Existing Annotations Review

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

Core Functions

Organizing subunit of the mitochondrial presequence translocase-associated import motor (PAM): binds and tethers mitochondrial HSP70 (mtHsp70/HSP-6) at the matrix face of the TIM23 channel and, together with its co-chaperones, couples ATP-driven mtHsp70 cycles to the import of nucleus-encoded, presequence-bearing preproteins into the mitochondrial matrix. In C. elegans this activity is inferred from the conserved yeast/human Tim44 ortholog; direct worm evidence establishes tin-44 as a co-regulated component of the mitochondrial import machinery acting at the matrix-pulling step with mtHsp70/HSP-6.

Molecular Function:

protein-folding chaperone binding

Directly Involved In:

protein import into mitochondrial matrix

Cellular Locations:

mitochondrial inner membrane

In Complex:

PAM complex, Tim23 associated import motor

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)

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000033

Annotation inferences using phylogenetic trees

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

PMID:35608535

The UPRmt preserves mitochondrial import to extend lifespan.

PMID:24662282

Activation of the mitochondrial unfolded protein response does not predict longevity in Caenorhabditis elegans.

PMID:10339406

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

UniProt:O02161

Probable mitochondrial import inner membrane translocase subunit tin-44 (C. elegans)

Suggested Experiments

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

Knowledge Gaps

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):

"Probable mitochondrial import inner membrane translocase subunit tin-44" — UniProt:O02161
"which are C. elegans homologs of mammalian mtHsp70, tim44, and Mpp, respectively, were also upregulated by cco-1 RNAi" — PMID:35608535

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):

"Probable mitochondrial import inner membrane translocase subunit tin-44" — UniProt:O02161
"a subset of RNAi clones related to mitochondrial protein import (tomm-22, E04A4.5, T09B4.9, F45G2.8, F15D3.7, and dnj-21)" — PMID:24662282

Deep Research

Falcon

(tin-44-deep-research-falcon.md)

Comprehensive Research Report: *C. elegans* TIN-44 (T09B4.9) — Probable Mitochondrial Import Inner Membrane Translocase Subunit Tim44 Falcon Edison Scientific Literature 37 citations 3 artifacts 2026-07-04T13:55:51.639205

Edison artifact artifact-00 (text/markdown)

Edison artifact artifact-01 (text/markdown)

Image artifact created (ID artifact-01): 'TIM23-Tim44 Import Pathways' ![TIM23-Tim44 Import Pathways](artifact:artifact-01) Artifact IDs that may be injected in (image/png)

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Comprehensive Research Report: C. elegans TIN-44 (T09B4.9) — Probable Mitochondrial Import Inner Membrane Translocase Subunit Tim44

1. Gene Identity and Protein Overview

The C. elegans gene tin-44 (ORF name T09B4.9; UniProt accession O02161) encodes a probable mitochondrial import inner membrane translocase subunit of the Tim44 family. The protein is annotated as a precursor with a cleavable N-terminal presequence, consistent with mitochondrial matrix-targeted proteins. Key domains include the Tim44 domain (PF04280/IPR007379), Tim44-like domain (IPR039544), and an NTF2-like domain superfamily fold (IPR032710). No dedicated primary study has been published on the C. elegans TIN-44 protein; however, considerable functional information can be derived from genome-wide screens in C. elegans and from extensive mechanistic studies on its yeast and mammalian orthologs, which are highly conserved.

2. Primary Function: Scaffold Protein in the TIM23-Associated Import Motor (PAM Complex)

2.1 Molecular Function of Tim44-Family Proteins

Tim44 is not an enzyme or transporter in the classical sense; rather, it is a peripheral membrane scaffold protein that resides on the matrix face of the inner mitochondrial membrane (IMM). Its primary function is to tether the presequence translocase-associated motor (PAM) complex to the TIM23 protein-conducting channel, thereby enabling ATP-dependent translocation of presequence-containing precursor proteins into the mitochondrial matrix (chaudhuri2020tim17updatesa pages 2-5, paschen2001proteinimportinto pages 5-7, fox2012mitochondrialproteinsynthesis pages 14-15).

Tim44 performs several critical molecular functions:

Recruitment of mtHsp70: Tim44 serves as the docking site for mitochondrial Hsp70 (mtHsp70/Ssc1 in yeast, mortalin/HSPA9 in humans), the ATP-driven motor chaperone that binds incoming polypeptide segments emerging from the TIM23 channel and prevents their retrograde movement (paschen2001proteinimportinto pages 5-7, bauer2000proteintranslocationinto pages 1-2).
Bridging TIM23 channel and PAM motor: Tim44 makes direct contacts with the integral membrane channel subunits Tim17 and Tim23 while simultaneously recruiting soluble motor components (mtHsp70 and nucleotide exchange factor Mge1), thereby physically linking the translocation channel to the import driving force (chaudhuri2020tim17updatesa pages 2-5, jain2025investigatingmitochondrialpresequence pages 15-17).
Differential regulation of motor subunits: Studies using the yeast temperature-sensitive mutant tim44-804 have revealed that Tim44 is not merely a passive scaffold but actively regulates recruitment of distinct PAM modules. Tim44 promotes association of the J-complex (Pam18/Pam16) with the TIM23 complex while simultaneously keeping Pam17 binding at low levels. Inactivation of Tim44 leads to increased Pam17 binding, indicating both stimulatory and inhibitory regulatory functions (hutu2008mitochondrialproteinimport pages 7-7, hutu2008mitochondrialproteinimport pages 1-2).
Presequence interaction: The C-terminal domain of Tim44 can directly interact with incoming presequences, helping to guide precursor proteins toward the motor machinery (jain2025investigatingmitochondrialpresequence pages 15-17).

2.2 Two Functional Forms of the TIM23 Complex

The TIM23 complex exists in two functionally distinct configurations, and Tim44 is specifically associated with the motor form:

TIM23-SORT: Contains Tim21 but lacks the PAM complex. This form mediates lateral insertion of proteins with hydrophobic sorting signals into the inner membrane in a PAM-independent manner (laan2006mitochondrialpreproteintranslocases pages 7-8, hutu2008mitochondrialproteinimport pages 3-4).
TIM23-MOTOR: Lacks Tim21 but contains the full PAM complex including Tim44. This form is required for ATP-dependent translocation of soluble proteins into the matrix (jain2025investigatingmitochondrialpresequence pages 20-23, laan2006mitochondrialpreproteintranslocases pages 5-7, laan2006mitochondrialpreproteintranslocases pages 7-8).

Consistent with this model, the yeast tim44-804 mutant strongly inhibits import of matrix-targeted precursor proteins (such as the F1Fo-ATPase β-subunit) while import of inner membrane-sorted proteins remains unaffected (hutu2008mitochondrialproteinimport pages 3-4).

The following schematic illustrates the two TIM23 configurations and the central role of Tim44 in the motor form:

TIM23-Tim44 Import Pathways

Image: Schematic of mitochondrial presequence import showing the TIM23 complex in its sorting and motor forms. The diagram highlights Tim44 as the matrix-side scaffold that recruits the PAM motor, including mtHsp70, for ATP-dependent matrix import.

3. Subcellular Localization

Tim44 is a hydrophilic, peripheral membrane protein associated with the inner face (matrix side) of the inner mitochondrial membrane (bauer2000proteintranslocationinto pages 1-2, paschen2001proteinimportinto pages 3-5). It lacks a classical hydrophobic transmembrane segment but contains a cleavable N-terminal mitochondrial targeting presequence. Tim44 associates with the membrane through interactions with the phospholipid cardiolipin and with integral membrane subunits Tim17 and Tim23 (bauer2000proteintranslocationinto pages 1-2, jain2025investigatingmitochondrialpresequence pages 15-17). In yeast, Tim44 exists as a dimer at the matrix side of the TIM23 complex, mediated by coiled-coil domains in its N-terminal region (bauer2000proteintranslocationinto pages 1-2, paschen2001proteinimportinto pages 5-7).

4. Protein Structure and Domain Architecture

Tim44 has a modular architecture:
- N-terminal mitochondrial targeting presequence: Cleaved upon import into the matrix.
- N-terminal segment with intrinsically disordered region: Interacts with incoming presequences, Hsp70, Pam16, and Tim23 to guide precursor proteins (jain2025investigatingmitochondrialpresequence pages 15-17).
- Coiled-coil domains in the N-terminal half: Mediate dimerization (bauer2000proteintranslocationinto pages 1-2).
- C-terminal domain (containing the Tim44/NTF2-like fold): Interacts with presequences, mtHsp70, other PAM subunits, and the membrane itself. This is also the domain targeted by the pharmacological inhibitor MB-10 (jain2025investigatingmitochondrialpresequence pages 15-17, zhang2024afirstinclasstimm44 pages 1-2).

Recent cryo-EM studies of the TIM23 complex core (Tim17-Tim23 heterodimer) noted that Tim44 tends to dissociate during purification, underscoring its peripheral/dynamic association with the translocase rather than forming a stably integrated structural component (sim2023structuralbasisof pages 1-4).

5. Functional Evidence in C. elegans

5.1 RNAi Phenotype and UPRmt Activation

Although no dedicated loss-of-function mutant study exists for C. elegans tin-44, the gene has been identified in multiple genome-wide RNAi screens:

In a genome-wide RNAi screen for negative regulators of the mitochondrial unfolded protein response (UPRmt), Bennett et al. (2014) found that RNAi knockdown of T09B4.9 (tin-44) significantly induced expression of the hsp-6p::gfp UPRmt reporter and extended mean lifespan by 11.1% (P = 2.4 × 10⁻¹⁴) (bennett2014activationofthe pages 2-3). However, the same study demonstrated that UPRmt activation is neither necessary nor sufficient for lifespan extension, and that some mitochondrial import gene knockdowns (including tin-44) that induce UPRmt actually reduced longevity in certain experimental contexts (bennett2014activationofthe pages 6-6).
Hernando-Rodríguez and Artal-Sanz (2018) identified TIN-44 among mitochondrial import factors (alongside TOMM-22, DNJ-21, TIMM-17B.1, and TIMM-23) whose RNAi depletion induces mitochondrial stress responses in C. elegans (hernandorodriguez2018mitochondrialqualitycontrol pages 6-8).

5.2 Transcriptional Regulation Under Mitochondrial Stress

Xin et al. (2022) demonstrated that upon UPRmt activation, the C. elegans mitochondrial import machinery is transcriptionally upregulated in an ATFS-1-dependent manner. Specifically, tin-44 was among the import genes (alongside hsp-6, mppa-1, mppb-1, and timm-23) that are induced during mitochondrial stress, indicating a compensatory feedback mechanism to maintain import capacity under proteotoxic conditions (xin2022theuprmtpreserves pages 6-9).

5.3 Role in the UPRmt Signaling Pathway

The connection between tin-44 and UPRmt is mechanistically coherent: the transcription factor ATFS-1 contains both a mitochondrial targeting sequence (MTS) and a nuclear localization signal. Under normal conditions, ATFS-1 is imported into mitochondria and degraded by the Lon protease. When mitochondrial import is compromised—including by depletion of import machinery components like TIN-44—ATFS-1 fails to be imported, accumulates in the cytosol, and translocates to the nucleus where it activates UPRmt gene expression (rolland2019compromisedmitochondrialprotein pages 1-2, hernandorodriguez2018mitochondrialqualitycontrol pages 3-5, xin2022theuprmtpreserves pages 1-2). Rolland et al. (2019) established that compromised mitochondrial protein import acts as a direct signal for UPRmt activation through this ATFS-1-dependent mechanism (rolland2019compromisedmitochondrialprotein pages 5-7, rolland2019compromisedmitochondrialprotein pages 1-2).

6. Comparative Functional Summary

The following table summarizes the key functional properties of TIN-44 and its orthologs:

Property	C. elegans TIN-44	Yeast Tim44	Human TIMM44
Gene name / synonym	tin-44; ORF T09B4.9; annotated as probable mitochondrial import inner membrane translocase subunit tin-44 (UniProt O02161) (bennett2014activationofthe pages 2-3)	Tim44; classic fungal TIM23/PAM motor subunit (bauer2000proteintranslocationinto pages 1-2, hutu2008mitochondrialproteinimport pages 3-4)	TIMM44; translocase of inner mitochondrial membrane 44 (OpenTargets Search: -TIMM44, zhang2024afirstinclasstimm44 pages 1-2)
UniProt ID	O02161 (user-provided target identity; consistent with T09B4.9/tin-44 annotation)	Not directly established in gathered evidence	Not directly established in gathered evidence
Protein family	Tim44 family; inferred metazoan ortholog of mitochondrial import motor scaffold protein (bennett2014activationofthe pages 2-3, xin2022theuprmtpreserves pages 6-9)	Tim44 family; docking/scaffold component of the PAM import motor associated with TIM23 (paschen2001proteinimportinto pages 5-7, laan2006mitochondrialpreproteintranslocases pages 5-7)	TIMM44/Tim44 family; PAM-associated mitochondrial protein import factor (zhang2024afirstinclasstimm44 pages 1-2, michaelis2022proteinimportmotor pages 5-6)
Subcellular localization	Mitochondrial inner membrane import machinery; function inferred on the matrix side of the inner membrane from orthology and import-related phenotypes (bennett2014activationofthe pages 2-3, xin2022theuprmtpreserves pages 6-9)	Peripheral inner mitochondrial membrane protein on the matrix face; hydrophilic, membrane-associated, can interact with cardiolipin (bauer2000proteintranslocationinto pages 1-2, paschen2001proteinimportinto pages 3-5)	Mitochondrial inner membrane import machinery / mitochondrial fraction; part of PAM/TIM23-associated import system (zhang2024afirstinclasstimm44 pages 1-2, zhang2024afirstinclasstimm44 pages 4-6)
Primary function	Likely scaffold/adaptor for matrix protein import through TIM23-PAM; required for efficient mitochondrial protein import, and its knockdown activates UPRmt (bennett2014activationofthe pages 2-3, xin2022theuprmtpreserves pages 6-9, rolland2019compromisedmitochondrialprotein pages 1-2)	Scaffold/docking protein that recruits mtHsp70 and links the TIM23 channel to the PAM motor to drive ATP-dependent import of presequence-containing proteins into the matrix (chaudhuri2020tim17updatesa pages 2-5, paschen2001proteinimportinto pages 5-7)	Essential mitochondrial import factor; supports pre-protein import, mitochondrial integrity, respiration/ATP production, and in cancer cells supports Akt-mTOR-linked growth programs (zhang2024afirstinclasstimm44 pages 1-2, zhang2024afirstinclasstimm44 pages 12-13, zhang2024afirstinclasstimm44 pages 10-12)
Complex membership	Inferred member of the TIM23/PAM mitochondrial import machinery in worms (bennett2014activationofthe pages 2-3, xin2022theuprmtpreserves pages 6-9)	Part of TIM23MOTOR/PAM; distinguishes matrix-import motor form from TIM23SORT form (jain2025investigatingmitochondrialpresequence pages 20-23, laan2006mitochondrialpreproteintranslocases pages 5-7, laan2006mitochondrialpreproteintranslocases pages 7-8)	Component of the PAM complex associated with TIM23 (zhang2024afirstinclasstimm44 pages 1-2, michaelis2022proteinimportmotor pages 5-6)
Key interactions	Functional association with mitochondrial import machinery and UPRmt signaling; transcriptionally co-upregulated with import genes such as timm-23, mppa-1, mppb-1 during UPRmt (xin2022theuprmtpreserves pages 6-9)	Interacts with mtHsp70, Mge1, Tim17, Tim23, Pam16/Pam18/Pam17, incoming presequences, and cardiolipin; recruits motor modules to the translocase (hutu2008mitochondrialproteinimport pages 7-7, hutu2008mitochondrialproteinimport pages 1-2, jain2025investigatingmitochondrialpresequence pages 15-17)	Associated with TIMM23/TIM23, PAM components, and functionally linked to mitochondrial import and mitophagy signaling; MB-10 binds the C-terminal domain of TIMM44 (zhang2024afirstinclasstimm44 pages 1-2, zhang2024afirstinclasstimm44 pages 4-6, michaelis2022proteinimportmotor pages 5-6)
Loss-of-function phenotype (RNAi / mutant)	RNAi induces hsp-6p::gfp UPRmt reporter and in one genome-wide screen increased mean lifespan by 11.1%; import impairment is consistent with mitochondrial stress signaling (bennett2014activationofthe pages 2-3, bennett2014activationofthe pages 6-6). Reviews also place TIN-44 among import factors whose depletion induces mitochondrial stress and can reduce lifespan in some contexts (hernandorodriguez2018mitochondrialqualitycontrol pages 6-8)	Essential for matrix-targeted protein import; temperature-sensitive tim44-804 strongly inhibits import of matrix-destined precursors while sparing inner-membrane sorting, showing a selective motor defect rather than gross TIM23 destruction (hutu2008mitochondrialproteinimport pages 3-4)	Genetic depletion or pharmacologic blockade (MB-10/MitoBloCK-10) causes mitochondrial depolarization, ROS increase, ATP reduction, apoptosis, and strong inhibition of bladder cancer cell growth; PAM sequestration involving TIMM44 can also reduce import and trigger mitophagy (zhang2024afirstinclasstimm44 pages 1-2, zhang2024afirstinclasstimm44 pages 6-8, michaelis2022proteinimportmotor pages 5-6)
Disease associations	No direct worm disease annotation; chiefly used as a model for mitochondrial import stress and UPRmt biology (bennett2014activationofthe pages 2-3, rolland2019compromisedmitochondrialprotein pages 1-2)	Not applicable as a human disease gene; chiefly a mechanistic model for mitochondrial import (paschen2001proteinimportinto pages 5-7, hutu2008mitochondrialproteinimport pages 3-4)	Open Targets reports associations with neurodegenerative disease, Parkinson disease, Alzheimer disease, multiple sclerosis, and lysosomal storage disease; recent work also implicates TIMM44 as a potential bladder cancer therapeutic target (OpenTargets Search: -TIMM44, zhang2024afirstinclasstimm44 pages 1-2)

Table: This table compares key functional properties of C. elegans TIN-44 with its yeast and human orthologs. It highlights the conserved role of Tim44-family proteins in TIM23/PAM-mediated mitochondrial protein import and summarizes organism-specific phenotypes and disease relevance.

7. Broader Biological Context and Pathways

7.1 Mitochondrial Protein Import Pathway

TIN-44 functions within the presequence import pathway (TIM23 pathway), the major route by which nuclear-encoded mitochondrial matrix proteins are imported. The pathway involves: (1) cytosolic recognition by chaperones; (2) passage through the TOM complex in the outer membrane; (3) transfer to the TIM23 complex in the inner membrane, driven by the membrane potential (Δψ); and (4) ATP-dependent pulling into the matrix by the PAM motor, in which Tim44 serves as the essential scaffold tethering mtHsp70 to the channel exit (paschen2001proteinimportinto pages 5-7, laan2006mitochondrialpreproteintranslocases pages 5-7, fox2012mitochondrialproteinsynthesis pages 11-13).

7.2 Mitophagy Signaling

In mammalian cells, the PAM complex containing TIMM44 has been implicated in mitophagy regulation. Michaelis et al. (2022) demonstrated that during mitochondrial protein misfolding, PAM components including TIMM44 dissociate from the TIM complex, reducing protein import and triggering mitophagy even in the absence of membrane depolarization. This establishes a novel mitophagy induction mechanism that is independent of the canonical PINK1 stabilization pathway (michaelis2022proteinimportmotor pages 5-6).

7.3 Disease Relevance of the Tim44 Family

Human TIMM44 has been associated with several disease contexts:

Cancer: TIMM44 is significantly overexpressed in bladder cancer tissues and cells. The first-in-class TIMM44 blocker MB-10 (MitoBloCK-10) binds the C-terminal domain of TIMM44, disrupts mitochondrial function (causing depolarization, oxidative stress, and ATP reduction), inhibits the Akt-mTOR pathway, and potently suppresses bladder cancer cell growth both in vitro and in vivo (zhang2024afirstinclasstimm44 pages 1-2, zhang2024afirstinclasstimm44 pages 12-13, zhang2024afirstinclasstimm44 pages 6-8).
Neurodegenerative diseases: OpenTargets database reports associations between TIMM44 and neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and multiple sclerosis, primarily through CRISPRi screening evidence in astrocyte models (OpenTargets Search: -TIMM44).
Diabetes: TIMM44 has been studied in the context of diabetic kidney disease and mitochondrial dysfunction in diabetes. Timm44 transgenic mice were used to study the effects of enhanced mitochondrial import on diabetic nephropathy (OpenTargets Search: -TIMM44).

8. Summary

C. elegans tin-44 (T09B4.9, UniProt O02161) encodes the nematode ortholog of the Tim44 family of mitochondrial protein import scaffold proteins. Based on extensive conservation within the Tim44 family and direct experimental evidence from C. elegans RNAi studies, TIN-44 functions as a peripheral inner mitochondrial membrane scaffold protein that tethers the PAM import motor to the TIM23 presequence translocase channel on the matrix side. Its primary role is to recruit mtHsp70 to the channel exit, enabling ATP-dependent vectorial translocation of presequence-containing precursor proteins into the mitochondrial matrix. TIN-44 is specifically required for the TIM23-MOTOR form of the translocase that drives matrix import, but not for the TIM23-SORT pathway that mediates lateral insertion into the inner membrane.

In C. elegans, depletion of tin-44 by RNAi compromises mitochondrial protein import, which activates the mitochondrial unfolded protein response (UPRmt) through the ATFS-1 transcription factor pathway and can modulate lifespan. Conversely, UPRmt activation transcriptionally upregulates tin-44 expression as part of a compensatory response to maintain import capacity. The human ortholog TIMM44 has emerged as a potential therapeutic target in cancer, with the first-in-class blocker MB-10 demonstrating preclinical efficacy against bladder cancer. The Tim44 protein family thus represents a conserved, essential component of mitochondrial biogenesis whose dysfunction intersects with aging, stress signaling, and disease.

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 26 citations.
(paschen2001proteinimportinto pages 5-7): Stefan A. Paschen and Walter Neupert. Protein import into mitochondria. IUBMB Life, 52:101-112, Sep 2001. URL: https://doi.org/10.1080/15216540152845894, doi:10.1080/15216540152845894. This article has 150 citations and is from a peer-reviewed journal.
(fox2012mitochondrialproteinsynthesis pages 14-15): Thomas D Fox. Mitochondrial protein synthesis, import, and assembly. Genetics, 192:1203-1234, Dec 2012. URL: https://doi.org/10.1534/genetics.112.141267, doi:10.1534/genetics.112.141267. This article has 283 citations and is from a domain leading peer-reviewed journal.
(bauer2000proteintranslocationinto pages 1-2): Matthias F Bauer, Sabine Hofmann, Walter Neupert, and Michael Brunner. Protein translocation into mitochondria: the role of tim complexes. Trends in cell biology, 10 1:25-31, Jan 2000. URL: https://doi.org/10.1016/s0962-8924(99)01684-0, doi:10.1016/s0962-8924(99)01684-0. This article has 334 citations and is from a domain leading peer-reviewed journal.
(jain2025investigatingmitochondrialpresequence pages 15-17): Naintara Jain. Investigating Mitochondrial Presequence Import. PhD thesis, University Goettingen, 2025. URL: https://doi.org/10.53846/goediss-11596, doi:10.53846/goediss-11596.
(hutu2008mitochondrialproteinimport pages 7-7): Dana P. Hutu, Bernard Guiard, Agnieszka Chacinska, Dorothea Becker, Nikolaus Pfanner, Peter Rehling, and Martin van der Laan. Mitochondrial protein import motor: differential role of tim44 in the recruitment of pam17 and j-complex to the presequence translocase. Molecular biology of the cell, 19 6:2642-9, Jun 2008. URL: https://doi.org/10.1091/mbc.e07-12-1226, doi:10.1091/mbc.e07-12-1226. This article has 104 citations and is from a domain leading peer-reviewed journal.
(hutu2008mitochondrialproteinimport pages 1-2): Dana P. Hutu, Bernard Guiard, Agnieszka Chacinska, Dorothea Becker, Nikolaus Pfanner, Peter Rehling, and Martin van der Laan. Mitochondrial protein import motor: differential role of tim44 in the recruitment of pam17 and j-complex to the presequence translocase. Molecular biology of the cell, 19 6:2642-9, Jun 2008. URL: https://doi.org/10.1091/mbc.e07-12-1226, doi:10.1091/mbc.e07-12-1226. This article has 104 citations and is from a domain leading peer-reviewed journal.
(laan2006mitochondrialpreproteintranslocases pages 7-8): Martin van der Laan, Michael Rissler, and Peter Rehling. Mitochondrial preprotein translocases as dynamic molecular machines. FEMS yeast research, 6 6:849-61, Sep 2006. URL: https://doi.org/10.1111/j.1567-1364.2006.00134.x, doi:10.1111/j.1567-1364.2006.00134.x. This article has 76 citations and is from a peer-reviewed journal.
(hutu2008mitochondrialproteinimport pages 3-4): Dana P. Hutu, Bernard Guiard, Agnieszka Chacinska, Dorothea Becker, Nikolaus Pfanner, Peter Rehling, and Martin van der Laan. Mitochondrial protein import motor: differential role of tim44 in the recruitment of pam17 and j-complex to the presequence translocase. Molecular biology of the cell, 19 6:2642-9, Jun 2008. URL: https://doi.org/10.1091/mbc.e07-12-1226, doi:10.1091/mbc.e07-12-1226. This article has 104 citations and is from a domain leading peer-reviewed journal.
(jain2025investigatingmitochondrialpresequence pages 20-23): Naintara Jain. Investigating Mitochondrial Presequence Import. PhD thesis, University Goettingen, 2025. URL: https://doi.org/10.53846/goediss-11596, doi:10.53846/goediss-11596.
(laan2006mitochondrialpreproteintranslocases pages 5-7): Martin van der Laan, Michael Rissler, and Peter Rehling. Mitochondrial preprotein translocases as dynamic molecular machines. FEMS yeast research, 6 6:849-61, Sep 2006. URL: https://doi.org/10.1111/j.1567-1364.2006.00134.x, doi:10.1111/j.1567-1364.2006.00134.x. This article has 76 citations and is from a peer-reviewed journal.
(paschen2001proteinimportinto pages 3-5): Stefan A. Paschen and Walter Neupert. Protein import into mitochondria. IUBMB Life, 52:101-112, Sep 2001. URL: https://doi.org/10.1080/15216540152845894, doi:10.1080/15216540152845894. This article has 150 citations and is from a peer-reviewed journal.
(zhang2024afirstinclasstimm44 pages 1-2): Lifeng Zhang, Xiaokai Shi, Lei Zhang, Yuanyuan Mi, Li Zuo, and Shenglin Gao. A first-in-class timm44 blocker inhibits bladder cancer cell growth. Cell Death & Disease, Mar 2024. URL: https://doi.org/10.1038/s41419-024-06585-x, doi:10.1038/s41419-024-06585-x. This article has 11 citations and is from a peer-reviewed journal.
(sim2023structuralbasisof pages 1-4): Sue Im Sim, Yuanyuan Chen, Diane L. Lynch, James C. Gumbart, and Eunyong Park. Structural basis of mitochondrial protein import by the tim23 complex. Nature, 621:620-626, Jun 2023. URL: https://doi.org/10.1038/s41586-023-06239-6, doi:10.1038/s41586-023-06239-6. This article has 113 citations and is from a highest quality peer-reviewed journal.
(bennett2014activationofthe pages 2-3): Christopher F. Bennett, Helen Vander Wende, Marissa Simko, Shannon Klum, Sarah Barfield, Haeri Choi, Victor V. Pineda, and Matt Kaeberlein. Activation of the mitochondrial unfolded protein response does not predict longevity in caenorhabditis elegans. Nature communications, 5:3483-3483, Mar 2014. URL: https://doi.org/10.1038/ncomms4483, doi:10.1038/ncomms4483. This article has 272 citations and is from a highest quality peer-reviewed journal.
(bennett2014activationofthe pages 6-6): Christopher F. Bennett, Helen Vander Wende, Marissa Simko, Shannon Klum, Sarah Barfield, Haeri Choi, Victor V. Pineda, and Matt Kaeberlein. Activation of the mitochondrial unfolded protein response does not predict longevity in caenorhabditis elegans. Nature communications, 5:3483-3483, Mar 2014. URL: https://doi.org/10.1038/ncomms4483, doi:10.1038/ncomms4483. This article has 272 citations and is from a highest quality peer-reviewed journal.
(hernandorodriguez2018mitochondrialqualitycontrol pages 6-8): Blanca Hernando-Rodríguez and Marta Artal-Sanz. Mitochondrial quality control mechanisms and the phb (prohibitin) complex. Cells, Nov 2018. URL: https://doi.org/10.3390/cells7120238, doi:10.3390/cells7120238. This article has 93 citations.
(xin2022theuprmtpreserves pages 6-9): Nan Xin, Jenni Durieux, Chunxia Yang, Suzanne Wolff, Hyun-Eui Kim, and Andrew Dillin. The uprmt preserves mitochondrial import to extend lifespan. May 2022. URL: https://doi.org/10.1083/jcb.202201071, doi:10.1083/jcb.202201071. This article has 64 citations and is from a highest quality peer-reviewed journal.
(rolland2019compromisedmitochondrialprotein pages 1-2): Stéphane G. Rolland, Sandra Schneid, Melanie Schwarz, Elisabeth Rackles, Christian Fischer, Simon Haeussler, Saroj G. Regmi, Assa Yeroslaviz, Bianca Habermann, Dejana Mokranjac, Eric Lambie, and Barbara Conradt. Compromised mitochondrial protein import acts as a signal for uprmt. Cell Reports, 28:1659-1669.e5, Aug 2019. URL: https://doi.org/10.1016/j.celrep.2019.07.049, doi:10.1016/j.celrep.2019.07.049. This article has 184 citations and is from a highest quality peer-reviewed journal.
(hernandorodriguez2018mitochondrialqualitycontrol pages 3-5): Blanca Hernando-Rodríguez and Marta Artal-Sanz. Mitochondrial quality control mechanisms and the phb (prohibitin) complex. Cells, Nov 2018. URL: https://doi.org/10.3390/cells7120238, doi:10.3390/cells7120238. This article has 93 citations.
(xin2022theuprmtpreserves pages 1-2): Nan Xin, Jenni Durieux, Chunxia Yang, Suzanne Wolff, Hyun-Eui Kim, and Andrew Dillin. The uprmt preserves mitochondrial import to extend lifespan. May 2022. URL: https://doi.org/10.1083/jcb.202201071, doi:10.1083/jcb.202201071. This article has 64 citations and is from a highest quality peer-reviewed journal.
(rolland2019compromisedmitochondrialprotein pages 5-7): Stéphane G. Rolland, Sandra Schneid, Melanie Schwarz, Elisabeth Rackles, Christian Fischer, Simon Haeussler, Saroj G. Regmi, Assa Yeroslaviz, Bianca Habermann, Dejana Mokranjac, Eric Lambie, and Barbara Conradt. Compromised mitochondrial protein import acts as a signal for uprmt. Cell Reports, 28:1659-1669.e5, Aug 2019. URL: https://doi.org/10.1016/j.celrep.2019.07.049, doi:10.1016/j.celrep.2019.07.049. This article has 184 citations and is from a highest quality peer-reviewed journal.
(OpenTargets Search: -TIMM44): Open Targets Query (-TIMM44, 5 results). Buniello, A. et al. (2025). Open Targets Platform: facilitating therapeutic hypotheses building in drug discovery. Nucleic Acids Research.
(michaelis2022proteinimportmotor pages 5-6): Jonas Benjamin Michaelis, Melinda Elaine Brunstein, Süleyman Bozkurt, Ludovico Alves, Martin Wegner, Manuel Kaulich, Christian Pohl, and Christian Münch. Protein import motor complex reacts to mitochondrial misfolding by reducing protein import and activating mitophagy. Nature Communications, Sep 2022. URL: https://doi.org/10.1038/s41467-022-32564-x, doi:10.1038/s41467-022-32564-x. This article has 86 citations and is from a highest quality peer-reviewed journal.
(zhang2024afirstinclasstimm44 pages 4-6): Lifeng Zhang, Xiaokai Shi, Lei Zhang, Yuanyuan Mi, Li Zuo, and Shenglin Gao. A first-in-class timm44 blocker inhibits bladder cancer cell growth. Cell Death & Disease, Mar 2024. URL: https://doi.org/10.1038/s41419-024-06585-x, doi:10.1038/s41419-024-06585-x. This article has 11 citations and is from a peer-reviewed journal.
(zhang2024afirstinclasstimm44 pages 12-13): Lifeng Zhang, Xiaokai Shi, Lei Zhang, Yuanyuan Mi, Li Zuo, and Shenglin Gao. A first-in-class timm44 blocker inhibits bladder cancer cell growth. Cell Death & Disease, Mar 2024. URL: https://doi.org/10.1038/s41419-024-06585-x, doi:10.1038/s41419-024-06585-x. This article has 11 citations and is from a peer-reviewed journal.
(zhang2024afirstinclasstimm44 pages 10-12): Lifeng Zhang, Xiaokai Shi, Lei Zhang, Yuanyuan Mi, Li Zuo, and Shenglin Gao. A first-in-class timm44 blocker inhibits bladder cancer cell growth. Cell Death & Disease, Mar 2024. URL: https://doi.org/10.1038/s41419-024-06585-x, doi:10.1038/s41419-024-06585-x. This article has 11 citations and is from a peer-reviewed journal.
(zhang2024afirstinclasstimm44 pages 6-8): Lifeng Zhang, Xiaokai Shi, Lei Zhang, Yuanyuan Mi, Li Zuo, and Shenglin Gao. A first-in-class timm44 blocker inhibits bladder cancer cell growth. Cell Death & Disease, Mar 2024. URL: https://doi.org/10.1038/s41419-024-06585-x, doi:10.1038/s41419-024-06585-x. This article has 11 citations and is from a peer-reviewed journal.
(fox2012mitochondrialproteinsynthesis pages 11-13): Thomas D Fox. Mitochondrial protein synthesis, import, and assembly. Genetics, 192:1203-1234, Dec 2012. URL: https://doi.org/10.1534/genetics.112.141267, doi:10.1534/genetics.112.141267. This article has 283 citations and is from a domain leading peer-reviewed journal.

Artifacts

Citations

jain2025investigatingmitochondrialpresequence pages 15-17
hutu2008mitochondrialproteinimport pages 3-4
bauer2000proteintranslocationinto pages 1-2
sim2023structuralbasisof pages 1-4
bennett2014activationofthe pages 2-3
bennett2014activationofthe pages 6-6
hernandorodriguez2018mitochondrialqualitycontrol pages 6-8
xin2022theuprmtpreserves pages 6-9
michaelis2022proteinimportmotor pages 5-6
paschen2001proteinimportinto pages 5-7
fox2012mitochondrialproteinsynthesis pages 14-15
hutu2008mitochondrialproteinimport pages 7-7
hutu2008mitochondrialproteinimport pages 1-2
laan2006mitochondrialpreproteintranslocases pages 7-8
jain2025investigatingmitochondrialpresequence pages 20-23
laan2006mitochondrialpreproteintranslocases pages 5-7
paschen2001proteinimportinto pages 3-5
rolland2019compromisedmitochondrialprotein pages 1-2
hernandorodriguez2018mitochondrialqualitycontrol pages 3-5
xin2022theuprmtpreserves pages 1-2
rolland2019compromisedmitochondrialprotein pages 5-7
fox2012mitochondrialproteinsynthesis pages 11-13
TIM23-Tim44 Import Pathways
https://doi.org/10.3390/biom10121643,
https://doi.org/10.1080/15216540152845894,
https://doi.org/10.1534/genetics.112.141267,
https://doi.org/10.1016/s0962-8924(99
https://doi.org/10.53846/goediss-11596,
https://doi.org/10.1091/mbc.e07-12-1226,
https://doi.org/10.1111/j.1567-1364.2006.00134.x,
https://doi.org/10.1038/s41419-024-06585-x,
https://doi.org/10.1038/s41586-023-06239-6,
https://doi.org/10.1038/ncomms4483,
https://doi.org/10.3390/cells7120238,
https://doi.org/10.1083/jcb.202201071,
https://doi.org/10.1016/j.celrep.2019.07.049,
https://doi.org/10.1038/s41467-022-32564-x,

📚 Additional Documentation

Notes

(tin-44-notes.md)

tin-44 (C. elegans) — research notes

Gene: tin-44 (WormBase T09B4.9, WBGene00020383); UniProt O02161 (TIM44_CAEEL).
Ortholog of human TIMM44 / yeast Tim44. Protein Existence level PE=3 (Inferred from
homology) — the worm protein's own biochemistry has NOT been directly characterized.

Summary of what this gene is

TIM44 is the central organizing subunit of the presequence translocase-associated import
motor (PAM), the ATP-driven engine on the matrix side of the TIM23 (presequence) translocase
that pulls nucleus-encoded, presequence-bearing preproteins across the mitochondrial inner
membrane into the matrix. TIM44 docks on the matrix face of the TIM23 channel and
recruits/tethers mitochondrial HSP70 (mtHsp70; worm HSP-6) together with its regulatory
co-chaperones (J-protein Pam18/Tim14 = worm dnj-21, J-like Pam16/Tim16 = worm tim-16,
and nucleotide-exchange factor GrpE/Mge1), coupling cycles of mtHsp70 ATP binding/hydrolysis to
the vectorial (ratchet/motor) inward movement of the incoming polypeptide.

KNOWN (established)

Family/orthology. tin-44 belongs to the Tim44 family (InterPro IPR017303 Tim44;
Pfam PF04280; PANTHER PTHR10721 "MITOCHONDRIAL IMPORT INNER MEMBRANE TRANSLOCASE SUBUNIT
TIM44"). Contains an NTF2-like C-terminal domain fold (IPR032710). [UniProt:O02161]
Worm identity + import-machinery membership + co-regulation (direct worm data).
Xin et al. 2022 (worm, full text) explicitly identify tin-44 as the C. elegans homolog of
mammalian tim44 and place it in the matrix-pulling step of import:
PMID:35608535 and
PMID:35608535. i.e. tin-44 is transcriptionally co-induced with the rest of the TIM/TOM
import machinery upon activation of the mitochondrial UPR (UPRmt).
Conserved PAM/import-motor mechanism (by-similarity basis). In yeast/human, matrix import
requires Tim23/Tim17/Tim44 acting with mtHsp70:
PMID:10339406. Human Tim44 topology differs from yeast:
PMID:10339406.
UniProt curated FUNCTION/SUBUNIT (by similarity to human Q07914).
FUNCTION: "Essential component of the PAM complex ... Recruits mitochondrial HSP70 to drive
protein translocation into the matrix using ATP as an energy source." SUBUNIT: "Probable
component of the PAM complex at least composed of a mitochondrial HSP70 protein, GrpE, tin-44,
tim-16 and tim-14/dnj-21. The complex interacts with the tim-23 component of the TIM23
complex." SUBCELLULAR LOCATION: "Mitochondrion inner membrane {ECO:0000305}". [UniProt:O02161]

NOT known (knowledge gaps)

The molecular activity of worm TIN-44 has never been directly measured. Its recruitment/
tethering of mtHsp70 (HSP-6), its role as the PAM scaffold, and the coupling of mtHsp70 ATPase
cycles to matrix translocation are all inferred from yeast/human Tim44. The only direct worm
evidence is transcriptional co-regulation (PMID:35608535); there is no worm biochemistry,
interaction, structure, or import-assay data on the TIN-44 protein itself. (UniProt keeps the
name "Probable mitochondrial import inner membrane translocase subunit" and all FUNCTION/
SUBUNIT annotations at evidence ECO:0000250 "by similarity".)
Submitochondrial topology of worm TIN-44 is unverified. It is annotated to the
mitochondrial inner membrane, but the human ortholog is matrix-localized and only loosely
membrane-associated (PMID:10339406); whether worm TIN-44 is peripheral on the matrix face or
more tightly membrane-anchored is untested.
Loss-of-function phenotype / essentiality in C. elegans not characterized in the cached
literature. (Mammalian TIMM44 is essential; worm phenotype not established here.)

Existing GOA annotations (6) — review plan

GO:0001405 PAM complex, Tim23 associated import motor (CC, part_of, IBA) → ACCEPT (core complex membership)
GO:0030150 protein import into mitochondrial matrix (BP, involved_in, IBA) → ACCEPT (core BP)
GO:0051087 protein-folding chaperone binding (MF, enables, IBA) → ACCEPT (core MF — binds/tethers mtHsp70)
GO:0005743 mitochondrial inner membrane (CC, located_in, IEA) → ACCEPT (core location; see topology caveat)
GO:0030150 protein import into mitochondrial matrix (BP, involved_in, IEA/InterPro) → ACCEPT (same correct BP, InterPro2GO)
GO:0051087 protein-folding chaperone binding (MF, enables, IEA/InterPro) → ACCEPT (same correct MF, InterPro2GO)

No protein binding-type uninformative MF present. All 6 annotations are consistent with the
Tim44 family assignment and the conserved PAM function; none contradicted by worm data.

Key references

PMID:35608535 — Xin et al., "The UPRmt preserves mitochondrial import to extend lifespan"
(worm, full text). Only direct-worm reference for tin-44: homolog identity + import-machinery
membership + UPRmt co-induction. HIGH relevance.
PMID:10339406 — Bauer et al. 1999, "Genetic and structural characterization of the human
mitochondrial inner membrane translocase" (abstract only). Establishes the conserved
Tim23/Tim17/Tim44 + mtHsp70 matrix-import mechanism and human Tim44 matrix/loose-membrane
topology. MEDIUM relevance (by-similarity basis).
UniProt:O02161 — Swiss-Prot record; curated FUNCTION/SUBUNIT (by similarity to human
TIMM44 Q07914) and inner-membrane location.
PMID:24662282 — Bennett et al. 2014, "Activation of the mitochondrial unfolded protein
response does not predict longevity in C. elegans" (worm, full text). Direct worm RNAi data:
PMID:24662282 and PMID:24662282.
tin-44 (T09B4.9) was an hsp-6p::gfp UPRmt-inducing clone and is grouped with the
lifespan-SHORTENING import knockdowns. HIGH relevance.

Deep research provenance / falcon verification

Falcon (Edison) deep research completed at 2026-07-04T13:55 (1210 s, 37 citations):
tin-44-deep-research-falcon.md. Verified as a real Edison output (proper frontmatter,
real artifacts, real papers e.g. xin2022theuprmtpreserves = PMID:35608535,
bennett2014activationofthe = PMID:24662282). The perplexity-lite fallback failed (401
quota), but the falcon file itself is genuine.
Falcon fact-check caveat: the falcon report asserted tin-44 RNAi "extended mean lifespan
by 11.1% (P = 2.4 × 10⁻¹⁴)". This is a falcon ERROR — the cited Bennett 2014 full text
instead places tin-44/T09B4.9 among RNAi clones that SIGNIFICANTLY REDUCED lifespan. The
falcon claim was NOT used; only verbatim, verified quotes from the cached paper were used.

📄 View Raw YAML

id: O02161
gene_symbol: tin-44
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:6239
  label: Caenorhabditis elegans
description: >-
  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.
existing_annotations:
- term:
    id: GO:0001405
    label: PAM complex, Tim23 associated import motor
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: part_of
  review:
    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.
    action: ACCEPT
    supported_by:
    - reference_id: UniProt:O02161
      supporting_text: Essential component of the PAM complex
    - reference_id: PMID:35608535
      supporting_text: >-
        the precursor proteins are pulled into the matrix by TIM44 and mtHSP70 (HSP-6)
- term:
    id: GO:0030150
    label: protein import into mitochondrial matrix
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: involved_in
  review:
    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).
    action: ACCEPT
    supported_by:
    - reference_id: UniProt:O02161
      supporting_text: >-
        translocation of transit peptide-containing proteins from the inner membrane into the
        mitochondrial matrix in an ATP-dependent manner
    - reference_id: PMID:24662282
      supporting_text: >-
        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
    - reference_id: PMID:10339406
      supporting_text: >-
        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
- term:
    id: GO:0051087
    label: protein-folding chaperone binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  qualifier: enables
  review:
    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.
    action: ACCEPT
    supported_by:
    - reference_id: UniProt:O02161
      supporting_text: >-
        Recruits mitochondrial HSP70 to drive protein translocation into the matrix using ATP
        as an energy source
    - reference_id: PMID:35608535
      supporting_text: >-
        the precursor proteins are pulled into the matrix by TIM44 and mtHSP70 (HSP-6)
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    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.
    action: ACCEPT
    supported_by:
    - reference_id: UniProt:O02161
      supporting_text: Mitochondrion inner membrane
    knowledge_gaps:
    - gap_statement: >-
        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.
      boundary: >-
        UniProt assigns tin-44 to the mitochondrion inner membrane (ECO:0000305). For the human
        ortholog, biochemical characterization showed hTim44 is matrix-localized and, unlike
        yeast Tim44, only loosely membrane-associated (Bauer et al. 1999), so the family's
        membrane disposition is not uniform across species and has never been measured in worm.
      gap_kind:
      - BIOLOGY
      dark_aspect: RESIDUAL_SUBGAP
      status: OPEN
      significance: >-
        Whether TIN-44 is membrane-embedded or a peripheral/matrix protein affects how its
        recruitment of mtHsp70 to the channel is interpreted and how import-motor assembly is
        modeled in C. elegans.
      resolution: >-
        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.
      provenance:
      - reference_id: PMID:10339406
        supporting_text: >-
          hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated
          with the inner membrane
      - reference_id: UniProt:O02161
        supporting_text: Mitochondrion inner membrane
- term:
    id: GO:0030150
    label: protein import into mitochondrial matrix
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: involved_in
  review:
    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.
    action: ACCEPT
    supported_by:
    - reference_id: UniProt:O02161
      supporting_text: >-
        translocation of transit peptide-containing proteins from the inner membrane into the
        mitochondrial matrix in an ATP-dependent manner
- term:
    id: GO:0051087
    label: protein-folding chaperone binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    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.
    action: ACCEPT
    supported_by:
    - reference_id: UniProt:O02161
      supporting_text: >-
        Recruits mitochondrial HSP70 to drive protein translocation into the matrix using ATP
        as an energy source
core_functions:
- description: >-
    Organizing subunit of the mitochondrial presequence translocase-associated import motor
    (PAM): binds and tethers mitochondrial HSP70 (mtHsp70/HSP-6) at the matrix face of the
    TIM23 channel and, together with its co-chaperones, couples ATP-driven mtHsp70 cycles to
    the import of nucleus-encoded, presequence-bearing preproteins into the mitochondrial
    matrix. In C. elegans this activity is inferred from the conserved yeast/human Tim44
    ortholog; direct worm evidence establishes tin-44 as a co-regulated component of the
    mitochondrial import machinery acting at the matrix-pulling step with mtHsp70/HSP-6.
  molecular_function:
    id: GO:0051087
    label: protein-folding chaperone binding
  directly_involved_in:
  - id: GO:0030150
    label: protein import into mitochondrial matrix
  locations:
  - id: GO:0005743
    label: mitochondrial inner membrane
  in_complex:
    id: GO:0001405
    label: PAM complex, Tim23 associated import motor
  supported_by:
  - reference_id: UniProt:O02161
    supporting_text: >-
      Recruits mitochondrial HSP70 to drive protein translocation into the matrix using ATP as
      an energy source
  - reference_id: PMID:35608535
    supporting_text: >-
      the precursor proteins are pulled into the matrix by TIM44 and mtHSP70 (HSP-6)
proposed_new_terms: []
knowledge_gaps:
- gap_statement: >-
    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.
  boundary: >-
    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.
  gap_kind:
  - BIOLOGY
  - CURATION
  dark_aspect: MF_DARK
  status: OPEN
  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.
  resolution: >-
    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:
  - reference_id: UniProt:O02161
    supporting_text: Probable mitochondrial import inner membrane translocase subunit tin-44
  - reference_id: PMID:35608535
    supporting_text: >-
      which are C. elegans homologs of mammalian mtHsp70, tim44, and Mpp, respectively, were
      also upregulated by cco-1 RNAi
- gap_statement: >-
    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.
  boundary: >-
    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.
  gap_kind:
  - BIOLOGY
  dark_aspect: RESIDUAL_SUBGAP
  status: OPEN
  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.
  resolution: >-
    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:
  - reference_id: UniProt:O02161
    supporting_text: Probable mitochondrial import inner membrane translocase subunit tin-44
  - reference_id: PMID:24662282
    supporting_text: >-
      a subset of RNAi clones related to mitochondrial protein import (tomm-22, E04A4.5,
      T09B4.9, F45G2.8, F15D3.7, and dnj-21)
suggested_questions:
- question: >-
    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?
  experts: []
- question: >-
    Is tin-44 essential in C. elegans, and how does a genetic null differ from RNAi knockdown
    in developmental and mitochondrial-import phenotypes?
  experts: []
- question: >-
    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?
  experts: []
suggested_experiments:
- hypothesis: >-
    C. elegans TIN-44 is a bona fide import-motor subunit that binds mtHsp70 (HSP-6) and is
    required for matrix protein import.
  description: >-
    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.
  experiment_type: biochemical interaction / in-organello import assay
- hypothesis: >-
    tin-44 is essential for C. elegans viability, like mammalian TIMM44.
  description: >-
    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.
  experiment_type: genetics / phenotypic analysis
references:
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO terms
  findings: []
- id: GO_REF:0000033
  title: Annotation inferences using phylogenetic trees
  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: PMID:35608535
  title: The UPRmt preserves mitochondrial import to extend lifespan.
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Only direct-worm reference for tin-44 (full text, PMC-available). Explicitly identifies
      tin-44 as the C. elegans homolog of mammalian tim44, places it at the matrix-pulling step
      of import with mtHSP70/HSP-6, and shows it is transcriptionally co-induced with the rest
      of the TIM/TOM import machinery upon UPRmt activation. Quoted sentences are verbatim from
      the cached full text. Note: the worm data here are transcriptional, not biochemical.
- id: PMID:24662282
  title: >-
    Activation of the mitochondrial unfolded protein response does not predict longevity in
    Caenorhabditis elegans.
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      Genome-wide hsp-6p::gfp UPRmt RNAi screen (full text, verified). Directly assays tin-44:
      T09B4.9 (TIM44) is recovered as a UPRmt-inducing clone, grouped with the TIM23-complex
      import components, and is among the mitochondrial-import knockdowns that shorten lifespan.
      This is the strongest direct worm loss-of-function evidence that tin-44 acts in
      mitochondrial protein import. NOTE: a falcon deep-research summary claimed tin-44 RNAi
      "extended lifespan by 11.1%"; the paper's verified text instead groups tin-44 with the
      lifespan-SHORTENING import knockdowns, so that falcon claim was not used.
- id: PMID:10339406
  title: >-
    Genetic and structural characterization of the human mitochondrial inner membrane
    translocase.
  reference_review:
    relevance: MEDIUM
    correctness: VERIFIED
    review_notes: >-
      Human/yeast Tim44 characterization (abstract only). Establishes the conserved
      Tim23/Tim17/Tim44 + mtHsp70 matrix-import mechanism that is the by-similarity basis for
      the worm annotations, and reports that human Tim44 is matrix-localized and only loosely
      membrane-associated — the source for the submitochondrial-topology knowledge gap.
- id: UniProt:O02161
  title: Probable mitochondrial import inner membrane translocase subunit tin-44 (C. elegans)
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: >-
      UniProt/Swiss-Prot record. Source of the curated FUNCTION (essential PAM component that
      recruits mtHsp70 to drive ATP-dependent matrix translocation) and SUBUNIT (PAM composed
      of mtHsp70, GrpE, tin-44, tim-16, tim-14/dnj-21; interacts with tim-23), the
      inner-membrane location, and the Tim44-family assignment. All FUNCTION/SUBUNIT statements
      are ECO:0000250 "by similarity" (PE=3), the basis for the by-orthology framing and the
      knowledge gaps.

tin-44

Gene Description

Existing Annotations Review

Core Functions

References

Suggested Questions for Experts

Suggested Experiments

Knowledge Gaps

Deep Research

Falcon

Comprehensive Research Report: C. elegans TIN-44 (T09B4.9) — Probable Mitochondrial Import Inner Membrane Translocase Subunit Tim44

1. Gene Identity and Protein Overview

2. Primary Function: Scaffold Protein in the TIM23-Associated Import Motor (PAM Complex)

2.1 Molecular Function of Tim44-Family Proteins

2.2 Two Functional Forms of the TIM23 Complex

3. Subcellular Localization

4. Protein Structure and Domain Architecture

5. Functional Evidence in C. elegans

5.1 RNAi Phenotype and UPRmt Activation

5.2 Transcriptional Regulation Under Mitochondrial Stress

5.3 Role in the UPRmt Signaling Pathway

6. Comparative Functional Summary

7. Broader Biological Context and Pathways

7.1 Mitochondrial Protein Import Pathway

7.2 Mitophagy Signaling

7.3 Disease Relevance of the Tim44 Family

8. Summary

Artifacts

Citations

📚 Additional Documentation

Notes

tin-44 (C. elegans) — research notes

Summary of what this gene is

KNOWN (established)

NOT known (knowledge gaps)

Existing GOA annotations (6) — review plan

Key references

Deep research provenance / falcon verification

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