tin-44 encodes the C. elegans ortholog of TIM44 (human TIMM44 / yeast Tim44), a component of the mitochondrial protein-import machinery at the inner membrane. TIM44 is the central organizing subunit of the presequence translocase-associated import motor (PAM), the ATP-driven engine that pulls nucleus-encoded, presequence-bearing preproteins across the inner membrane into the matrix after they emerge from the TIM23 channel. Positioned on the matrix face of the TIM23 translocase, TIM44 recruits and tethers mitochondrial HSP70 (mtHsp70; HSP-6 in C. elegans) together with its regulatory co-chaperones — the J-protein Pam18/Tim14 (dnj-21), the J-like Pam16/Tim16 (tim-16), and the nucleotide-exchange factor GrpE/Mge1 — so that cycles of mtHsp70 ATP binding and hydrolysis are coupled to the vectorial, ratchet-like inward movement of the incoming polypeptide. Through this motor, TIM44/tin-44 supports import of the many nuclear-encoded matrix and inner-membrane proteins that build and maintain mitochondria. In C. elegans, tin-44 is transcriptionally co-induced with the rest of the TIM/TOM import machinery (including mtHsp70/hsp-6 and the mitochondrial processing peptidase subunits) when the mitochondrial unfolded protein response is activated, and its RNAi knockdown behaves as a mitochondrial protein-import defect.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0001405 PAM complex, Tim23 associated import motor | IBA GO_REF:0000033 | ACCEPT | Summary: Core cellular-component / complex-membership annotation. tin-44 is the C. elegans Tim44 ortholog (Tim44 family, IPR017303) and, like its yeast/human counterparts, a subunit of the presequence translocase-associated import motor (PAM) that operates on the matrix side of the TIM23 complex. The direct worm evidence places tin-44 in the matrix-pulling step of import together with mtHsp70/HSP-6. Supporting Evidence: UniProt:O02161 Essential component of the PAM complex PMID:35608535 the precursor proteins are pulled into the matrix by TIM44 and mtHSP70 (HSP-6) |
| GO:0030150 protein import into mitochondrial matrix | IBA GO_REF:0000033 | ACCEPT | Summary: Core biological-process annotation. As the PAM import-motor organizer, tin-44 functions in the ATP-dependent import of presequence-bearing preproteins across the inner membrane into the mitochondrial matrix. Direct worm genetics supports this process assignment: Bennett et al. group tin-44/T09B4.9 (TIM44) with the TIM23-complex components that transport proteins into the inner membrane and matrix, and tin-44 RNAi behaves as a mitochondrial-import knockdown (activating the UPRmt and shortening lifespan). Supporting Evidence: UniProt:O02161 translocation of transit peptide-containing proteins from the inner membrane into the mitochondrial matrix in an ATP-dependent manner PMID:24662282 T09B4.9 (TIM44), F45G2.8 (TIM16), F15D3.7 (TIM23), and dnj-21 (TIM14), which function in the TIM23 complex that transports proteins into the inner membrane and the matrix PMID:10339406 translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p |
| GO:0051087 protein-folding chaperone binding | IBA GO_REF:0000033 | ACCEPT | Summary: Core molecular-function annotation and the most specific MF available for a Tim44-family protein. tin-44's defining biochemical activity is binding/tethering the mitochondrial HSP70 chaperone (worm HSP-6) at the import channel so that mtHsp70's ATPase cycles power translocation; "protein-folding chaperone binding" captures this mtHsp70-binding activity. This is an informative MF, not an uninformative "protein binding" term. Supporting Evidence: UniProt:O02161 Recruits mitochondrial HSP70 to drive protein translocation into the matrix using ATP as an energy source PMID:35608535 the precursor proteins are pulled into the matrix by TIM44 and mtHSP70 (HSP-6) |
| GO:0005743 mitochondrial inner membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Correct core subcellular location: tin-44 acts at the mitochondrial inner membrane as part of the TIM23-associated import motor, consistent with the UniProt Swiss-Prot location. The precise submitochondrial disposition of the worm protein (integral vs. peripheral/matrix-side) is unverified — see the annotation-level knowledge gap — but the inner-membrane compartment assignment is appropriate. Supporting Evidence: UniProt:O02161 Mitochondrion inner membrane Knowledge gap: The submitochondrial topology of C. elegans TIN-44 is unverified. It is assigned to the mitochondrial inner membrane by ortholog/subcellular-mapping inference, but whether the worm protein is an integral inner-membrane protein, a peripheral protein on the matrix face of the TIM23 channel, or (as reported for the human ortholog) a matrix-localized protein only loosely associated with the inner membrane has not been tested. OPEN BIOLOGY RESIDUAL_SUBGAP Resolve: Sub-mitochondrial fractionation (alkaline/carbonate extraction, protease-protection) of endogenous or tagged worm TIN-44 to determine integral vs. peripheral membrane association and matrix vs. inner-membrane localization. "hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane" — PMID:10339406 "Mitochondrion inner membrane" — UniProt:O02161 |
| GO:0030150 protein import into mitochondrial matrix | IEA GO_REF:0000002 | ACCEPT | Summary: Same core biological process as the IBA annotation above, here inferred electronically by InterPro2GO from the Tim44 family signature (IPR017303). Correct and consistent; redundant with the phylogenetic annotation but not wrong. Supporting Evidence: UniProt:O02161 translocation of transit peptide-containing proteins from the inner membrane into the mitochondrial matrix in an ATP-dependent manner |
| GO:0051087 protein-folding chaperone binding | IEA GO_REF:0000002 | ACCEPT | Summary: Same core molecular function as the IBA annotation above, here inferred electronically by InterPro2GO from the Tim44 family signature (IPR017303). Captures the conserved mtHsp70-binding activity of Tim44; correct and consistent. Supporting Evidence: UniProt:O02161 Recruits mitochondrial HSP70 to drive protein translocation into the matrix using ATP as an energy source |
Loading supporting content…
Download this section (compressed HTML)Q: Does C. elegans TIN-44 bind mitochondrial HSP70 (HSP-6) directly and tether it to the TIM23 channel, as demonstrated for yeast and human Tim44?
Q: Is tin-44 essential in C. elegans, and how does a genetic null differ from RNAi knockdown in developmental and mitochondrial-import phenotypes?
Q: Is worm TIN-44 an integral inner-membrane protein or a peripheral/matrix protein, given that the human ortholog is matrix-localized and only loosely membrane-associated?
Experiment: Reconstitute or co-immunoprecipitate recombinant/tagged worm TIN-44 with HSP-6 and test direct binding; deplete tin-44 (RNAi or null) and quantify import of a matrix-targeted reporter (e.g. su9-DHFR or MTS::GFP) relative to wild type.
Hypothesis: C. elegans TIN-44 is a bona fide import-motor subunit that binds mtHsp70 (HSP-6) and is required for matrix protein import.
Type: biochemical interaction / in-organello import assay
Experiment: Generate a tin-44 deletion allele (CRISPR) and score viability, developmental arrest, and UPRmt (hsp-6p::gfp) activation; compare with the milder phenotypes of outer-membrane receptor knockdowns.
Hypothesis: tin-44 is essential for C. elegans viability, like mammalian TIMM44.
Type: genetics / phenotypic analysis
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The molecular activity of C. elegans TIN-44 has never been directly measured. Its recruitment/tethering of mitochondrial HSP70 (HSP-6), its role as the scaffold that organizes the PAM import motor, and the coupling of mtHsp70 ATPase cycles to matrix translocation are all inferred from yeast and human Tim44. No worm biochemistry, protein-interaction, structural, or in-vitro import-assay data exist for the TIN-44 protein itself; the direct worm data are only genetic/transcriptional (RNAi that induces the UPRmt and stress co-regulation of the tin-44 transcript), which report import stress rather than the protein's activity.
OPEN BIOLOGYCURATION MF_DARK
What is known: It is firmly established that tin-44 is the C. elegans Tim44-family ortholog (IPR017303, PANTHER PTHR10721), that it is a component of the mitochondrial import machinery, that its RNAi knockdown behaves as an import-defect that activates the UPRmt (Bennett et al. 2014), and that it is transcriptionally co-regulated with mtHsp70/hsp-6 and the MPP subunits during the UPRmt (Xin et al. 2022). The PAM/import-motor mechanism — Tim44 as the membrane- associated mtHsp70 recruiter driving matrix translocation — is well characterized in yeast and human. What is missing is any assay of the worm protein's own activity.
Significance: tin-44 is treated as a housekeeping component of the mitochondrial import motor in worm UPRmt/aging studies, yet whether the worm protein reproduces the mtHsp70-tethering and import-motor-organizing activities of its orthologs — or has any organism-specific features — is untested. Anchoring the molecular function in worm data would strengthen the interpretation of the mitochondrial-proteostasis literature that relies on this gene.
What would resolve it: In vitro reconstitution / interaction assays with recombinant worm TIN-44 and HSP-6 (mtHsp70) to demonstrate direct binding and stimulation of import; tagging endogenous TIN-44 to test co-assembly with tim-23/tim-16/dnj-21 and requirement in an in-organello or in-vitro import assay.
Provenance (the field's own admissions):
Gap: The molecular consequences and essentiality of tin-44 loss in C. elegans have not been directly characterized. The only worm loss-of-function data are indirect: tin-44/T09B4.9 RNAi behaves as a mitochondrial-import knockdown that activates the UPRmt (hsp-6p::gfp) and shortens lifespan. No tin-44 null/deletion allele has been analyzed, its essentiality (the mammalian TIMM44 is essential) is untested, and no assay has directly measured how tin-44 depletion affects mitochondrial protein-import capacity or which preprotein classes depend on it.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: tin-44 was recovered as an hsp-6p::gfp UPRmt-inducing RNAi clone and grouped with the lifespan-shortening mitochondrial-import knockdowns (tomm-22, TIM17/TIM16/TIM23/TIM14) (Bennett et al. 2014); UniProt still classifies the protein at evidence level "Inferred from homology" with the hedged name "Probable" import-motor subunit. These readouts report import stress, not the direct requirement for, or activity of, TIN-44.
Significance: Establishing the null phenotype and a direct import requirement would convert tin-44 from a by-similarity assignment supported only by stress-reporter readouts into an experimentally grounded import-motor gene, and clarify its place in the C. elegans mitochondrial-import and UPRmt network.
What would resolve it: Characterize a tin-44 deletion/null allele for viability and developmental arrest, and quantify mitochondrial protein-import capacity on tin-44 depletion (e.g. MTS::GFP or su9-DHFR import) alongside UPRmt (hsp-6p::gfp) activation.
Provenance (the field's own admissions):
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)