1242/jcs.244632
https://doi.org/10.1126/sciadv.abd0263
https://doi.org/10.53846/goediss-10678
https://doi.org/10.1242/jcs.260060
https://doi.org/10.1042/bst20240450
https://doi.org/10.3390/biom10121643,
https://doi.org/10.1242/jcs.244632,
https://doi.org/10.1093/molbev/msm031,
https://doi.org/10.1242/jcs.260060,
https://doi.org/10.1126/sciadv.abd0263,
https://doi.org/10.1042/bst20240450,
https://doi.org/10.53846/goediss-10678,
https://doi.org/10.1101/2023.05.29.542777,

📄 View Raw YAML

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.
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Tim9 acts as a **chaperone/escort factor** that prevents aggregation of hydrophobic precursors in the aqueous IMS and transfers them from **TOM** to **TIM22**.

- 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.
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Tim9 is located in the **mitochondrial IMS**

- 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. Falcon deep
      research independently confirms the Tim9-Tim10 heterohexamer with 3:3 stoichiometry
      (~70 kDa soluble complex).
    supported_by:
    - reference_id: PMID:9822593
      supporting_text: >-
        Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex.
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Tim9 forms the canonical **Tim9–Tim10 heterohexamer** and also participates in a **Tim9–Tim10–Tim12 docking complex** linked to TIM22.
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Tim9:Tim10 complex stoichiometry is **3:3**; the TIM22-associated complex is **Tim9–Tim10–Tim12 = 3:2:1**; the soluble Tim9/Tim10 complex is about **~70 kDa**

- 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
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Small TIM proteins (including Tim9) are imported through TOM and undergo oxidative folding in the IMS via the **MIA (mitochondrial import and assembly) pathway**, which introduces disulfides in the conserved CX3C motifs

# --- 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. Falcon deep research notes that within the
      complex Tim10 provides the key substrate-binding elements (Tim9 alone does not bind
      the AAC carrier), and that the obligate Tim9-Tim10 partnership forms an essential
      heterohexamer -- the functionally meaningful interaction, not generic protein binding.
    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.
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Tim9 and Tim10 assemble into an essential heterohexameric Tim9/10 complex** that chaperones hydrophobic preproteins from TOM to TIM22
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Biochemical substrate-binding evidence indicates that **Tim10** (alone) can bind the **ATP/ADP carrier (AAC)** similarly to the Tim9–Tim10 complex, whereas **Tim9 alone does not bind AAC**, supporting a model where Tim10 provides key substrate-binding elements within the 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.
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        the conserved **CX3C** cysteines form **two intramolecular disulfide bonds** important for structural integrity

# --- 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.
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Tim9 acts as a **chaperone/escort factor** that prevents aggregation of hydrophobic precursors in the aqueous IMS and transfers them from **TOM** to **TIM22**.
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        TIM9·10 recognizes hydrophobic clients primarily through a **hydrophobic cleft** formed by chaperone helices

# --- 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
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Tim9 is best viewed as a **specialized, soluble IMS holdase chaperone**
    - reference_id: file:yeast/TIM9/TIM9-deep-research-falcon.md
      supporting_text: |-
        Small TIMs can also assist trafficking of some outer-membrane β-barrel proteins toward the SAM complex

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.
- id: file:yeast/TIM9/TIM9-deep-research-falcon.md
  title: Falcon deep research on TIM9 (Edison Scientific Literature)
  findings:
  - statement: >-
      Tim9 is a small TIM family soluble IMS chaperone (holdase) whose primary role is
      chaperone-like binding and transfer of hydrophobic precursors, not catalysis or
      transport.
    reference_section_type: OTHER
    supporting_text: |-
      Tim9 is best viewed as a **specialized, soluble IMS holdase chaperone**
  - statement: >-
      Tim9 is located in the mitochondrial intermembrane space, between TOM-mediated
      entry and TIM22-mediated inner-membrane insertion.
    reference_section_type: OTHER
    supporting_text: |-
      Tim9 is located in the **mitochondrial IMS**
  - statement: >-
      Tim9 forms the canonical Tim9-Tim10 heterohexamer (3:3 stoichiometry, ~70 kDa
      soluble complex) and a Tim9-Tim10-Tim12 (3:2:1) docking complex linked to TIM22.
    reference_section_type: OTHER
    supporting_text: |-
      Tim9:Tim10 complex stoichiometry is **3:3**; the TIM22-associated complex is **Tim9–Tim10–Tim12 = 3:2:1**; the soluble Tim9/Tim10 complex is about **~70 kDa**
  - statement: >-
      Tim9 acts as a chaperone/escort factor that prevents aggregation of hydrophobic
      precursors in the aqueous IMS and transfers them from TOM to TIM22.
    reference_section_type: OTHER
    supporting_text: |-
      Tim9 acts as a **chaperone/escort factor** that prevents aggregation of hydrophobic precursors in the aqueous IMS and transfers them from **TOM** to **TIM22**.
  - statement: >-
      Within the complex, Tim10 provides the key substrate-binding elements; Tim9 alone
      does not bind the AAC carrier (consistent with UniProt noting Tim9 has a strong
      structural role relative to Tim10).
    reference_section_type: OTHER
    supporting_text: |-
      Biochemical substrate-binding evidence indicates that **Tim10** (alone) can bind the **ATP/ADP carrier (AAC)** similarly to the Tim9–Tim10 complex, whereas **Tim9 alone does not bind AAC**, supporting a model where Tim10 provides key substrate-binding elements within the complex
  - statement: >-
      The Tim9-Tim10 complex recognizes hydrophobic clients through a hydrophobic cleft
      formed by chaperone helices.
    reference_section_type: OTHER
    supporting_text: |-
      TIM9·10 recognizes hydrophobic clients primarily through a **hydrophobic cleft** formed by chaperone helices
  - statement: >-
      Small TIMs including Tim9 can also assist trafficking of some outer-membrane
      beta-barrel proteins toward the SAM complex.
    reference_section_type: OTHER
    supporting_text: |-
      Small TIMs can also assist trafficking of some outer-membrane β-barrel proteins toward the SAM complex
  - statement: >-
      Tim9 is itself imported via TOM and oxidatively folded in the IMS by the MIA
      pathway, which introduces the two intramolecular disulfides in its conserved CX3C
      motifs (rather than stable metal coordination in the mature form).
    reference_section_type: OTHER
    supporting_text: |-
      Small TIM proteins (including Tim9) are imported through TOM and undergo oxidative folding in the IMS via the **MIA (mitochondrial import and assembly) pathway**, which introduces disulfides in the conserved CX3C motifs
  - statement: >-
      Tim9 and Tim10 assemble into an essential heterohexameric Tim9/10 complex;
      unassembled small TIMs can be degraded by Yme1, with assembly being protective.
    reference_section_type: OTHER
    supporting_text: |-
      Tim9 and Tim10 assemble into an essential heterohexameric Tim9/10 complex** that chaperones hydrophobic preproteins from TOM to TIM22
  - statement: >-
      Conserved-residue mutants tim9-19 (E52G) and tim9-3 (V40A + S60P) abolish a
      detectable Tim9-Tim10 heterohexamer, linking these residues to complex integrity
      and import function.
    reference_section_type: OTHER
    supporting_text: |-
      tim9-19 (E52G)** and **tim9-3 (V40A + S60P)** show **no detectable Tim9–Tim10 heterohexamer** in detergent-solubilized mitochondria

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

Gene Description

Existing Annotations Review

Core Functions

References

Tags

Deep Research

Falcon

Research Report: Functional Annotation of S. cerevisiae TIM9 (UniProt O74700)

0) Mandatory identity verification (disambiguation)

1) Key concepts, definitions, and current understanding

1.1 Small TIM chaperones (definition)

1.2 TIM22 “carrier pathway” and Tim9’s central role

1.3 Localization and biogenesis (MIA pathway)

2) Molecular function, complexes, substrates, and mechanism

2.1 Complex composition and stoichiometry

2.2 Substrate specificity: carrier proteins and other hydrophobic clients

2.3 Mechanism (handoff from TOM to TIM22)

3) Structural basis of function (client binding and complex stability)

3.1 Core fold and conserved motifs

3.2 Hexamer architecture and interfaces

3.3 Genetic evidence linking structure to function

3.4 Client-binding clefts and specificity (molecular biophysics)

4) Recent developments (prioritizing 2023–2024)

4.1 2023: TIM22 pathway is integrated with mitochondrial quality control via Yme1

4.2 2024: Review synthesis—small TIM assembly state is surveilled by Yme1

4.3 2024: Updated structural interpretations of small TIM docking to TIM22

5) Current applications and real-world implementations

6) Expert opinions and authoritative synthesis

7) Evidence map (summary table)

8) Key references (URLs and publication dates)

9) Limitations and gaps

Artifacts

Citations

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