| 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. (pqac-00000000, pqac-00000013) | 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. (pqac-00000002, pqac-00000004, pqac-00000015) | 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. (pqac-00000002, pqac-00000004, pqac-00000012, pqac-00000015) | 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. (pqac-00000002, pqac-00000006, pqac-00000014, pqac-00000015) | 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. (pqac-00000002, pqac-00000005, pqac-00000008, pqac-00000016) | 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**. (pqac-00000001, pqac-00000006, pqac-00000007, pqac-00000016) | 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. (pqac-00000002, pqac-00000011, pqac-00000012, pqac-00000013) | 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. (pqac-00000000, pqac-00000001, pqac-00000010) | 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. (pqac-00000007, pqac-00000016) | 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.*