Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0030943 mitochondrion targeting sequence binding	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for mitochondrion targeting sequence binding. TIM22 does bind targeting signals, but specifically internal targeting signals of carrier proteins, not the classical N-terminal matrix targeting presequences. The term GO:0030943 is defined broadly as "Binding to a mitochondrion targeting sequence, a specific peptide sequence that acts as a signal to localize the protein within the mitochondrion" -- this is broad enough to encompass internal targeting signals. Kovermann et al. (PMID:11864609) demonstrated that reconstituted Tim22 forms a channel that "specifically responds to an internal targeting signal, but not to presequences." The IBA annotation is phylogenetically sound and the term is acceptable, though imprecise about the type of targeting signal recognized. Reason: GO:0030943 is defined broadly enough to cover internal targeting signal binding. TIM22 has been experimentally shown to recognize internal targeting signals of carrier proteins (PMID:11864609). While a more specific term for "internal mitochondrial targeting signal binding" would be ideal, GO:0030943 is the best available term and the IBA annotation is phylogenetically correct. Supporting Evidence: PMID:11864609 The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences
GO:0042721 TIM22 mitochondrial import inner membrane insertion complex	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for TIM22 complex membership. TIM22 is the essential core channel subunit of the TIM22 complex. Multiple studies confirm this: Kerscher et al. (PMID:10637294) and Koehler et al. (PMID:10648604) identified TIM22 as a core integral membrane subunit, and Rehling et al. (PMID:12637749) showed it forms the twin-pore translocase. UniProt describes it as "Essential core component of the TIM22 complex." Reason: TIM22 is the defining, essential core subunit of the TIM22 complex. This is one of the most well-established annotations for this gene, supported by extensive experimental evidence (PMID:10637294, PMID:10648604, PMID:12637749) and phylogenetically sound. Supporting Evidence: PMID:10637294 Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane PMID:10648604 The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p
GO:0045039 protein insertion into mitochondrial inner membrane	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for protein insertion into mitochondrial inner membrane. This is the core biological process of TIM22. Sirrenberg et al. (PMID:8955274) first showed that "Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane." Kovermann et al. (PMID:11864609) demonstrated TIM22 forms the channel, and Rehling et al. (PMID:12637749) showed the twin-pore translocase mediates insertion. Reason: This is the primary biological process of TIM22. Extensively supported by experimental evidence from multiple groups (PMID:8955274, PMID:11864609, PMID:12637749, PMID:32591483). The IBA annotation is phylogenetically sound. Supporting Evidence: PMID:8955274 Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane PMID:11864609 The protein insertion complex of the mitochondrial inner membrane is crucial for import of the numerous multitopic membrane proteins with internal targeting signals
GO:0008320 protein transmembrane transporter activity	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for protein transmembrane transporter activity. GO:0008320 is defined as "Enables the transfer of a protein from one side of a membrane to the other." TIM22 does transfer proteins across/into the inner membrane. However, it is more precise to say TIM22 mediates insertion of polytopic membrane proteins INTO the membrane rather than transfer across it. The term is acceptable as the best available MF term for TIM22's core activity -- it forms a channel that enables protein translocation through the membrane. The IBA is phylogenetically sound. Reason: GO:0008320 is the best available MF term for the protein translocation channel activity of TIM22. Kovermann et al. (PMID:11864609) demonstrated that reconstituted TIM22 forms a "hydrophilic, high-conductance channel with distinct opening states and pore diameters" that mediates protein transfer. The IBA is phylogenetically correct. Supporting Evidence: PMID:11864609 Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters
GO:0071806 protein transmembrane transport	IEA GO_REF:0000108	ACCEPT	Summary: IEA annotation inferred from GO:0008320 (protein transmembrane transporter activity) via logical inference. Since TIM22 enables protein transmembrane transporter activity, it follows that it is involved in protein transmembrane transport. This is a reasonable computational inference. Reason: Logically inferred from the IBA/IDA-supported GO:0008320 annotation. TIM22 mediates protein transmembrane transport (specifically insertion of carrier proteins into the inner membrane). The inference is sound, and this BP term is broader but not incorrect.
GO:0005743 mitochondrial inner membrane	IEA GO_REF:0000044	ACCEPT	Summary: IEA annotation from UniProt subcellular location mapping. TIM22 is an integral multi-pass protein of the mitochondrial inner membrane. This is abundantly supported by experimental evidence (PMID:8955274, PMID:10397776, PMID:10648604, PMID:32591483). Reason: Correct localization. TIM22 is an integral inner membrane protein with multiple transmembrane helices. Redundant with experimental IDA annotations but not incorrect.
GO:0015031 protein transport	IEA GO_REF:0000043	ACCEPT	Summary: IEA annotation from UniProt keyword mapping (Protein transport, Translocation). GO:0015031 "protein transport" is a broad parent term. TIM22 is involved in protein transport, specifically insertion of carrier proteins into the inner membrane (GO:0045039). This IEA is not wrong but is very general. Reason: Correct but general. GO:0045039 (protein insertion into mitochondrial inner membrane) is the more specific and informative term, which is already annotated via IBA, IDA, and IMP. This IEA annotation to the parent term is redundant but acceptable.
GO:0042721 TIM22 mitochondrial import inner membrane insertion complex	IEA GO_REF:0000002	ACCEPT	Summary: IEA annotation from InterPro mapping (IPR039175 TIM22). TIM22 is the core subunit of this complex. Redundant with IBA and IDA annotations for the same term. Reason: Correct. InterPro domain IPR039175 is specific to TIM22 family members. Redundant with experimental annotations but not incorrect.
GO:0045039 protein insertion into mitochondrial inner membrane	IEA GO_REF:0000002	ACCEPT	Summary: IEA annotation from InterPro mapping (IPR039175 TIM22). Redundant with IBA, IDA, and IMP annotations for the same term. Reason: Correct. Redundant with experimental annotations but the InterPro mapping is sound.
GO:0005515 protein binding	IPI PMID:17099692 The Tim21 binding domain connects the preprotein translocase...	REMOVE	Summary: IPI annotation for protein binding based on interaction with Tim21 (P53220), from Albrecht et al. (PMID:17099692). That paper studied the Tim21 binding domain and its connections between TOM and TIM complexes. The interaction between TIM22 and TIM21 is documented in IntAct. However, GO:0005515 "protein binding" is uninformative -- it does not tell us what the interaction means functionally. Reason: GO:0005515 "protein binding" is a vague term that provides no functional information. Per curation guidelines, this term should be avoided. The interaction with Tim21 is better captured by the CC annotation to the TIM22 complex (GO:0042721). Furthermore, Albrecht et al. (PMID:17099692) focused on Tim21's role in connecting TOM and TIM23 complexes; the Tim22-Tim21 interaction is secondary and does not inform TIM22's core function. Supporting Evidence: PMID:17099692 Tim21, a subunit of the presequence translocase consisting of a membrane anchor and a carboxy-terminal domain exposed to the intermembrane space, directly connects the TOM and TIM23 complexes by binding to the intermembrane space domain of the Tom22 receptor
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 from Koehler et al. (PMID:10648604). That study characterized the TIM22 complex and showed Tim22p is an integral membrane subunit of the inner membrane-localized 300-kDa complex. Reason: Experimentally demonstrated. TIM22 is an integral inner membrane protein, confirmed by co-migration with the 300-kDa TIM22 complex on nondenaturing gels (PMID:10648604) and by topology studies (UniProt). Supporting Evidence: PMID:10648604 a 300-kDa complex in the inner membrane, the TIM22 complex
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 from Koehler et al. (PMID:10648604). This paper identified Tim18p as a new subunit of the TIM22 complex "that mediates insertion of imported proteins into the yeast mitochondrial inner membrane." Tim22p was established as the core channel subunit in this complex. Reason: Direct experimental evidence that the TIM22 complex containing Tim22p mediates protein insertion into the inner membrane (PMID:10648604). Supporting Evidence: PMID:10648604 Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex
GO:0005198 structural molecule activity	IDA PMID:32591483 Conserved regions of budding yeast Tim22 have a role in stru...	ACCEPT	Summary: IDA annotation for structural molecule activity from Kumar et al. (PMID:32591483). That study showed that conserved regions of Tim22 (IMS domain and TM4) are "critically required for interactions with the membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase." Impairment of TIM22 complex assembly influenced translocase activity and cell viability. The structural role is real -- Tim22 is the scaffold around which the complex assembles. Reason: GO:0005198 "structural molecule activity" is defined as "The action of a molecule that contributes to the structural integrity of a complex." Kumar et al. (PMID:32591483) demonstrated that Tim22 regions are critical for maintaining the architecture and structural integrity of the TIM22 complex by mediating interactions with Tim54, Tim18, and Sdh3. This is a legitimate annotation -- Tim22 is both the channel and the structural core around which the complex assembles. Supporting Evidence: PMID:32591483 the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are critically required for interactions with the membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase
GO:0005198 structural molecule activity	IMP PMID:32591483 Conserved regions of budding yeast Tim22 have a role in stru...	ACCEPT	Summary: IMP annotation for structural molecule activity from the same study (PMID:32591483). Mutations in conserved TIM22 regions disrupted complex assembly, demonstrating the structural role via mutant phenotype. Reason: Mutant analysis in Kumar et al. (PMID:32591483) showed that impairment of TIM22 regions disrupts complex assembly and stability, confirming the structural role by IMP evidence. Complements the IDA annotation above. Supporting Evidence: PMID:32591483 impairment of TIM22 complex assembly influences its translocase activity, the mitochondrial network, and the viability of cells lacking mitochondrial DNA
GO:0005743 mitochondrial inner membrane	IDA PMID:32591483 Conserved regions of budding yeast Tim22 have a role in stru...	ACCEPT	Summary: IDA annotation for inner membrane localization from Kumar et al. (PMID:32591483). That study confirmed Tim22 is active in the inner membrane, as expected for the core channel subunit. The GOA qualifier is "is_active_in." Reason: Tim22 is an integral multi-pass protein of the mitochondrial inner membrane, confirmed by multiple studies. This IDA is consistent with all other evidence. Supporting Evidence: PMID:32591483 the TIM22 complex enables the integration of complex membrane proteins with internal targeting signals into the inner membrane
GO:0045039 protein insertion into mitochondrial inner membrane	IDA PMID:32591483 Conserved regions of budding yeast Tim22 have a role in stru...	ACCEPT	Summary: IDA annotation for protein insertion from Kumar et al. (PMID:32591483). Direct assays showed Tim22 is critically required for the translocase activity of the TIM22 complex. Reason: Direct experimental evidence from Kumar et al. (PMID:32591483). Tim22 is the core channel; its conserved regions are required for translocase activity. Supporting Evidence: PMID:32591483 the TIM22 complex enables the integration of complex membrane proteins with internal targeting signals into the inner membrane
GO:0045039 protein insertion into mitochondrial inner membrane	IMP PMID:32591483 Conserved regions of budding yeast Tim22 have a role in stru...	ACCEPT	Summary: IMP annotation for protein insertion from Kumar et al. (PMID:32591483). Mutant analysis of conserved Tim22 regions showed impaired translocase activity. Reason: Mutant phenotype evidence from Kumar et al. (PMID:32591483). Disruption of conserved Tim22 regions impaired protein insertion activity of the TIM22 complex. Supporting Evidence: PMID:32591483 impairment of TIM22 complex assembly influences its translocase activity
GO:0005739 mitochondrion	HDA PMID:24769239 Quantitative variations of the mitochondrial proteome and ph...	ACCEPT	Summary: HDA annotation for mitochondrion localization from Renvoise et al. (PMID:24769239), a quantitative mitochondrial proteomics study. TIM22 was identified as a mitochondrial protein by high-throughput mass spectrometry. Reason: Correct. TIM22 is a mitochondrial protein. The HDA evidence from proteomics (PMID:24769239) is a broader CC term than the more specific "mitochondrial inner membrane" (GO:0005743) but is not incorrect.
GO:0005739 mitochondrion	HDA PMID:16823961 Toward the complete yeast mitochondrial proteome: multidimen...	ACCEPT	Summary: HDA annotation for mitochondrion localization from Reinders et al. (PMID:16823961), a comprehensive yeast mitochondrial proteome analysis using multidimensional separation techniques. Reason: Correct. TIM22 was identified in the yeast mitochondrial proteome (PMID:16823961). Redundant with other CC annotations but acceptable.
GO:0008320 protein transmembrane transporter activity	IDA PMID:11864609 Tim22, the essential core of the mitochondrial protein inser...	ACCEPT	Summary: IDA annotation for protein transmembrane transporter activity from Kovermann et al. (PMID:11864609). This landmark paper demonstrated that reconstituted Tim22 forms a "hydrophilic, high-conductance channel with distinct opening states and pore diameters" that is "voltage-activated and specifically responds to an internal targeting signal." The paper showed Tim22 is "the only essential membrane-integrated subunit of the complex" and that "a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component." Reason: Strong experimental evidence. Kovermann et al. (PMID:11864609) performed reconstitution and electrophysiology experiments demonstrating that Tim22 alone forms a functional protein-translocating channel. This is the definitive evidence for TIM22's MF. Supporting Evidence: PMID:11864609 Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters. The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences PMID:11864609 a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component
GO:0030943 mitochondrion targeting sequence binding	IDA PMID:11864609 Tim22, the essential core of the mitochondrial protein inser...	ACCEPT	Summary: IDA annotation for mitochondrion targeting sequence binding from Kovermann et al. (PMID:11864609). The study showed that the Tim22 channel "specifically responds to an internal targeting signal, but not to presequences." This demonstrates signal-gated channel activity where TIM22 recognizes internal targeting signals of carrier proteins. Reason: Experimental evidence from electrophysiology showing signal-gated response to internal targeting signals (PMID:11864609). GO:0030943 is the best available term, though it does not distinguish internal signals from presequences. TIM22 specifically binds internal signals, not N-terminal presequences. Supporting Evidence: PMID:11864609 The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences
GO:0042721 TIM22 mitochondrial import inner membrane insertion complex	IDA PMID:10637294 Tim18p is a new component of the Tim54p-Tim22p translocon in...	ACCEPT	Summary: IDA annotation from Kerscher et al. (PMID:10637294). This study identified TIM18 as a new component of the Tim54p-Tim22p translocon and demonstrated that Tim22p is part of the approximately 300-kDa complex by blue native electrophoresis and co-immunoprecipitation. Reason: Direct biochemical evidence (co-IP, BN-PAGE) demonstrating Tim22p as a core component of the TIM22 complex (PMID:10637294). Supporting Evidence: PMID:10637294 Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an approximately 300-kDa complex after blue native electrophoresis
GO:0042721 TIM22 mitochondrial import inner membrane insertion complex	IDA PMID:10648604 Tim18p, a new subunit of the TIM22 complex that mediates ins...	ACCEPT	Summary: IDA annotation from Koehler et al. (PMID:10648604). The study characterized the TIM22 complex composition, confirming Tim22p as a core integral membrane subunit alongside Tim54p, with peripheral subunits Tim9p, Tim10p, and Tim12p. Reason: Biochemical characterization of the TIM22 complex demonstrating Tim22p as a core subunit (PMID:10648604). Supporting Evidence: PMID:10648604 The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p
GO:0045039 protein insertion into mitochondrial inner membrane	IMP PMID:10648604 Tim18p, a new subunit of the TIM22 complex that mediates ins...	ACCEPT	Summary: IMP annotation from Koehler et al. (PMID:10648604). Deletion of Tim18p (a complex partner) impaired import of precursor proteins, demonstrating the role of the TIM22 complex in protein insertion by mutant phenotype. Reason: Mutant phenotype evidence supporting the role of the TIM22 complex in protein insertion (PMID:10648604). Although the mutation was in Tim18p rather than Tim22p directly, it demonstrates the functional requirement of the complex. Supporting Evidence: PMID:10648604 It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex
GO:0045039 protein insertion into mitochondrial inner membrane	IMP PMID:8955274 Import of carrier proteins into the mitochondrial inner memb...	ACCEPT	Summary: IMP annotation from the founding paper by Sirrenberg et al. (PMID:8955274). This study first identified Tim22 as required for import of ADP/ATP carrier family members into the inner membrane, and showed that Tim22 is in an assembly distinct from the Tim23-Tim17 complex. Reason: Foundational experimental evidence. Sirrenberg et al. (PMID:8955274) first demonstrated that "Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane" and that "Import of proteins of the AAC family is independent of Tim23." Supporting Evidence: PMID:8955274 Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane. Members of the yeast AAC family are synthesized without matrix targeting signals PMID:8955274 Import of proteins of the AAC family is independent of Tim23, and import of matrix targeting signals containing preproteins is independent of Tim22
GO:0005743 mitochondrial inner membrane	TAS Reactome:R-SCE-1252258	ACCEPT	Summary: TAS annotation from Reactome pathway R-SCE-1252258 "TIM22 complex inserts proteins into inner membrane." TIM22 is indeed located in the inner membrane. Reason: Correct localization. Reactome pathway annotation is consistent with all experimental evidence.
GO:0005758 mitochondrial intermembrane space	TAS Reactome:R-SCE-1252255	MARK AS OVER ANNOTATED	Summary: TAS annotation from Reactome pathway R-SCE-1252255 "TOM40:TOM70 complex translocates proteins from the cytosol to the mitochondrial intermembrane space." This Reactome pathway describes the TOM-mediated import step, not TIM22 function per se. TIM22 does have domains exposed to the IMS (the N-terminal and C-terminal regions per UniProt topology), so there is some basis for IMS localization, but the primary localization is the inner membrane itself. This appears to be a Reactome annotation associating TIM22 with a pathway step where it is a participant rather than indicating its primary localization. Reason: TIM22 is an integral inner membrane protein, not an IMS protein. While it has IMS-exposed domains (N-terminal and C-terminal regions per UniProt topology), its primary localization is the inner membrane (GO:0005743), which is already well-annotated. The Reactome pathway R-SCE-1252255 describes TOM-mediated translocation to the IMS, not TIM22's localization. Annotating TIM22 to the IMS is misleading.
GO:0005758 mitochondrial intermembrane space	TAS Reactome:R-SCE-1252258	MARK AS OVER ANNOTATED	Summary: TAS annotation from Reactome pathway R-SCE-1252258 "TIM22 complex inserts proteins into inner membrane." Same concern as the other IMS annotation -- TIM22 is an integral inner membrane protein with IMS-exposed domains, but its primary CC is the inner membrane. Reason: TIM22 is an integral inner membrane protein. While the TIM22 complex operates at the interface between the IMS and the inner membrane, and Tim22 has IMS-exposed domains, annotating it to GO:0005758 (mitochondrial intermembrane space) suggests it is a soluble IMS protein, which is incorrect. The inner membrane annotation (GO:0005743) is the correct and sufficient CC term.
GO:0005758 mitochondrial intermembrane space	TAS Reactome:R-SCE-1252259	MARK AS OVER ANNOTATED	Summary: TAS annotation from Reactome pathway R-SCE-1252259 "TIM9:TIM10 binds hydrophobic proteins." This pathway describes the action of the small TIM chaperones (Tim9-Tim10) in the IMS, not TIM22 itself. TIM22 is listed as a participant in the broader mitochondrial import pathway but its primary localization is the inner membrane. Reason: Same rationale as other IMS annotations. TIM22 is an inner membrane protein, not an IMS protein. This Reactome pathway (R-SCE-1252259) describes TIM9:TIM10 function in the IMS, and TIM22 is merely a downstream partner.
GO:0005829 cytosol	TAS Reactome:R-SCE-1252255	REMOVE	Summary: TAS annotation from Reactome pathway R-SCE-1252255. This likely reflects that TIM22 precursor protein passes through the cytosol during its own import. However, mature TIM22 is an integral inner membrane protein and is not a cytosolic protein. Reason: TIM22 is not a cytosolic protein. It is an integral multi-pass protein of the mitochondrial inner membrane. While the TIM22 precursor transiently exists in the cytosol during its own biogenesis (like all nuclear-encoded mitochondrial proteins), the mature protein resides exclusively in the inner membrane. The Reactome pathway R-SCE-1252255 describes TOM-mediated import from the cytosol, but this does not make TIM22 a cytosolic protein.

Core Functions

TIM22 is the essential voltage-activated, signal-gated channel subunit of the TIM22 translocase that mediates insertion of multi-pass transmembrane proteins into the mitochondrial inner membrane. Tim22 has a 4-transmembrane helix architecture with conserved charged residues (E140, D190, K127, K169) critical for function and a C42-C141 disulfide bond (Zhang et al. 2021, DOI:10.1038/s41422-020-00399-0). The channel specifically recognizes internal targeting signals of carrier proteins but not N-terminal presequences, and is driven by the membrane potential (Delta-psi). The precise translocation mechanism remains debated: cryo-EM did not capture an open pore state, suggesting Tim22 may operate via conformationally gated pore formation in distinct functional states or via an insertase-like mechanism that lowers the energetic barrier for helix insertion by altering local bilayer properties.

Molecular Function:

protein transmembrane transporter activity

Directly Involved In:

protein insertion into mitochondrial inner membrane

Cellular Locations:

mitochondrial inner membrane

In Complex:

TIM22 mitochondrial import inner membrane insertion complex

Supporting Evidence:

PMID:11864609
Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters. The channel is voltage-activated and specifically responds to an internal targeting signal
PMID:8955274
Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane
DOI:10.1038/s41422-020-00399-0
Tim22 has four transmembrane helices ... a disulfide bond between C42 and C141 ... conserved charged residues implicated in function (E140, D190, K127, K169)

TIM22 serves as the structural core of the TIM22 complex, with its conserved regions required for interactions with Tim54, Tim18, and Sdh3 that maintain the functional architecture of the translocase.

Molecular Function:

structural molecule activity

Directly Involved In:

protein insertion into mitochondrial inner membrane

Cellular Locations:

mitochondrial inner membrane

In Complex:

TIM22 mitochondrial import inner membrane insertion complex

Supporting Evidence:

PMID:32591483
the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are critically required for interactions with the membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase

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: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:0000108

Automatic assignment of GO terms using logical inference, based on on inter-ontology links

PMID:8955274

Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22.

First identification of Tim22 as required for import of ADP/ATP carrier family members into the mitochondrial inner membrane.

"Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane"
Tim22 is in a complex distinct from the Tim23-Tim17 complex, and import of carrier proteins is independent of Tim23.

"Import of proteins of the AAC family is independent of Tim23, and import of matrix targeting signals containing preproteins is independent of Tim22"

PMID:10637294

Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane.

Tim18p identified as a new subunit of the Tim54p-Tim22p complex by genetic suppression and biochemical co-purification.

"Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and Tim18p, along with Tim54p and Tim22p, is detected in an approximately 300-kDa complex after blue native electrophoresis"

PMID:10648604

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

The TIM22 complex contains integral membrane subunits Tim22p and Tim54p, plus peripheral subunits Tim9p, Tim10p, and Tim12p.

"The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p"
Tim18p functions in assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion.

"Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane"

PMID:11864609

Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel.

Tim22 is the only essential membrane-integrated subunit of the protein insertion complex and forms a channel when reconstituted.

"Tim22 is the only essential membrane-integrated subunit of the complex. Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters"
The Tim22 channel is voltage-activated and specifically gated by internal targeting signals, not by N-terminal presequences.

"The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences"
Tim22 combines signal recognition, channel formation, and energy transduction in one component.

"a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component"

PMID:12637749

Protein insertion into the mitochondrial inner membrane by a twin-pore translocase.

The TIM22 complex forms a twin-pore translocase that uses membrane potential in two voltage-dependent steps for protein insertion.

PMID:16823961

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

TIM22 identified in the yeast mitochondrial proteome by multidimensional LC-MS/MS analysis.

PMID:17099692

The Tim21 binding domain connects the preprotein translocases of both mitochondrial membranes.

Tim21 connects the TOM and TIM23 complexes. TIM22-Tim21 interaction detected by IntAct.

"Tim21, a subunit of the presequence translocase consisting of a membrane anchor and a carboxy-terminal domain exposed to the intermembrane space, directly connects the TOM and TIM23 complexes"

PMID:24769239

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

TIM22 detected in mitochondrial proteome across fermentative and respiratory conditions.

PMID:32591483

Conserved regions of budding yeast Tim22 have a role in structural organization of the carrier translocase.

The IMS and TM4 regions of Tim22 are critically required for interactions with Tim54, Tim18, and Sdh3, maintaining the functional architecture of the TIM22 translocase.

"the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are critically required for interactions with the membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase"
TM1 and TM2 regions mediate Tim18 interaction; TM3 is required for Sdh3 interaction.

"the TM1 and TM2 regions of Tim22 are important for association with Tim18, whereas TM3 is exclusively required for the interaction with Sdh3"
Impairment of TIM22 complex assembly affects translocase activity, mitochondrial network integrity, and viability.

"impairment of TIM22 complex assembly influences its translocase activity, the mitochondrial network, and the viability of cells lacking mitochondrial DNA"

DOI:10.1038/s41422-020-00399-0

Structure of the mitochondrial TIM22 complex from yeast

Cryo-EM structure of the endogenous yeast TIM22 complex at 3.8 angstrom resolution reveals a 7-subunit architecture with Tim22 possessing 4 transmembrane helices, a C42-C141 disulfide bond, and conserved charged residues (E140, D190, K127, K169) critical for function.
The hexameric small-Tim ring (Tim9/Tim10/Tim12) is tilted approximately 45 degrees relative to the membrane plane.
The captured cryo-EM state does not display a clear open translocation pore, suggesting Tim22 may operate via conformationally gated states or an insertase-like mechanism that alters local bilayer properties.

DOI:10.1242/jcs.260060

Functional crosstalk between the TIM22 complex and YME1 machinery maintains mitochondrial proteostasis and integrity

Yme1 i-AAA protease is required for stability of the TIM22 complex and for proteostasis of TIM22 pathway substrates; a small non-stoichiometric pool of Yme1 associates transiently with TIM22.
Loss of Yme1 leads to accumulation of TIM22 substrates outside mitochondria, altered mitochondrial morphology, and compromised respiration; reducing TIM22 activity (e.g., deleting Tim18) reduced cargo accumulation in yme1-delta contexts.

DOI:10.1042/bst20240450

Role of Yme1 in mitochondrial protein homeostasis: from regulation of protein import, OXPHOS function to lipid synthesis and mitochondrial dynamics

Mini-review synthesizing evidence that Yme1 contributes to TIM22 pathway robustness, including degradation of misfolded small Tim chaperones with differential susceptibility of Tim10 vs Tim9, and that loss of Yme1 reduces TIM22 stability and import efficiency especially under heat stress.

DOI:10.1016/j.celrep.2024.113772

Identification of MIMAS, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria

MIMAS is a ~3 MDa inner-membrane mega-assembly comprising metabolite carriers (including Aac2) and respiratory chain biogenesis factors, demonstrating that canonical TIM22 cargo proteins physically co-reside in large multifunctional assemblies.

Reactome:R-SCE-1252255

TOM40:TOM70 complex translocates proteins from the cytosol to the mitochondrial intermembrane space

Reactome:R-SCE-1252258

TIM22 complex inserts proteins into inner membrane

Reactome:R-SCE-1252259

TIM9:TIM10 binds hydrophobic proteins

📚 Additional Documentation

Deep Research Falcon

(TIM22-deep-research-falcon.md)

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template_file: templates/gene_research_go_focused.md
template_variables:
organism: yeast
gene_id: TIM22
gene_symbol: TIM22
uniprot_accession: Q12328
protein_description: 'RecName: Full=Mitochondrial import inner membrane translocase
subunit TIM22;'
gene_info: Name=TIM22; OrderedLocusNames=YDL217C; ORFNames=D0884;
organism_full: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
protein_family: Belongs to the Tim17/Tim22/Tim23 family. .
protein_domains: TIM22. (IPR039175); Tim17 (PF02466)
provider_config:
timeout: 600
max_retries: 3
parameters:
allowed_domains: []
temperature: 0.1
citation_count: 30

Question

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.

Target Gene/Protein Identity (from UniProt):

UniProt Accession: Q12328
Protein Description: RecName: Full=Mitochondrial import inner membrane translocase subunit TIM22;
Gene Information: Name=TIM22; OrderedLocusNames=YDL217C; ORFNames=D0884;
Organism (full): Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
Protein Family: Belongs to the Tim17/Tim22/Tim23 family. .
Key Domains: TIM22. (IPR039175); Tim17 (PF02466)

MANDATORY VERIFICATION STEPS:

Check if the gene symbol "TIM22" matches the protein description above
Verify the organism is correct: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
Check if protein family/domains align with what you find in literature
If you find literature for a DIFFERENT gene with the same or similar symbol, STOP

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'TIM22' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information

Research Target:

Please provide a comprehensive research report on the gene TIM22 (gene ID: TIM22, UniProt: Q12328) in yeast.

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.

Output

Question: You are an expert researcher providing comprehensive, well-cited information.

Provide detailed information focusing on:
1. Key concepts and definitions with current understanding
2. Recent developments and latest research (prioritize 2023-2024 sources)
3. Current applications and real-world implementations
4. Expert opinions and analysis from authoritative sources
5. Relevant statistics and data from recent studies

Format as a comprehensive research report with proper citations. Include URLs and publication dates where available.
Always prioritize recent, authoritative sources and provide specific citations for all major claims.

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.

Target Gene/Protein Identity (from UniProt):

UniProt Accession: Q12328
Protein Description: RecName: Full=Mitochondrial import inner membrane translocase subunit TIM22;
Gene Information: Name=TIM22; OrderedLocusNames=YDL217C; ORFNames=D0884;
Organism (full): Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
Protein Family: Belongs to the Tim17/Tim22/Tim23 family. .
Key Domains: TIM22. (IPR039175); Tim17 (PF02466)

MANDATORY VERIFICATION STEPS:

Check if the gene symbol "TIM22" matches the protein description above
Verify the organism is correct: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast).
Check if protein family/domains align with what you find in literature
If you find literature for a DIFFERENT gene with the same or similar symbol, STOP

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Please provide a comprehensive research report on the gene TIM22 (gene ID: TIM22, UniProt: Q12328) in yeast.

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.

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.

Title: Functional Annotation Research Report — TIM22 (Saccharomyces cerevisiae S288c; UniProt Q12328; YDL217C)

Publication date of this report: 2026-02-20.

Target verification (mandatory)

Identity confirmed for the requested target: TIM22 in budding yeast Saccharomyces cerevisiae (strain S288c), corresponding to the mitochondrial inner membrane translocase subunit Tim22 and the Tim17/Tim22/Tim23 family. The yeast Tim22 is repeatedly described in yeast-focused primary literature as the essential core subunit of the TIM22 “carrier translocase” (inner-membrane insertase) that works together with yeast-specific partner subunits Tim54, Tim18, and Sdh3 plus small Tim chaperones (Tim9/Tim10/Tim12). This matches the UniProt-provided identity and distinguishes the yeast system from mammalian TIMM22 assemblies that contain Tim29 and AGK and lack Tim54/Tim18. (kumar2023functionalcrosstalkbetween pages 1-2, zhang2021structureofthe pages 1-2, qi2021cryoemstructureof pages 1-3)

Key concepts and definitions (current understanding)

2.1. What Tim22 is

Tim22 (gene: TIM22; locus: YDL217C) is the core, multi-pass inner mitochondrial membrane (IMM) subunit of the yeast TIM22 complex, which functions as the dedicated inner-membrane insertase/translocase for hydrophobic, multi-pass “carrier pathway” substrates. (kumar2023functionalcrosstalkbetween pages 1-2, zhang2021structureofthe pages 1-2)

2.2. The TIM22 pathway (carrier pathway)

In the “carrier pathway,” hydrophobic multi-pass precursors are first imported through the translocase of the outer membrane (TOM) and then escorted through the intermembrane space (IMS) by soluble/IMS small Tim chaperones (notably Tim9/Tim10, with Tim12 involved in docking), which deliver substrates to the TIM22 translocase for insertion into the IMM. (zhang2021structureofthe pages 1-2, kan2024roleofyme1 pages 3-4)

2.3. Canonical vs non-canonical TIM22 cargo

Canonical TIM22 cargos are metabolite carrier proteins with six transmembrane segments (e.g., Aac2/Pet9 ADP/ATP carrier; Pic/Pic2 phosphate carrier; Dic1 dicarboxylate carrier). (kumar2023functionalcrosstalkbetween pages 1-2, kumar2020conservedregionsof pages 12-15)

Non-canonical cargo spectrum has expanded beyond six-TM carriers. In yeast-focused discussions and experiments, the TIM22 pathway is described as also supporting insertion/biogenesis of some translocase subunits with four transmembrane segments (Tim17, Tim22, Tim23) and of “unconventional” clients such as mitochondrial pyruvate carrier subunits (MPCs; fewer TM helices) and proteins with odd numbers of transmembrane segments. (kumar2023functionalcrosstalkbetween pages 1-2, kumar2022functionalcrosstalkbetween pages 30-32)

Molecular composition, localization, and structural organization

3.1. Complex composition (yeast)

A cryo-EM structure of the endogenous S. cerevisiae TIM22 complex defines seven subunits: Tim22, Tim18, Tim54, Sdh3, Tim9, Tim10, and Tim12. (Cell Research; published Sep 2021; https://doi.org/10.1038/s41422-020-00399-0) (zhang2021structureofthe pages 1-2)

Genetic/biochemical work describes the TIM22 machinery as a ~300 kDa complex comprising a membrane module (Tim22, Tim54, Tim18, Sdh3) and a peripheral module consisting of small Tim chaperones (Tim9, Tim10, Tim12). (Journal of Cell Science; published Jul 2020; https://doi.org/10.1242/jcs.244632) (kumar2020conservedregionsof pages 1-4)

3.2. Subunit roles

Tim22 is described as the central core/channel subunit; Tim54 functions as an adaptor for docking small Tim chaperones; Tim18 and Sdh3 contribute to complex stability and assembly. (kumar2023functionalcrosstalkbetween pages 1-2, kumar2020conservedregionsof pages 1-4)

In the yeast cryo-EM model, Tim54 is membrane-anchored and appears positioned to help hold the small Tim ring in a tilted conformation, consistent with a role in organizing substrate delivery. (zhang2021structureofthe pages 2-3)

3.3. Localization and topology

Tim22 is a multi-pass IMM protein whose functional activity is in the inner membrane, with key interactions on the IMS side where small Tim chaperones deliver substrates from TOM. (zhang2021structureofthe pages 1-2)

In yeast TIM22 cryo-EM, Tim22 is a four-transmembrane (4-TM) subunit; Tim18 and Sdh3 each have three TMs; Tim54 is anchored by an N-terminal TM (residues 37–71). (zhang2021structureofthe pages 1-2)

Figure-based evidence for the arrangement of Tim22, Tim54, Tim18, Sdh3 and the small Tim hexameric ring is shown in the overall architecture of the yeast TIM22 complex (Figure 1). (zhang2021structureofthe media 0583fe89)

3.4. Structural understanding and mechanistic implications

Yeast TIM22 cryo-EM was solved at 3.8 Å resolution, revealing: (i) a hexameric small-Tim ring with a ~45° tilt; (ii) a Tim22 disulfide bond between C42 and C141; and (iii) a Tim22 transmembrane surface enriched in conserved charged residues implicated in function (E140, D190, K127, K169). (zhang2021structureofthe pages 1-2)

Notably, the yeast structure did not display a clear translocation pore in the captured state. The authors discuss that this may represent an idle/intermediate conformation, and they outline alternative mechanistic models (e.g., potential pore formation in other states and/or an insertase-like mechanism that lowers the energetic barrier for helix insertion by altering local bilayer properties). (zhang2021structureofthe pages 1-2, zhang2021structureofthe pages 2-3)

Primary function and pathway placement

4.1. Primary function

Tim22’s primary function is to act as the essential core element of the TIM22 carrier translocase responsible for the insertion/biogenesis of hydrophobic multi-pass inner-membrane proteins (especially metabolite carriers) into the mitochondrial inner membrane. Tim22 is not an enzyme and does not catalyze a chemical reaction; rather, it functions as an insertase/translocase channel component in protein import. (kumar2023functionalcrosstalkbetween pages 1-2, kumar2023functionalcrosstalkbetween pages 15-16)

4.2. Substrate specificity (functional annotation)

Based on yeast genetic/biochemical evidence, representative canonical substrates include six-TM metabolite carriers: Pic/Pic2, Aac2/Pet9, and Dic1. (kumar2023functionalcrosstalkbetween pages 1-2, kumar2020conservedregionsof pages 12-15)

Non-canonical substrates/cargo classes supported in the retrieved sources include:

• TIM22-pathway insertion/biogenesis of some translocase components with ~4 TM helices: Tim17, Tim22, and Tim23. (kumar2023functionalcrosstalkbetween pages 1-2, kumar2020conservedregionsof pages 1-4)

• Mitochondrial pyruvate carrier subunits (MPCs) as unconventional cargos (consistent with a broadened TIM22 client spectrum). (kumar2023functionalcrosstalkbetween pages 1-2, kumar2022functionalcrosstalkbetween pages 30-32)

4.3. Interacting partners and pathway integration

Within mitochondria, TIM22 pathway operation is integrated with:

• Small Tim chaperone system (Tim9/Tim10/Tim12; also Tim8/Tim13 in related contexts), which ferries substrates in the IMS and docks them to the membrane insertase. (kumar2023functionalcrosstalkbetween pages 1-2, kan2024roleofyme1 pages 3-4)

• Porin/VDAC (Por1), reported to interact with the TIM22 complex to promote carrier biogenesis. (kumar2020conservedregionsof pages 1-4)

• MICOS, reported in the context of TIM22-mediated carrier biogenesis (association of TIM22 with MICOS). (kumar2023functionalcrosstalkbetween pages 10-11)

• Respiratory chain component Sdh3, which is both a complex II membrane subunit and a TIM22 component (dual-role integration between metabolism/respiration and protein import). (kumar2023functionalcrosstalkbetween pages 1-2)

Recent developments and latest research (prioritizing 2023–2024)

5.1. TIM22–Yme1 proteostasis crosstalk (2023 primary literature)

A 2023 Journal of Cell Science study reported functional and genetic crosstalk between the TIM22 carrier translocase and the i-AAA protease Yme1 in S. cerevisiae. Key points:

• Yme1 is required for stability of the TIM22 complex and for proteostasis of TIM22 pathway substrates. (Journal of Cell Science; published Jan 2023; https://doi.org/10.1242/jcs.260060) (kumar2023functionalcrosstalkbetween pages 1-2)

• A small, non-stoichiometric pool of Yme1 associates transiently with TIM22 (Yme1 interacting with Tim18-FLAG), supporting a direct physical proximity between import machinery and quality control protease. (kumar2023functionalcrosstalkbetween pages 10-11)

• Loss of Yme1 leads to accumulation of TIM22 substrates outside mitochondria, altered mitochondrial morphology, increased mtDNA transfer, and compromised respiration; reducing TIM22 activity (e.g., deleting Tim18) reduced cargo accumulation and promoted survival in yme1Δ contexts. (kumar2023functionalcrosstalkbetween pages 10-11)

Interpretation/expert analysis: This body of work supports a modern view of Tim22 not only as a constitutive import factor but as a node in a regulated proteostasis network, where import capacity and inner-membrane quality control are functionally coupled and can be genetically tuned to mitigate mitochondrial stress caused by substrate overload. (kumar2023functionalcrosstalkbetween pages 10-11, kan2024roleofyme1 pages 3-4)

5.2. 2024 authoritative mini-review synthesis (Biochemical Society Transactions)

A 2024 Biochemical Society Transactions mini-review synthesizes recent evidence that Yme1 contributes to TIM22 pathway robustness, including degradation of misfolded/unassembled small Tim chaperones (notably differential susceptibility of Tim10 vs Tim9), and that loss of Yme1 reduces TIM22 stability and import efficiency especially under heat stress. (published Jun 2024; https://doi.org/10.1042/bst20240450) (kan2024roleofyme1 pages 3-4, kan2024roleofyme1 pages 2-3)

5.3. 2024 discovery of a mitochondrial mega-assembly containing multiple carrier substrates (MIMAS)

A 2024 Cell Reports paper identified MIMAS, a ~3 MDa inner-membrane mega-assembly comprising metabolite carriers (including Aac2 and others) and many respiratory chain biogenesis factors, suggesting that carrier proteins (TIM22 pathway products) can physically co-reside in large multifunctional assemblies with metabolic and respiratory biogenesis modules. (published Mar 2024; https://doi.org/10.1016/j.celrep.2024.113772) (horten2024identificationofmimas pages 3-5, horten2024identificationofmimas pages 1-3)

Important limitation: the extracted text does not explicitly state that Tim22/TIM22 is a structural component of MIMAS; it documents that several carrier proteins that are canonical TIM22 cargos (e.g., AAC/Aac2) are present and crosslink to other MIMAS components, but the mechanistic connection to TIM22-mediated insertion is inferential rather than directly demonstrated in the excerpts retrieved here. (horten2024identificationofmimas pages 5-6, horten2024identificationofmimas pages 3-5)

Current applications and real-world implementations

6.1. TIM22 modulation as a strategy to manage mitochondrial import stress (yeast model application)

In yeast, genetic modulation of TIM22 pathway activity (e.g., deleting the TIM22 accessory subunit Tim18) was implemented to relieve the accumulation burden of TIM22 substrates in Yme1-deficient cells, suppressing defects in mitochondrial morphology and respiration-associated phenotypes. This is a concrete experimental “implementation” of TIM22 pathway tuning as an intervention in organelle proteostasis. (kumar2023functionalcrosstalkbetween pages 10-11)

6.2. Mitochondrial import engineering for allotopic expression (2024 Genetics)

A 2024 Genetics study evaluated practical import/biogenesis-factor overexpression strategies to improve internalization of an allotopically expressed Cox2W56R precursor in yeast. It reported that overproduction of Cox20, Oxa1, and Pse1 facilitated Cox2W56R internalization and improved respiratory growth, whereas overproducing TIM22 components had only limited benefit and overproducing TIM23-related components was detrimental. (published Apr 2024; https://doi.org/10.1093/genetics/iyae058) (nietopanqueva2024identificationoffactors pages 1-2)

Implication: While TIM22 is central for carrier insertion, simply increasing TIM22 components may not generically improve import of engineered membrane proteins (such as allotopically expressed Cox2), emphasizing pathway/substrate specificity in mitochondrial engineering approaches. (nietopanqueva2024identificationoffactors pages 1-2)

6.3. Methods for monitoring inner-membrane insertion pathways (context)

Recent methodological work emphasizes assays adaptable to monitoring import into the inner membrane via TIM22 as part of mechanistic mitochondrial protein import studies (Methods in Enzymology; 2024; https://doi.org/10.1016/bs.mie.2024.07.033). (Note: detailed method performance metrics were not extracted in the current evidence set.) (kan2024roleofyme1 pages 3-4)

Quantitative/statistical highlights (recent studies)

7.1. TIM22 complex and chaperone module sizes

• Yeast TIM22 complex migrates at ~300 kDa by BN-PAGE (immunodecorated for Tim54 and Tim22). (kumar2023functionalcrosstalkbetween pages 7-7)

• Small Tim chaperone module Tim9/Tim10 is described as a soluble heterohexameric complex of ~70 kDa in the IMS. (kumar2020conservedregionsof pages 1-4)

7.2. Structural metrics

• Yeast TIM22 cryo-EM resolution: 3.8 Å. (zhang2021structureofthe pages 1-2)

• Small Tim ring orientation: ~45° tilt relative to the membrane. (zhang2021structureofthe pages 1-2)

• Tim54 membrane anchor: N-terminal TM spanning residues 37–71. (zhang2021structureofthe pages 1-2)

7.3. Functional residues and phenotype-linked data

• Tim22 disulfide: C42–C141 in yeast (between TM1 and TM2). (zhang2021structureofthe pages 1-2)

• Conserved charged residues implicated in function: E140, D190, K127, K169; single mutants (E140A or K127A) severely impair growth; combined mutants can be essentially lethal. (zhang2021structureofthe pages 1-2)

7.4. Recent quantitative experimental conditions from 2023 TIM22–Yme1 study

• Stress dependence: effects compared at 30°C vs 37°C, with pronounced destabilization of TIM22 complex at 37°C in yme1Δ background. (kumar2023functionalcrosstalkbetween pages 7-7)

• Replication and sampling: n=3 biological replicates; ≥50 cells scored for mitochondrial morphology analyses. (kumar2023functionalcrosstalkbetween pages 7-7, kumar2023functionalcrosstalkbetween pages 10-11)

• Additional parameters: cycloheximide (CYH) used at 100 ng/mL in stress/translation-modulation experiments; mtDNA staining at 10 µM SYTO18 for 15 min. (kumar2023functionalcrosstalkbetween pages 7-7)

7.5. MIMAS quantitative parameters (2024 Cell Reports)

• MIMAS complex size: ~3 MDa, resolving into peaks at ~3.3 and ~2.5 MDa (MIMAS-A and MIMAS-B). (horten2024identificationofmimas pages 3-5, horten2024identificationofmimas pages 6-8)

• Detergent and salt sensitivity: stable over digitonin:protein ratios 4:1 to 20:1; moderately destabilized at ≥150 mM NaCl; dissociated by dodecylmaltoside or Triton X-100. (horten2024identificationofmimas pages 5-6, horten2024identificationofmimas pages 3-5)

Expert opinions and authoritative analysis (synthesis across sources)

8.1. TIM22 as a regulated proteostasis node

The 2023 primary study and the 2024 mini-review converge on the interpretation that TIM22 pathway throughput and assembly are actively monitored by inner-membrane quality control (Yme1), with substrate overload and small-Tim chaperone stability being key stress-sensitive points; modulating TIM22 can alleviate proteostatic stress in specific genetic contexts (yme1Δ). (kumar2023functionalcrosstalkbetween pages 10-11, kan2024roleofyme1 pages 3-4)

8.2. Structural interpretation: pore vs insertase-like action

High-resolution structural work in yeast provides a framework for mechanistic inference: while Tim22 is a 4-TM core component, the captured endogenous complex lacked a clear open pore, supporting the view that TIM22 may operate through conformationally gated or transient translocation states and/or via an insertion mechanism that locally remodels bilayer energetics to facilitate helix insertion. (zhang2021structureofthe pages 1-2, zhang2021structureofthe pages 2-3)

Practical functional annotation summary (for databases/curation)

Gene/protein: TIM22 (YDL217C; UniProt Q12328)

Cellular component: mitochondrial inner membrane; TIM22 carrier translocase complex; IMS-facing interfaces with small Tim chaperones. (zhang2021structureofthe pages 1-2)

Molecular function: essential core subunit of the TIM22 inner-membrane insertase/translocase; mediates insertion of multi-pass hydrophobic proteins (especially metabolite carriers) into the IMM; participates in regulated proteostasis via functional linkage to Yme1. (kumar2023functionalcrosstalkbetween pages 15-16, kumar2023functionalcrosstalkbetween pages 10-11)

Biological process: mitochondrial protein import—carrier pathway; assembly/biogenesis of mitochondrial metabolite carriers; inner-membrane proteostasis coupling. (zhang2021structureofthe pages 1-2, kumar2023functionalcrosstalkbetween pages 10-11)

Canonical substrates (examples from yeast): Aac2/Pet9 (ADP/ATP carrier), Pic/Pic2 (phosphate carrier), Dic1 (dicarboxylate carrier). (kumar2023functionalcrosstalkbetween pages 1-2)

Noncanonical substrates (examples mentioned): Tim17/Tim22/Tim23 (selected 4-TM translocase subunits), mitochondrial pyruvate carrier subunits (MPC). (kumar2023functionalcrosstalkbetween pages 1-2)

Interacting partners/subunits: Tim54 (adaptor/docking), Tim18 & Sdh3 (stability/assembly), small Tim chaperones Tim9/Tim10/Tim12 (handoff/docking); additional associations reported with porin/VDAC and MICOS; transient association with Yme1 for proteostasis linkage. (kumar2020conservedregionsof pages 1-4, kumar2023functionalcrosstalkbetween pages 10-11)

Key references (with URLs and publication dates)

• Zhang Y. et al. “Structure of the mitochondrial TIM22 complex from yeast.” Cell Research. Published Sep 2021. https://doi.org/10.1038/s41422-020-00399-0 (zhang2021structureofthe pages 1-2)

• Kumar A. et al. “Conserved regions of budding yeast Tim22 have a role in structural organization of the carrier translocase.” Journal of Cell Science. Published Jul 2020. https://doi.org/10.1242/jcs.244632 (kumar2020conservedregionsof pages 1-4)

• Kumar A. et al. “Functional crosstalk between the TIM22 complex and YME1 machinery maintains mitochondrial proteostasis and integrity.” Journal of Cell Science. Published Jan 2023. https://doi.org/10.1242/jcs.260060 (kumar2023functionalcrosstalkbetween pages 10-11)

• Kan K.T. et al. “Role of Yme1 in mitochondrial protein homeostasis: from regulation of protein import, OXPHOS function to lipid synthesis and mitochondrial dynamics.” Biochemical Society Transactions. Published Jun 2024. https://doi.org/10.1042/bst20240450 (kan2024roleofyme1 pages 3-4)

• Horten P. et al. “Identification of MIMAS, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria.” Cell Reports. Published Mar 2024. https://doi.org/10.1016/j.celrep.2024.113772 (horten2024identificationofmimas pages 3-5)

• Nieto-Panqueva F. et al. “Identification of factors limiting the allotopic production of the Cox2 subunit of yeast cytochrome c oxidase.” Genetics. Published Apr 2024. https://doi.org/10.1093/genetics/iyae058 (nietopanqueva2024identificationoffactors pages 1-2)

Figure citation

• Overall architecture of the yeast TIM22 complex (structural context for Tim22/Tim54/Tim18/Sdh3 and small Tim hexamer) is shown in Figure 1 of Zhang et al. (2021). (zhang2021structureofthe media 0583fe89)

References

(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 8 citations and is from a domain leading peer-reviewed journal.
(zhang2021structureofthe pages 1-2): Yutong Zhang, Xiaomin Ou, Xuezheng Wang, Dongjie Sun, Xueyin Zhou, Xiaofei Wu, Qing Li, and Long Li. Structure of the mitochondrial tim22 complex from yeast. Cell Research, 31:366-368, Sep 2021. URL: https://doi.org/10.1038/s41422-020-00399-0, doi:10.1038/s41422-020-00399-0. This article has 53 citations and is from a domain leading peer-reviewed journal.
(qi2021cryoemstructureof pages 1-3): L. Qi, Qiang Wang, Z. Guan, Yan Wu, Cuicui Shen, Sixing Hong, Jianbo Cao, Xing Zhang, Chuangye Yan, and P. Yin. Cryo-em structure of the human mitochondrial translocase tim22 complex. Cell Research, 31:369-372, Dec 2021. URL: https://doi.org/10.1038/s41422-020-00400-w, doi:10.1038/s41422-020-00400-w. This article has 84 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 7 citations and is from a peer-reviewed journal.
(kumar2020conservedregionsof pages 12-15): 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.
(kumar2022functionalcrosstalkbetween pages 30-32): Abhishek Kumar and Patrick D’Silva. Functional crosstalk between the carrier translocase machinery and yme1 complex maintains mitochondrial proteostasis and integrity. bioRxiv, Feb 2022. URL: https://doi.org/10.1101/2022.02.03.478940, doi:10.1101/2022.02.03.478940. This article has 1 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.
(zhang2021structureofthe pages 2-3): Yutong Zhang, Xiaomin Ou, Xuezheng Wang, Dongjie Sun, Xueyin Zhou, Xiaofei Wu, Qing Li, and Long Li. Structure of the mitochondrial tim22 complex from yeast. Cell Research, 31:366-368, Sep 2021. URL: https://doi.org/10.1038/s41422-020-00399-0, doi:10.1038/s41422-020-00399-0. This article has 53 citations and is from a domain leading peer-reviewed journal.
(zhang2021structureofthe media 0583fe89): Yutong Zhang, Xiaomin Ou, Xuezheng Wang, Dongjie Sun, Xueyin Zhou, Xiaofei Wu, Qing Li, and Long Li. Structure of the mitochondrial tim22 complex from yeast. Cell Research, 31:366-368, Sep 2021. URL: https://doi.org/10.1038/s41422-020-00399-0, doi:10.1038/s41422-020-00399-0. This article has 53 citations and is from a domain leading peer-reviewed journal.
(kumar2023functionalcrosstalkbetween pages 15-16): 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 8 citations and is from a domain leading peer-reviewed journal.
(kumar2023functionalcrosstalkbetween pages 10-11): 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 8 citations and is from a domain leading peer-reviewed journal.
(kan2024roleofyme1 pages 2-3): 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 7 citations and is from a peer-reviewed journal.
(horten2024identificationofmimas pages 3-5): Patrick Horten, Kuo Song, Joshua Garlich, Robert Hardt, Lilia Colina-Tenorio, Susanne E. Horvath, Uwe Schulte, Bernd Fakler, Martin van der Laan, Thomas Becker, Rosemary A. Stuart, Nikolaus Pfanner, and Heike Rampelt. Identification of mimas, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria. Cell Reports, 43:113772, Mar 2024. URL: https://doi.org/10.1016/j.celrep.2024.113772, doi:10.1016/j.celrep.2024.113772. This article has 6 citations and is from a highest quality peer-reviewed journal.
(horten2024identificationofmimas pages 1-3): Patrick Horten, Kuo Song, Joshua Garlich, Robert Hardt, Lilia Colina-Tenorio, Susanne E. Horvath, Uwe Schulte, Bernd Fakler, Martin van der Laan, Thomas Becker, Rosemary A. Stuart, Nikolaus Pfanner, and Heike Rampelt. Identification of mimas, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria. Cell Reports, 43:113772, Mar 2024. URL: https://doi.org/10.1016/j.celrep.2024.113772, doi:10.1016/j.celrep.2024.113772. This article has 6 citations and is from a highest quality peer-reviewed journal.
(horten2024identificationofmimas pages 5-6): Patrick Horten, Kuo Song, Joshua Garlich, Robert Hardt, Lilia Colina-Tenorio, Susanne E. Horvath, Uwe Schulte, Bernd Fakler, Martin van der Laan, Thomas Becker, Rosemary A. Stuart, Nikolaus Pfanner, and Heike Rampelt. Identification of mimas, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria. Cell Reports, 43:113772, Mar 2024. URL: https://doi.org/10.1016/j.celrep.2024.113772, doi:10.1016/j.celrep.2024.113772. This article has 6 citations and is from a highest quality peer-reviewed journal.
(nietopanqueva2024identificationoffactors pages 1-2): Felipe Nieto-Panqueva, Miriam Vázquez-Acevedo, Patrice P Hamel, and Diego González-Halphen. Identification of factors limiting the allotopic production of the cox2 subunit of yeast cytochrome c oxidase. Genetics, Apr 2024. URL: https://doi.org/10.1093/genetics/iyae058, doi:10.1093/genetics/iyae058. This article has 2 citations and is from a domain leading peer-reviewed journal.
(kumar2023functionalcrosstalkbetween pages 7-7): 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 8 citations and is from a domain leading peer-reviewed journal.
(horten2024identificationofmimas pages 6-8): Patrick Horten, Kuo Song, Joshua Garlich, Robert Hardt, Lilia Colina-Tenorio, Susanne E. Horvath, Uwe Schulte, Bernd Fakler, Martin van der Laan, Thomas Becker, Rosemary A. Stuart, Nikolaus Pfanner, and Heike Rampelt. Identification of mimas, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria. Cell Reports, 43:113772, Mar 2024. URL: https://doi.org/10.1016/j.celrep.2024.113772, doi:10.1016/j.celrep.2024.113772. This article has 6 citations and is from a highest quality peer-reviewed journal.

Citations

zhang2021structureofthe pages 1-2
kumar2020conservedregionsof pages 1-4
zhang2021structureofthe pages 2-3
kumar2023functionalcrosstalkbetween pages 10-11
kumar2023functionalcrosstalkbetween pages 1-2
nietopanqueva2024identificationoffactors pages 1-2
kumar2023functionalcrosstalkbetween pages 7-7
horten2024identificationofmimas pages 3-5
qi2021cryoemstructureof pages 1-3
kumar2020conservedregionsof pages 12-15
kumar2022functionalcrosstalkbetween pages 30-32
kumar2023functionalcrosstalkbetween pages 15-16
horten2024identificationofmimas pages 1-3
horten2024identificationofmimas pages 5-6
horten2024identificationofmimas pages 6-8
https://doi.org/10.1038/s41422-020-00399-0
https://doi.org/10.1242/jcs.244632
https://doi.org/10.1242/jcs.260060
https://doi.org/10.1042/bst20240450
https://doi.org/10.1016/j.celrep.2024.113772
https://doi.org/10.1093/genetics/iyae058
https://doi.org/10.1016/bs.mie.2024.07.033
https://doi.org/10.1242/jcs.260060,
https://doi.org/10.1038/s41422-020-00399-0,
https://doi.org/10.1038/s41422-020-00400-w,
https://doi.org/10.1042/bst20240450,
https://doi.org/10.1242/jcs.244632,
https://doi.org/10.1101/2022.02.03.478940,
https://doi.org/10.1016/j.celrep.2024.113772,
https://doi.org/10.1093/genetics/iyae058,

📄 View Raw YAML

id: Q12328
gene_symbol: TIM22
product_type: PROTEIN
status: IN_PROGRESS
taxon:
  id: NCBITaxon:559292
  label: Saccharomyces cerevisiae
description: >-
  TIM22 is the essential core channel-forming subunit of the TIM22 complex in the
  mitochondrial inner membrane. It forms a voltage-activated, signal-gated twin-pore
  translocase that mediates the insertion of multi-pass transmembrane proteins into the
  inner membrane. Cryo-EM of the endogenous yeast TIM22 complex at 3.8 angstrom
  resolution (Zhang et al. 2021, doi:10.1038/s41422-020-00399-0) revealed a 7-subunit
  architecture (Tim22, Tim54, Tim18, Sdh3, Tim9, Tim10, Tim12) with Tim22 possessing 4
  transmembrane helices, a C42-C141 disulfide bond between TM1 and TM2, and conserved
  charged residues (E140, D190, K127, K169) critical for function -- single mutants
  such as E140A or K127A severely impair growth. The hexameric small-Tim chaperone ring
  (Tim9/Tim10/Tim12) sits tilted approximately 45 degrees relative to the membrane
  plane. Notably, the cryo-EM structure did not reveal a clear open translocation pore
  in the captured state, fueling debate about whether TIM22 operates via
  conformationally gated pore formation in distinct functional states or via an
  insertase-like mechanism that lowers the energetic barrier for helix insertion by
  altering local bilayer properties. Canonical TIM22 substrates are 6-TM metabolite
  carriers (e.g., Aac2/Pet9 ADP/ATP carrier, Pic/Pic2 phosphate carrier, Dic1
  dicarboxylate carrier), but the substrate spectrum extends to non-canonical cargos
  including 4-TM translocase subunits (Tim17, Tim22, Tim23) and mitochondrial pyruvate
  carrier (MPC) subunits with fewer transmembrane segments. TIM22 recognizes internal
  targeting signals (not N-terminal presequences) and uses the membrane potential
  (Delta-psi) as external driving force. It is distinct from the TIM23 complex, which
  imports presequence-containing matrix proteins. Recent work has revealed functional
  crosstalk between the TIM22 complex and the Yme1 i-AAA protease quality control
  machinery: Yme1 is required for stability of the TIM22 complex and for proteostasis
  of TIM22 pathway substrates, with a small transient pool of Yme1 associating with
  TIM22 (Kumar et al. 2023, doi:10.1242/jcs.260060). Loss of Yme1 leads to
  accumulation of TIM22 substrates outside mitochondria and compromised respiration.
  Substrate delivery to the TIM22 complex is performed by the small TIM chaperones
  (Tim9-Tim10, Tim8-Tim13) in the intermembrane space.
existing_annotations:
- term:
    id: GO:0030943
    label: mitochondrion targeting sequence binding
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      IBA annotation for mitochondrion targeting sequence binding. TIM22 does bind
      targeting signals, but specifically internal targeting signals of carrier
      proteins, not the classical N-terminal matrix targeting presequences. The term
      GO:0030943 is defined broadly as "Binding to a mitochondrion targeting sequence,
      a specific peptide sequence that acts as a signal to localize the protein within
      the mitochondrion" -- this is broad enough to encompass internal targeting
      signals. Kovermann et al. (PMID:11864609) demonstrated that reconstituted Tim22
      forms a channel that "specifically responds to an internal targeting signal, but
      not to presequences." The IBA annotation is phylogenetically sound and the term
      is acceptable, though imprecise about the type of targeting signal recognized.
    action: ACCEPT
    reason: >-
      GO:0030943 is defined broadly enough to cover internal targeting signal binding.
      TIM22 has been experimentally shown to recognize internal targeting signals of
      carrier proteins (PMID:11864609). While a more specific term for "internal
      mitochondrial targeting signal binding" would be ideal, GO:0030943 is the best
      available term and the IBA annotation is phylogenetically correct.
    supported_by:
    - reference_id: PMID:11864609
      supporting_text: >-
        The channel is voltage-activated and specifically responds to an internal
        targeting signal, but not to presequences
- term:
    id: GO:0042721
    label: TIM22 mitochondrial import inner membrane insertion complex
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      IBA annotation for TIM22 complex membership. TIM22 is the essential core
      channel subunit of the TIM22 complex. Multiple studies confirm this: Kerscher
      et al. (PMID:10637294) and Koehler et al. (PMID:10648604) identified TIM22 as
      a core integral membrane subunit, and Rehling et al. (PMID:12637749) showed it
      forms the twin-pore translocase. UniProt describes it as "Essential core
      component of the TIM22 complex."
    action: ACCEPT
    reason: >-
      TIM22 is the defining, essential core subunit of the TIM22 complex. This is one
      of the most well-established annotations for this gene, supported by extensive
      experimental evidence (PMID:10637294, PMID:10648604, PMID:12637749) and
      phylogenetically sound.
    supported_by:
    - reference_id: PMID:10637294
      supporting_text: >-
        Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial
        inner membrane
    - 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:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      IBA annotation for protein insertion into mitochondrial inner membrane. This is
      the core biological process of TIM22. Sirrenberg et al. (PMID:8955274) first
      showed that "Tim22 is required for the import of proteins of the mitochondrial
      ADP/ATP carrier (AAC) family into the inner membrane." Kovermann et al.
      (PMID:11864609) demonstrated TIM22 forms the channel, and Rehling et al.
      (PMID:12637749) showed the twin-pore translocase mediates insertion.
    action: ACCEPT
    reason: >-
      This is the primary biological process of TIM22. Extensively supported by
      experimental evidence from multiple groups (PMID:8955274, PMID:11864609,
      PMID:12637749, PMID:32591483). The IBA annotation is phylogenetically sound.
    supported_by:
    - reference_id: PMID:8955274
      supporting_text: >-
        Tim22 is required for the import of proteins of the mitochondrial ADP/ATP
        carrier (AAC) family into the inner membrane
    - reference_id: PMID:11864609
      supporting_text: >-
        The protein insertion complex of the mitochondrial inner membrane is crucial
        for import of the numerous multitopic membrane proteins with internal targeting
        signals
- term:
    id: GO:0008320
    label: protein transmembrane transporter activity
  evidence_type: IBA
  original_reference_id: GO_REF:0000033
  review:
    summary: >-
      IBA annotation for protein transmembrane transporter activity. GO:0008320 is
      defined as "Enables the transfer of a protein from one side of a membrane to
      the other." TIM22 does transfer proteins across/into the inner membrane. However,
      it is more precise to say TIM22 mediates insertion of polytopic membrane proteins
      INTO the membrane rather than transfer across it. The term is acceptable as the
      best available MF term for TIM22's core activity -- it forms a channel that
      enables protein translocation through the membrane. The IBA is phylogenetically
      sound.
    action: ACCEPT
    reason: >-
      GO:0008320 is the best available MF term for the protein translocation channel
      activity of TIM22. Kovermann et al. (PMID:11864609) demonstrated that
      reconstituted TIM22 forms a "hydrophilic, high-conductance channel with distinct
      opening states and pore diameters" that mediates protein transfer. The IBA is
      phylogenetically correct.
    supported_by:
    - reference_id: PMID:11864609
      supporting_text: >-
        Reconstituted Tim22 forms a hydrophilic, high-conductance channel with
        distinct opening states and pore diameters
- term:
    id: GO:0071806
    label: protein transmembrane transport
  evidence_type: IEA
  original_reference_id: GO_REF:0000108
  review:
    summary: >-
      IEA annotation inferred from GO:0008320 (protein transmembrane transporter
      activity) via logical inference. Since TIM22 enables protein transmembrane
      transporter activity, it follows that it is involved in protein transmembrane
      transport. This is a reasonable computational inference.
    action: ACCEPT
    reason: >-
      Logically inferred from the IBA/IDA-supported GO:0008320 annotation. TIM22
      mediates protein transmembrane transport (specifically insertion of carrier
      proteins into the inner membrane). The inference is sound, and this BP term is
      broader but not incorrect.
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  review:
    summary: >-
      IEA annotation from UniProt subcellular location mapping. TIM22 is an integral
      multi-pass protein of the mitochondrial inner membrane. This is abundantly
      supported by experimental evidence (PMID:8955274, PMID:10397776, PMID:10648604,
      PMID:32591483).
    action: ACCEPT
    reason: >-
      Correct localization. TIM22 is an integral inner membrane protein with multiple
      transmembrane helices. Redundant with experimental IDA annotations but not
      incorrect.
- term:
    id: GO:0015031
    label: protein transport
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: >-
      IEA annotation from UniProt keyword mapping (Protein transport, Translocation).
      GO:0015031 "protein transport" is a broad parent term. TIM22 is involved in
      protein transport, specifically insertion of carrier proteins into the inner
      membrane (GO:0045039). This IEA is not wrong but is very general.
    action: ACCEPT
    reason: >-
      Correct but general. GO:0045039 (protein insertion into mitochondrial inner
      membrane) is the more specific and informative term, which is already annotated
      via IBA, IDA, and IMP. This IEA annotation to the parent term is redundant but
      acceptable.
- term:
    id: GO:0042721
    label: TIM22 mitochondrial import inner membrane insertion complex
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: >-
      IEA annotation from InterPro mapping (IPR039175 TIM22). TIM22 is the core
      subunit of this complex. Redundant with IBA and IDA annotations for the same
      term.
    action: ACCEPT
    reason: >-
      Correct. InterPro domain IPR039175 is specific to TIM22 family members.
      Redundant with experimental annotations but not incorrect.
- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  review:
    summary: >-
      IEA annotation from InterPro mapping (IPR039175 TIM22). Redundant with IBA, IDA,
      and IMP annotations for the same term.
    action: ACCEPT
    reason: >-
      Correct. Redundant with experimental annotations but the InterPro mapping is
      sound.
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:17099692
  review:
    summary: >-
      IPI annotation for protein binding based on interaction with Tim21 (P53220),
      from Albrecht et al. (PMID:17099692). That paper studied the Tim21 binding
      domain and its connections between TOM and TIM complexes. The interaction
      between TIM22 and TIM21 is documented in IntAct. However, GO:0005515 "protein
      binding" is uninformative -- it does not tell us what the interaction means
      functionally.
    action: REMOVE
    reason: >-
      GO:0005515 "protein binding" is a vague term that provides no functional
      information. Per curation guidelines, this term should be avoided. The
      interaction with Tim21 is better captured by the CC annotation to the TIM22
      complex (GO:0042721). Furthermore, Albrecht et al. (PMID:17099692) focused on
      Tim21's role in connecting TOM and TIM23 complexes; the Tim22-Tim21 interaction
      is secondary and does not inform TIM22's core function.
    supported_by:
    - reference_id: PMID:17099692
      supporting_text: >-
        Tim21, a subunit of the presequence translocase consisting of a membrane
        anchor and a carboxy-terminal domain exposed to the intermembrane space,
        directly connects the TOM and TIM23 complexes by binding to the intermembrane
        space domain of the Tom22 receptor
- 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 from Koehler et al.
      (PMID:10648604). That study characterized the TIM22 complex and showed Tim22p
      is an integral membrane subunit of the inner membrane-localized 300-kDa complex.
    action: ACCEPT
    reason: >-
      Experimentally demonstrated. TIM22 is an integral inner membrane protein,
      confirmed by co-migration with the 300-kDa TIM22 complex on nondenaturing gels
      (PMID:10648604) and by topology studies (UniProt).
    supported_by:
    - reference_id: PMID:10648604
      supporting_text: >-
        a 300-kDa complex in the inner membrane, the TIM22 complex
- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IDA
  original_reference_id: PMID:10648604
  review:
    summary: >-
      IDA annotation from Koehler et al. (PMID:10648604). This paper identified
      Tim18p as a new subunit of the TIM22 complex "that mediates insertion of
      imported proteins into the yeast mitochondrial inner membrane." Tim22p was
      established as the core channel subunit in this complex.
    action: ACCEPT
    reason: >-
      Direct experimental evidence that the TIM22 complex containing Tim22p mediates
      protein insertion into the inner membrane (PMID:10648604).
    supported_by:
    - reference_id: PMID:10648604
      supporting_text: >-
        Import of carrier proteins from the cytoplasm into the mitochondrial inner
        membrane of yeast is mediated by a distinct system consisting of two soluble
        70-kDa protein complexes in the intermembrane space and a 300-kDa complex in
        the inner membrane, the TIM22 complex
- term:
    id: GO:0005198
    label: structural molecule activity
  evidence_type: IDA
  original_reference_id: PMID:32591483
  review:
    summary: >-
      IDA annotation for structural molecule activity from Kumar et al. (PMID:32591483).
      That study showed that conserved regions of Tim22 (IMS domain and TM4) are
      "critically required for interactions with the membrane-embedded subunits,
      including Tim54, Tim18, and Sdh3, and thereby maintain the functional
      architecture of the TIM22 translocase." Impairment of TIM22 complex assembly
      influenced translocase activity and cell viability. The structural role is
      real -- Tim22 is the scaffold around which the complex assembles.
    action: ACCEPT
    reason: >-
      GO:0005198 "structural molecule activity" is defined as "The action of a
      molecule that contributes to the structural integrity of a complex." Kumar et al.
      (PMID:32591483) demonstrated that Tim22 regions are critical for maintaining the
      architecture and structural integrity of the TIM22 complex by mediating
      interactions with Tim54, Tim18, and Sdh3. This is a legitimate annotation --
      Tim22 is both the channel and the structural core around which the complex
      assembles.
    supported_by:
    - reference_id: PMID:32591483
      supporting_text: >-
        the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are
        critically required for interactions with the membrane-embedded subunits,
        including Tim54, Tim18, and Sdh3, and thereby maintain the functional
        architecture of the TIM22 translocase
- term:
    id: GO:0005198
    label: structural molecule activity
  evidence_type: IMP
  original_reference_id: PMID:32591483
  review:
    summary: >-
      IMP annotation for structural molecule activity from the same study (PMID:32591483).
      Mutations in conserved TIM22 regions disrupted complex assembly, demonstrating
      the structural role via mutant phenotype.
    action: ACCEPT
    reason: >-
      Mutant analysis in Kumar et al. (PMID:32591483) showed that impairment of TIM22
      regions disrupts complex assembly and stability, confirming the structural role
      by IMP evidence. Complements the IDA annotation above.
    supported_by:
    - reference_id: PMID:32591483
      supporting_text: >-
        impairment of TIM22 complex assembly influences its translocase activity, the
        mitochondrial network, and the viability of cells lacking mitochondrial DNA
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: IDA
  original_reference_id: PMID:32591483
  review:
    summary: >-
      IDA annotation for inner membrane localization from Kumar et al. (PMID:32591483).
      That study confirmed Tim22 is active in the inner membrane, as expected for the
      core channel subunit. The GOA qualifier is "is_active_in."
    action: ACCEPT
    reason: >-
      Tim22 is an integral multi-pass protein of the mitochondrial inner membrane,
      confirmed by multiple studies. This IDA is consistent with all other evidence.
    supported_by:
    - reference_id: PMID:32591483
      supporting_text: >-
        the TIM22 complex enables the integration of complex membrane proteins with
        internal targeting signals into the inner membrane
- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IDA
  original_reference_id: PMID:32591483
  review:
    summary: >-
      IDA annotation for protein insertion from Kumar et al. (PMID:32591483). Direct
      assays showed Tim22 is critically required for the translocase activity of the
      TIM22 complex.
    action: ACCEPT
    reason: >-
      Direct experimental evidence from Kumar et al. (PMID:32591483). Tim22 is the
      core channel; its conserved regions are required for translocase activity.
    supported_by:
    - reference_id: PMID:32591483
      supporting_text: >-
        the TIM22 complex enables the integration of complex membrane proteins with
        internal targeting signals into the inner membrane
- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IMP
  original_reference_id: PMID:32591483
  review:
    summary: >-
      IMP annotation for protein insertion from Kumar et al. (PMID:32591483). Mutant
      analysis of conserved Tim22 regions showed impaired translocase activity.
    action: ACCEPT
    reason: >-
      Mutant phenotype evidence from Kumar et al. (PMID:32591483). Disruption of
      conserved Tim22 regions impaired protein insertion activity of the TIM22
      complex.
    supported_by:
    - reference_id: PMID:32591483
      supporting_text: >-
        impairment of TIM22 complex assembly influences its translocase activity
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: HDA
  original_reference_id: PMID:24769239
  review:
    summary: >-
      HDA annotation for mitochondrion localization from Renvoise et al.
      (PMID:24769239), a quantitative mitochondrial proteomics study. TIM22 was
      identified as a mitochondrial protein by high-throughput mass spectrometry.
    action: ACCEPT
    reason: >-
      Correct. TIM22 is a mitochondrial protein. The HDA evidence from proteomics
      (PMID:24769239) is a broader CC term than the more specific "mitochondrial
      inner membrane" (GO:0005743) but is not incorrect.
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: HDA
  original_reference_id: PMID:16823961
  review:
    summary: >-
      HDA annotation for mitochondrion localization from Reinders et al.
      (PMID:16823961), a comprehensive yeast mitochondrial proteome analysis using
      multidimensional separation techniques.
    action: ACCEPT
    reason: >-
      Correct. TIM22 was identified in the yeast mitochondrial proteome
      (PMID:16823961). Redundant with other CC annotations but acceptable.
- term:
    id: GO:0008320
    label: protein transmembrane transporter activity
  evidence_type: IDA
  original_reference_id: PMID:11864609
  review:
    summary: >-
      IDA annotation for protein transmembrane transporter activity from Kovermann
      et al. (PMID:11864609). This landmark paper demonstrated that reconstituted
      Tim22 forms a "hydrophilic, high-conductance channel with distinct opening
      states and pore diameters" that is "voltage-activated and specifically responds
      to an internal targeting signal." The paper showed Tim22 is "the only essential
      membrane-integrated subunit of the complex" and that "a protein insertion
      complex can combine three essential functions, signal recognition, channel
      formation, and energy transduction, in one central component."
    action: ACCEPT
    reason: >-
      Strong experimental evidence. Kovermann et al. (PMID:11864609) performed
      reconstitution and electrophysiology experiments demonstrating that Tim22 alone
      forms a functional protein-translocating channel. This is the definitive
      evidence for TIM22's MF.
    supported_by:
    - reference_id: PMID:11864609
      supporting_text: >-
        Reconstituted Tim22 forms a hydrophilic, high-conductance channel with
        distinct opening states and pore diameters. The channel is voltage-activated
        and specifically responds to an internal targeting signal, but not to
        presequences
    - reference_id: PMID:11864609
      supporting_text: >-
        a protein insertion complex can combine three essential functions, signal
        recognition, channel formation, and energy transduction, in one central
        component
- term:
    id: GO:0030943
    label: mitochondrion targeting sequence binding
  evidence_type: IDA
  original_reference_id: PMID:11864609
  review:
    summary: >-
      IDA annotation for mitochondrion targeting sequence binding from Kovermann et al.
      (PMID:11864609). The study showed that the Tim22 channel "specifically responds
      to an internal targeting signal, but not to presequences." This demonstrates
      signal-gated channel activity where TIM22 recognizes internal targeting signals
      of carrier proteins.
    action: ACCEPT
    reason: >-
      Experimental evidence from electrophysiology showing signal-gated response to
      internal targeting signals (PMID:11864609). GO:0030943 is the best available
      term, though it does not distinguish internal signals from presequences. TIM22
      specifically binds internal signals, not N-terminal presequences.
    supported_by:
    - reference_id: PMID:11864609
      supporting_text: >-
        The channel is voltage-activated and specifically responds to an internal
        targeting signal, but not to presequences
- term:
    id: GO:0042721
    label: TIM22 mitochondrial import inner membrane insertion complex
  evidence_type: IDA
  original_reference_id: PMID:10637294
  review:
    summary: >-
      IDA annotation from Kerscher et al. (PMID:10637294). This study identified
      TIM18 as a new component of the Tim54p-Tim22p translocon and demonstrated that
      Tim22p is part of the approximately 300-kDa complex by blue native
      electrophoresis and co-immunoprecipitation.
    action: ACCEPT
    reason: >-
      Direct biochemical evidence (co-IP, BN-PAGE) demonstrating Tim22p as a core
      component of the TIM22 complex (PMID:10637294).
    supported_by:
    - reference_id: PMID:10637294
      supporting_text: >-
        Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and
        Tim18p, along with Tim54p and Tim22p, is detected in an approximately 300-kDa
        complex after blue native electrophoresis
- term:
    id: GO:0042721
    label: TIM22 mitochondrial import inner membrane insertion complex
  evidence_type: IDA
  original_reference_id: PMID:10648604
  review:
    summary: >-
      IDA annotation from Koehler et al. (PMID:10648604). The study characterized the
      TIM22 complex composition, confirming Tim22p as a core integral membrane subunit
      alongside Tim54p, with peripheral subunits Tim9p, Tim10p, and Tim12p.
    action: ACCEPT
    reason: >-
      Biochemical characterization of the TIM22 complex demonstrating Tim22p as a
      core subunit (PMID:10648604).
    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:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IMP
  original_reference_id: PMID:10648604
  review:
    summary: >-
      IMP annotation from Koehler et al. (PMID:10648604). Deletion of Tim18p (a
      complex partner) impaired import of precursor proteins, demonstrating the role
      of the TIM22 complex in protein insertion by mutant phenotype.
    action: ACCEPT
    reason: >-
      Mutant phenotype evidence supporting the role of the TIM22 complex in protein
      insertion (PMID:10648604). Although the mutation was in Tim18p rather than
      Tim22p directly, it demonstrates the functional requirement of the complex.
    supported_by:
    - reference_id: PMID:10648604
      supporting_text: >-
        It also impairs the import of several precursor proteins into isolated
        mitochondria, and lowers the apparent mass of the TIM22 complex
- term:
    id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  evidence_type: IMP
  original_reference_id: PMID:8955274
  review:
    summary: >-
      IMP annotation from the founding paper by Sirrenberg et al. (PMID:8955274).
      This study first identified Tim22 as required for import of ADP/ATP carrier
      family members into the inner membrane, and showed that Tim22 is in an assembly
      distinct from the Tim23-Tim17 complex.
    action: ACCEPT
    reason: >-
      Foundational experimental evidence. Sirrenberg et al. (PMID:8955274) first
      demonstrated that "Tim22 is required for the import of proteins of the
      mitochondrial ADP/ATP carrier (AAC) family into the inner membrane" and that
      "Import of proteins of the AAC family is independent of Tim23."
    supported_by:
    - reference_id: PMID:8955274
      supporting_text: >-
        Tim22 is required for the import of proteins of the mitochondrial ADP/ATP
        carrier (AAC) family into the inner membrane. Members of the yeast AAC family
        are synthesized without matrix targeting signals
    - reference_id: PMID:8955274
      supporting_text: >-
        Import of proteins of the AAC family is independent of Tim23, and import of
        matrix targeting signals containing preproteins is independent of Tim22
- term:
    id: GO:0005743
    label: mitochondrial inner membrane
  evidence_type: TAS
  original_reference_id: Reactome:R-SCE-1252258
  review:
    summary: >-
      TAS annotation from Reactome pathway R-SCE-1252258 "TIM22 complex inserts
      proteins into inner membrane." TIM22 is indeed located in the inner membrane.
    action: ACCEPT
    reason: >-
      Correct localization. Reactome pathway annotation is consistent with all
      experimental evidence.
- term:
    id: GO:0005758
    label: mitochondrial intermembrane space
  evidence_type: TAS
  original_reference_id: Reactome:R-SCE-1252255
  review:
    summary: >-
      TAS annotation from Reactome pathway R-SCE-1252255 "TOM40:TOM70 complex
      translocates proteins from the cytosol to the mitochondrial intermembrane space."
      This Reactome pathway describes the TOM-mediated import step, not TIM22 function
      per se. TIM22 does have domains exposed to the IMS (the N-terminal and
      C-terminal regions per UniProt topology), so there is some basis for IMS
      localization, but the primary localization is the inner membrane itself. This
      appears to be a Reactome annotation associating TIM22 with a pathway step where
      it is a participant rather than indicating its primary localization.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      TIM22 is an integral inner membrane protein, not an IMS protein. While it has
      IMS-exposed domains (N-terminal and C-terminal regions per UniProt topology),
      its primary localization is the inner membrane (GO:0005743), which is already
      well-annotated. The Reactome pathway R-SCE-1252255 describes TOM-mediated
      translocation to the IMS, not TIM22's localization. Annotating TIM22 to the IMS
      is misleading.
- term:
    id: GO:0005758
    label: mitochondrial intermembrane space
  evidence_type: TAS
  original_reference_id: Reactome:R-SCE-1252258
  review:
    summary: >-
      TAS annotation from Reactome pathway R-SCE-1252258 "TIM22 complex inserts
      proteins into inner membrane." Same concern as the other IMS annotation -- TIM22
      is an integral inner membrane protein with IMS-exposed domains, but its primary
      CC is the inner membrane.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      TIM22 is an integral inner membrane protein. While the TIM22 complex operates
      at the interface between the IMS and the inner membrane, and Tim22 has
      IMS-exposed domains, annotating it to GO:0005758 (mitochondrial intermembrane
      space) suggests it is a soluble IMS protein, which is incorrect. The inner
      membrane annotation (GO:0005743) is the correct and sufficient CC term.
- 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 R-SCE-1252259 "TIM9:TIM10 binds
      hydrophobic proteins." This pathway describes the action of the small TIM
      chaperones (Tim9-Tim10) in the IMS, not TIM22 itself. TIM22 is listed as a
      participant in the broader mitochondrial import pathway but its primary
      localization is the inner membrane.
    action: MARK_AS_OVER_ANNOTATED
    reason: >-
      Same rationale as other IMS annotations. TIM22 is an inner membrane protein,
      not an IMS protein. This Reactome pathway (R-SCE-1252259) describes TIM9:TIM10
      function in the IMS, and TIM22 is merely a downstream partner.
- term:
    id: GO:0005829
    label: cytosol
  evidence_type: TAS
  original_reference_id: Reactome:R-SCE-1252255
  review:
    summary: >-
      TAS annotation from Reactome pathway R-SCE-1252255. This likely reflects
      that TIM22 precursor protein passes through the cytosol during its own import.
      However, mature TIM22 is an integral inner membrane protein and is not a
      cytosolic protein.
    action: REMOVE
    reason: >-
      TIM22 is not a cytosolic protein. It is an integral multi-pass protein of the
      mitochondrial inner membrane. While the TIM22 precursor transiently exists in
      the cytosol during its own biogenesis (like all nuclear-encoded mitochondrial
      proteins), the mature protein resides exclusively in the inner membrane. The
      Reactome pathway R-SCE-1252255 describes TOM-mediated import from the cytosol,
      but this does not make TIM22 a cytosolic protein.
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: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:0000108
  title: >-
    Automatic assignment of GO terms using logical inference, based on on
    inter-ontology links
  findings: []
- id: PMID:8955274
  title: >-
    Import of carrier proteins into the mitochondrial inner membrane mediated by
    Tim22.
  findings:
  - statement: >-
      First identification of Tim22 as required for import of ADP/ATP carrier family
      members into the mitochondrial inner membrane.
    supporting_text: >-
      Tim22 is required for the import of proteins of the mitochondrial ADP/ATP
      carrier (AAC) family into the inner membrane
  - statement: >-
      Tim22 is in a complex distinct from the Tim23-Tim17 complex, and import of
      carrier proteins is independent of Tim23.
    supporting_text: >-
      Import of proteins of the AAC family is independent of Tim23, and import of
      matrix targeting signals containing preproteins is independent of Tim22
- id: PMID:10637294
  title: >-
    Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial
    inner membrane.
  findings:
  - statement: >-
      Tim18p identified as a new subunit of the Tim54p-Tim22p complex by genetic
      suppression and biochemical co-purification.
    supporting_text: >-
      Tim54p and Tim22p can be coimmune precipitated with the Tim18 protein; and
      Tim18p, along with Tim54p and Tim22p, is detected in an approximately 300-kDa
      complex after blue native electrophoresis
- 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: >-
      The TIM22 complex contains integral membrane subunits Tim22p and Tim54p, plus
      peripheral subunits Tim9p, Tim10p, and Tim12p.
    supporting_text: >-
      The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p
      and the integral membrane subunits Tim22p and Tim54p
  - statement: >-
      Tim18p functions in assembly and stabilization of the TIM22 complex but does not
      directly participate in protein insertion.
    supporting_text: >-
      Tim18p functions in the assembly and stabilization of the TIM22 complex but
      does not directly participate in protein insertion into the inner membrane
- id: PMID:11864609
  title: >-
    Tim22, the essential core of the mitochondrial protein insertion complex, forms
    a voltage-activated and signal-gated channel.
  findings:
  - statement: >-
      Tim22 is the only essential membrane-integrated subunit of the protein insertion
      complex and forms a channel when reconstituted.
    supporting_text: >-
      Tim22 is the only essential membrane-integrated subunit of the complex.
      Reconstituted Tim22 forms a hydrophilic, high-conductance channel with
      distinct opening states and pore diameters
  - statement: >-
      The Tim22 channel is voltage-activated and specifically gated by internal
      targeting signals, not by N-terminal presequences.
    supporting_text: >-
      The channel is voltage-activated and specifically responds to an internal
      targeting signal, but not to presequences
  - statement: >-
      Tim22 combines signal recognition, channel formation, and energy transduction
      in one component.
    supporting_text: >-
      a protein insertion complex can combine three essential functions, signal
      recognition, channel formation, and energy transduction, in one central
      component
- id: PMID:12637749
  title: >-
    Protein insertion into the mitochondrial inner membrane by a twin-pore
    translocase.
  findings:
  - statement: >-
      The TIM22 complex forms a twin-pore translocase that uses membrane potential in
      two voltage-dependent steps for protein insertion.
- id: PMID:16823961
  title: >-
    Toward the complete yeast mitochondrial proteome: multidimensional separation
    techniques for mitochondrial proteomics.
  findings:
  - statement: >-
      TIM22 identified in the yeast mitochondrial proteome by multidimensional
      LC-MS/MS analysis.
- id: PMID:17099692
  title: >-
    The Tim21 binding domain connects the preprotein translocases of both
    mitochondrial membranes.
  findings:
  - statement: >-
      Tim21 connects the TOM and TIM23 complexes. TIM22-Tim21 interaction detected
      by IntAct.
    supporting_text: >-
      Tim21, a subunit of the presequence translocase consisting of a membrane
      anchor and a carboxy-terminal domain exposed to the intermembrane space,
      directly connects the TOM and TIM23 complexes
- id: PMID:24769239
  title: >-
    Quantitative variations of the mitochondrial proteome and phosphoproteome during
    fermentative and respiratory growth in Saccharomyces cerevisiae.
  findings:
  - statement: >-
      TIM22 detected in mitochondrial proteome across fermentative and respiratory
      conditions.
- id: PMID:32591483
  title: >-
    Conserved regions of budding yeast Tim22 have a role in structural organization
    of the carrier translocase.
  findings:
  - statement: >-
      The IMS and TM4 regions of Tim22 are critically required for interactions with
      Tim54, Tim18, and Sdh3, maintaining the functional architecture of the TIM22
      translocase.
    supporting_text: >-
      the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are
      critically required for interactions with the membrane-embedded subunits,
      including Tim54, Tim18, and Sdh3, and thereby maintain the functional
      architecture of the TIM22 translocase
  - statement: >-
      TM1 and TM2 regions mediate Tim18 interaction; TM3 is required for Sdh3
      interaction.
    supporting_text: >-
      the TM1 and TM2 regions of Tim22 are important for association with Tim18,
      whereas TM3 is exclusively required for the interaction with Sdh3
  - statement: >-
      Impairment of TIM22 complex assembly affects translocase activity, mitochondrial
      network integrity, and viability.
    supporting_text: >-
      impairment of TIM22 complex assembly influences its translocase activity, the
      mitochondrial network, and the viability of cells lacking mitochondrial DNA
- id: DOI:10.1038/s41422-020-00399-0
  title: >-
    Structure of the mitochondrial TIM22 complex from yeast
  findings:
  - statement: >-
      Cryo-EM structure of the endogenous yeast TIM22 complex at 3.8 angstrom
      resolution reveals a 7-subunit architecture with Tim22 possessing 4
      transmembrane helices, a C42-C141 disulfide bond, and conserved charged
      residues (E140, D190, K127, K169) critical for function.
  - statement: >-
      The hexameric small-Tim ring (Tim9/Tim10/Tim12) is tilted approximately 45
      degrees relative to the membrane plane.
  - statement: >-
      The captured cryo-EM state does not display a clear open translocation pore,
      suggesting Tim22 may operate via conformationally gated states or an
      insertase-like mechanism that alters local bilayer properties.
- id: DOI:10.1242/jcs.260060
  title: >-
    Functional crosstalk between the TIM22 complex and YME1 machinery maintains
    mitochondrial proteostasis and integrity
  findings:
  - statement: >-
      Yme1 i-AAA protease is required for stability of the TIM22 complex and for
      proteostasis of TIM22 pathway substrates; a small non-stoichiometric pool of
      Yme1 associates transiently with TIM22.
  - statement: >-
      Loss of Yme1 leads to accumulation of TIM22 substrates outside mitochondria,
      altered mitochondrial morphology, and compromised respiration; reducing TIM22
      activity (e.g., deleting Tim18) reduced cargo accumulation in yme1-delta
      contexts.
- id: DOI:10.1042/bst20240450
  title: >-
    Role of Yme1 in mitochondrial protein homeostasis: from regulation of protein
    import, OXPHOS function to lipid synthesis and mitochondrial dynamics
  findings:
  - statement: >-
      Mini-review synthesizing evidence that Yme1 contributes to TIM22 pathway
      robustness, including degradation of misfolded small Tim chaperones with
      differential susceptibility of Tim10 vs Tim9, and that loss of Yme1 reduces
      TIM22 stability and import efficiency especially under heat stress.
- id: DOI:10.1016/j.celrep.2024.113772
  title: >-
    Identification of MIMAS, a multifunctional mega-assembly integrating metabolic
    and respiratory biogenesis factors of mitochondria
  findings:
  - statement: >-
      MIMAS is a ~3 MDa inner-membrane mega-assembly comprising metabolite carriers
      (including Aac2) and respiratory chain biogenesis factors, demonstrating that
      canonical TIM22 cargo proteins physically co-reside in large multifunctional
      assemblies.
- id: Reactome:R-SCE-1252255
  title: >-
    TOM40:TOM70 complex translocates proteins from the cytosol to the mitochondrial
    intermembrane space
  findings: []
- id: Reactome:R-SCE-1252258
  title: TIM22 complex inserts proteins into inner membrane
  findings: []
- id: Reactome:R-SCE-1252259
  title: TIM9:TIM10 binds hydrophobic proteins
  findings: []
core_functions:
- description: >-
    TIM22 is the essential voltage-activated, signal-gated channel subunit of the
    TIM22 translocase that mediates insertion of multi-pass transmembrane proteins
    into the mitochondrial inner membrane. Tim22 has a 4-transmembrane helix
    architecture with conserved charged residues (E140, D190, K127, K169) critical
    for function and a C42-C141 disulfide bond (Zhang et al. 2021,
    DOI:10.1038/s41422-020-00399-0). The channel specifically recognizes internal
    targeting signals of carrier proteins but not N-terminal presequences, and is
    driven by the membrane potential (Delta-psi). The precise translocation mechanism
    remains debated: cryo-EM did not capture an open pore state, suggesting Tim22 may
    operate via conformationally gated pore formation in distinct functional states or
    via an insertase-like mechanism that lowers the energetic barrier for helix
    insertion by altering local bilayer properties.
  molecular_function:
    id: GO:0008320
    label: protein transmembrane transporter activity
  directly_involved_in:
  - id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  locations:
  - id: GO:0005743
    label: mitochondrial inner membrane
  in_complex:
    id: GO:0042721
    label: TIM22 mitochondrial import inner membrane insertion complex
  supported_by:
  - reference_id: PMID:11864609
    supporting_text: >-
      Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct
      opening states and pore diameters. The channel is voltage-activated and
      specifically responds to an internal targeting signal
  - reference_id: PMID:8955274
    supporting_text: >-
      Tim22 is required for the import of proteins of the mitochondrial ADP/ATP
      carrier (AAC) family into the inner membrane
  - reference_id: DOI:10.1038/s41422-020-00399-0
    supporting_text: >-
      Tim22 has four transmembrane helices ... a disulfide bond between C42 and C141
      ... conserved charged residues implicated in function (E140, D190, K127, K169)
- description: >-
    TIM22 serves as the structural core of the TIM22 complex, with its conserved
    regions required for interactions with Tim54, Tim18, and Sdh3 that maintain the
    functional architecture of the translocase.
  molecular_function:
    id: GO:0005198
    label: structural molecule activity
  directly_involved_in:
  - id: GO:0045039
    label: protein insertion into mitochondrial inner membrane
  locations:
  - id: GO:0005743
    label: mitochondrial inner membrane
  in_complex:
    id: GO:0042721
    label: TIM22 mitochondrial import inner membrane insertion complex
  supported_by:
  - reference_id: PMID:32591483
    supporting_text: >-
      the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are
      critically required for interactions with the membrane-embedded subunits,
      including Tim54, Tim18, and Sdh3, and thereby maintain the functional
      architecture of the TIM22 translocase

TIM22

Gene Description

Existing Annotations Review

Core Functions

TIM22 serves as the structural core of the TIM22 complex, with its conserved regions required for interactions with Tim54, Tim18, and Sdh3 that maintain the functional architecture of the translocase.

References

📚 Additional Documentation

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Gene Research for Functional Annotation

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Target Gene/Protein Identity (from UniProt):

MANDATORY VERIFICATION STEPS:

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Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

Target Gene/Protein Identity (from UniProt):

MANDATORY VERIFICATION STEPS:

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

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