id: Q9Y4W6 gene_symbol: AFG3L2 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: AFG3L2 is a mitochondrial inner membrane m-AAA protease subunit with AAA+ ATPase and zinc metalloendopeptidase domains exposed to the matrix side of the inner membrane. It assembles as AFG3L2 homohexamers and as AFG3L2-SPG7 heterohexamers that use ATP hydrolysis to unfold, translocate, and cleave mitochondrial substrates. Its major roles include protein quality control of newly synthesized or misassembled inner-membrane proteins, processing of selected mitochondrial proteins such as MRPL32 and PINK1, and regulatory degradation of substrates including EMRE/SMDT1, SLC25A39, SLC25A45, and TMBIM5/GHITM. Through these proteolytic activities AFG3L2 supports mitochondrial respiratory-chain assembly, calcium uniporter regulation, mitochondrial glutathione homeostasis, mitochondrial morphology, and neuronal maintenance. existing_annotations: - term: id: GO:0034982 label: mitochondrial protein processing evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - FB:FBgn0036702 - MGI:MGI:1916847 - MGI:MGI:1928277 - PANTHER:PTN000554085 - UniProtKB:Q9UQ90 - UniProtKB:Q9Y4W6 - WB:WBGene00004978 review: summary: AFG3L2 directly processes mitochondrial substrates, including MRPL32 and multiple inner membrane proteins/carriers. action: ACCEPT reason: Mitochondrial protein processing is a core biological role of the m-AAA protease alongside degradative quality control. supported_by: - reference_id: PMID:29932645 supporting_text: conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form - reference_id: PMID:41075794 supporting_text: SLC25A45-FLAG levels only increased in cells treated with siRNA targeting AFG3L2 - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - term: id: GO:0005745 label: m-AAA complex evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: part_of supporting_entities: - FB:FBgn0024992 - FB:FBgn0036702 - MGI:MGI:1916847 - MGI:MGI:1928277 - MGI:MGI:2385906 - PANTHER:PTN000554085 - SGD:S000000819 - SGD:S000004695 - UniProtKB:Q9UQ90 - UniProtKB:Q9Y4W6 review: summary: AFG3L2 is a constituent of the mitochondrial m-AAA protease complex, as homohexamers and as heterohexamers with SPG7. action: ACCEPT reason: The m-AAA complex is the correct cellular component context for AFG3L2 protease function. supported_by: - reference_id: PMID:31327635 supporting_text: The human m-AAA protease assembles as homohexamers of AFG3L2 subunits or heterohexamers comprising AFG3L2 subunits and subunits of the closely related homolog paraplegin (SPG7) - reference_id: PMID:14623864 supporting_text: paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables supporting_entities: - PANTHER:PTN008683692 - UniProtKB:Q9UQ90 - UniProtKB:Q9Y4W6 review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0004176 label: ATP-dependent peptidase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR000642 - InterPro:IPR005936 - InterPro:IPR011546 - InterPro:IPR037219 review: summary: ATP-dependent peptidase activity accurately combines the ATPase and protease aspects of AFG3L2 function. action: ACCEPT reason: The m-AAA protease uses ATP hydrolysis to unfold/translocate substrates and protease active sites to degrade them. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - reference_id: PMID:19748354 supporting_text: we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR000642 - InterPro:IPR005936 - InterPro:IPR011546 - InterPro:IPR037219 review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0005524 label: ATP binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR000642 - InterPro:IPR003959 - InterPro:IPR003960 - InterPro:IPR011546 - InterPro:IPR037219 review: summary: ATP binding is a true domain-associated property, but ATP hydrolysis is the informative catalytic annotation. action: KEEP_AS_NON_CORE reason: Keep as a non-core molecular feature; core function should emphasize ATP hydrolysis and ATP-dependent peptidase activity. supported_by: - reference_id: PMID:19748354 supporting_text: we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes - reference_id: PMID:31327635 supporting_text: ATP-dependent translocation to unfold and degrade targeted proteins - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: located_in supporting_entities: - UniProtKB:Q8JZQ2 - ensembl:ENSMUSP00000025408 - UniProtKB-SubCell:SL-0168 review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0006508 label: proteolysis evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: involved_in supporting_entities: - ARBA:ARBA00026291 - InterPro:IPR000642 - InterPro:IPR037219 review: summary: Generic proteolysis is correct but underspecified for the mitochondrial m-AAA protease role. action: MODIFY reason: The evidence supports mitochondrial substrate degradation and protein quality control, not merely generic proteolysis. proposed_replacement_terms: - id: GO:0035694 label: mitochondrial protein catabolic process supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - reference_id: file:projects/PROTEOSTASIS/reports/pn_projection/pn_projected_annotations.tsv supporting_text: This PN class groups mitochondrial protein-degradation pathways. GO mitochondrial protein catabolic process is the conservative shared target. - term: id: GO:0008270 label: zinc ion binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR011546 review: summary: Zinc binding is supported by the M41 metalloprotease active site but is not itself the core function. action: KEEP_AS_NON_CORE reason: Keep as a cofactor/domain feature and emphasize metalloendopeptidase activity as the core molecular function. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0016020 label: membrane evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: located_in supporting_entities: - InterPro:IPR005936 - InterPro:IPR011546 review: summary: Membrane localization is correct but too broad. action: MODIFY reason: AFG3L2 is specifically an integral mitochondrial inner membrane protein. proposed_replacement_terms: - id: GO:0005743 label: mitochondrial inner membrane supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0016887 label: ATP hydrolysis activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables supporting_entities: - InterPro:IPR003959 - InterPro:IPR003960 - RHEA:13065 review: summary: ATP hydrolysis is a core motor activity of the AAA+ ring that powers substrate unfolding and translocation. action: ACCEPT reason: This is directly supported by m-AAA ATPase assays and AFG3L2 structural work tying ATP-dependent translocation to substrate degradation. supported_by: - reference_id: PMID:19748354 supporting_text: we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes - reference_id: PMID:31327635 supporting_text: ATP-dependent translocation to unfold and degrade targeted proteins - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32814053 qualifier: enables supporting_entities: - UniProtKB:P42858 review: summary: The IPI row records binding to UniProtKB:P42858, but protein binding is too generic to describe AFG3L2 function. action: MARK_AS_OVER_ANNOTATED reason: Specific interactions should be interpreted through m-AAA complex membership, substrate degradation, calcium regulation, or other informative process terms rather than the generic protein binding MF term. supported_by: - reference_id: PMID:32814053 supporting_text: Interactome maps are valuable resources to elucidate protein function and disease mechanisms. - term: id: GO:0005745 label: m-AAA complex evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: part_of supporting_entities: - ARBA:ARBA00088390 - UniProtKB:Q8JZQ2 - ensembl:ENSMUSP00000025408 review: summary: AFG3L2 is a constituent of the mitochondrial m-AAA protease complex, as homohexamers and as heterohexamers with SPG7. action: ACCEPT reason: The m-AAA complex is the correct cellular component context for AFG3L2 protease function. supported_by: - reference_id: PMID:31327635 supporting_text: The human m-AAA protease assembles as homohexamers of AFG3L2 subunits or heterohexamers comprising AFG3L2 subunits and subunits of the closely related homolog paraplegin (SPG7) - reference_id: PMID:14623864 supporting_text: paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane - term: id: GO:0008237 label: metallopeptidase activity evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: enables supporting_entities: - UniProtKB:Q8JZQ2 - ensembl:ENSMUSP00000025408 review: summary: The annotation is directionally correct but too broad for AFG3L2. action: MODIFY reason: AFG3L2 is specifically supported as a metalloendopeptidase/m-AAA protease; use the more precise metalloendopeptidase activity term. proposed_replacement_terms: - id: GO:0004222 label: metalloendopeptidase activity supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0016485 label: protein processing evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: involved_in supporting_entities: - UniProtKB:Q8JZQ2 - ensembl:ENSMUSP00000025408 review: summary: Protein processing is true but should be represented with the existing mitochondrial protein processing term. action: MODIFY reason: AFG3L2 processing events occur in mitochondria and are already captured more specifically by mitochondrial protein processing. proposed_replacement_terms: - id: GO:0034982 label: mitochondrial protein processing supported_by: - reference_id: PMID:29932645 supporting_text: conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form - reference_id: PMID:41075794 supporting_text: SLC25A45-FLAG levels only increased in cells treated with siRNA targeting AFG3L2 - term: id: GO:0016540 label: protein autoprocessing evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: involved_in supporting_entities: - UniProtKB:Q8JZQ2 - ensembl:ENSMUSP00000025408 review: summary: Autoprocessing of the imported precursor is described for AFG3L2, but it is ancillary to the mature protease role. action: KEEP_AS_NON_CORE reason: Retain as non-core because the main biological function is degradation/processing of mitochondrial substrates. supported_by: - reference_id: file:human/AFG3L2/AFG3L2-uniprot.txt supporting_text: autocatalytic proteolytic processing to generate the proteolytically active mature form - term: id: GO:0051604 label: protein maturation evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: involved_in supporting_entities: - ARBA:ARBA00026902 - UniProtKB:Q8JZQ2 - ensembl:ENSMUSP00000025408 review: summary: Protein maturation is true for selected substrates but too generic. action: MODIFY reason: The evidence points to mitochondrial protein processing/maturation, especially MRPL32 and related mitochondrial substrates. proposed_replacement_terms: - id: GO:0034982 label: mitochondrial protein processing supported_by: - reference_id: PMID:29932645 supporting_text: conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form - reference_id: PMID:41075794 supporting_text: SLC25A45-FLAG levels only increased in cells treated with siRNA targeting AFG3L2 - term: id: GO:0110097 label: regulation of calcium import into the mitochondrion evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: involved_in supporting_entities: - UniProtKB:Q8JZQ2 - ensembl:ENSMUSP00000025408 review: summary: AFG3L2 regulates mitochondrial calcium import by degrading unassembled EMRE before MCU complex assembly. action: KEEP_AS_NON_CORE reason: This is a supported substrate-specific consequence of m-AAA proteolysis, but not the central gene-level function. supported_by: - reference_id: PMID:27642048 supporting_text: the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU - reference_id: PMID:28396416 supporting_text: mitochondrial mAAA proteases AFG3L2 and SPG7 rapidly degrade unassembled EMRE using the energy of ATP hydrolysis - term: id: GO:0005739 label: mitochondrion evidence_type: IDA original_reference_id: GO_REF:0000052 qualifier: located_in review: summary: Mitochondrial localization is correct but less specific than the established inner-membrane localization. action: MODIFY reason: The earliest localization work supports mitochondria, and later biochemical/structural work resolves AFG3L2 to the mitochondrial inner membrane. proposed_replacement_terms: - id: GO:0005743 label: mitochondrial inner membrane supported_by: - reference_id: PMID:10395799 supporting_text: Immunofluorescence studies revealed that AFG3L2 and paraplegin share a similar expression pattern and the same subcellular localization, the mitochondrial compartment. - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IMP original_reference_id: PMID:41075794 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0034982 label: mitochondrial protein processing evidence_type: IMP original_reference_id: PMID:41075794 qualifier: involved_in review: summary: AFG3L2 directly processes mitochondrial substrates, including MRPL32 and multiple inner membrane proteins/carriers. action: ACCEPT reason: Mitochondrial protein processing is a core biological role of the m-AAA protease alongside degradative quality control. supported_by: - reference_id: PMID:29932645 supporting_text: conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form - reference_id: PMID:41075794 supporting_text: SLC25A45-FLAG levels only increased in cells treated with siRNA targeting AFG3L2 - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: IDA original_reference_id: PMID:31327635 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005745 label: m-AAA complex evidence_type: IPI original_reference_id: PMID:31327635 qualifier: part_of review: summary: AFG3L2 is a constituent of the mitochondrial m-AAA protease complex, as homohexamers and as heterohexamers with SPG7. action: ACCEPT reason: The m-AAA complex is the correct cellular component context for AFG3L2 protease function. supported_by: - reference_id: PMID:31327635 supporting_text: The human m-AAA protease assembles as homohexamers of AFG3L2 subunits or heterohexamers comprising AFG3L2 subunits and subunits of the closely related homolog paraplegin (SPG7) - reference_id: PMID:14623864 supporting_text: paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane - term: id: GO:0034982 label: mitochondrial protein processing evidence_type: IDA original_reference_id: PMID:31327635 qualifier: involved_in review: summary: AFG3L2 directly processes mitochondrial substrates, including MRPL32 and multiple inner membrane proteins/carriers. action: ACCEPT reason: Mitochondrial protein processing is a core biological role of the m-AAA protease alongside degradative quality control. supported_by: - reference_id: PMID:29932645 supporting_text: conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form - reference_id: PMID:41075794 supporting_text: SLC25A45-FLAG levels only increased in cells treated with siRNA targeting AFG3L2 - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - term: id: GO:0005739 label: mitochondrion evidence_type: HTP original_reference_id: PMID:34800366 qualifier: located_in review: summary: Mitochondrial localization is correct but less specific than the established inner-membrane localization. action: MODIFY reason: The earliest localization work supports mitochondria, and later biochemical/structural work resolves AFG3L2 to the mitochondrial inner membrane. proposed_replacement_terms: - id: GO:0005743 label: mitochondrial inner membrane supported_by: - reference_id: PMID:10395799 supporting_text: Immunofluorescence studies revealed that AFG3L2 and paraplegin share a similar expression pattern and the same subcellular localization, the mitochondrial compartment. - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0141164 label: mitochondrial protein quality control evidence_type: IDA original_reference_id: PMID:26504172 qualifier: involved_in review: summary: AFG3L2 performs mitochondrial protein quality control, especially for newly synthesized or misassembled inner-membrane proteins. action: ACCEPT reason: This is a core process annotation supported by direct perturbation and mitochondrial translation/proteostasis studies. supported_by: - reference_id: PMID:26504172 supporting_text: The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism - reference_id: PMID:30683687 supporting_text: Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. - reference_id: PMID:34718584 supporting_text: defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex - reference_id: file:human/AFG3L2/AFG3L2-deep-research-falcon.md supporting_text: '**m-AAA proteases** are IMM-embedded ATP-dependent protease complexes that provide **protein quality control (PQC)** by selective removal/processing of **non-assembled** or **damaged** mitochondrial proteins' - term: id: GO:0141164 label: mitochondrial protein quality control evidence_type: IDA original_reference_id: PMID:30683687 qualifier: involved_in review: summary: AFG3L2 performs mitochondrial protein quality control, especially for newly synthesized or misassembled inner-membrane proteins. action: ACCEPT reason: This is a core process annotation supported by direct perturbation and mitochondrial translation/proteostasis studies. supported_by: - reference_id: PMID:26504172 supporting_text: The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism - reference_id: PMID:30683687 supporting_text: Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. - reference_id: PMID:34718584 supporting_text: defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex - term: id: GO:0141164 label: mitochondrial protein quality control evidence_type: IDA original_reference_id: PMID:34718584 qualifier: involved_in review: summary: AFG3L2 performs mitochondrial protein quality control, especially for newly synthesized or misassembled inner-membrane proteins. action: ACCEPT reason: This is a core process annotation supported by direct perturbation and mitochondrial translation/proteostasis studies. supported_by: - reference_id: PMID:26504172 supporting_text: The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism - reference_id: PMID:30683687 supporting_text: Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. - reference_id: PMID:34718584 supporting_text: defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:29545505 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:27642048 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:28396416 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: IDA original_reference_id: PMID:37917749 qualifier: is_active_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005745 label: m-AAA complex evidence_type: IDA original_reference_id: PMID:28396416 qualifier: part_of review: summary: AFG3L2 is a constituent of the mitochondrial m-AAA protease complex, as homohexamers and as heterohexamers with SPG7. action: ACCEPT reason: The m-AAA complex is the correct cellular component context for AFG3L2 protease function. supported_by: - reference_id: PMID:31327635 supporting_text: The human m-AAA protease assembles as homohexamers of AFG3L2 subunits or heterohexamers comprising AFG3L2 subunits and subunits of the closely related homolog paraplegin (SPG7) - reference_id: PMID:14623864 supporting_text: paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane - term: id: GO:0110097 label: regulation of calcium import into the mitochondrion evidence_type: IDA original_reference_id: PMID:27642048 qualifier: involved_in review: summary: AFG3L2 regulates mitochondrial calcium import by degrading unassembled EMRE before MCU complex assembly. action: KEEP_AS_NON_CORE reason: This is a supported substrate-specific consequence of m-AAA proteolysis, but not the central gene-level function. supported_by: - reference_id: PMID:27642048 supporting_text: the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU - reference_id: PMID:28396416 supporting_text: mitochondrial mAAA proteases AFG3L2 and SPG7 rapidly degrade unassembled EMRE using the energy of ATP hydrolysis - term: id: GO:0110097 label: regulation of calcium import into the mitochondrion evidence_type: IDA original_reference_id: PMID:28396416 qualifier: involved_in review: summary: AFG3L2 regulates mitochondrial calcium import by degrading unassembled EMRE before MCU complex assembly. action: KEEP_AS_NON_CORE reason: This is a supported substrate-specific consequence of m-AAA proteolysis, but not the central gene-level function. supported_by: - reference_id: PMID:27642048 supporting_text: the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU - reference_id: PMID:28396416 supporting_text: mitochondrial mAAA proteases AFG3L2 and SPG7 rapidly degrade unassembled EMRE using the energy of ATP hydrolysis - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:37917749 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:38157846 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: PMID:31327635 qualifier: is_active_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0030163 label: protein catabolic process evidence_type: IDA original_reference_id: PMID:37917749 qualifier: involved_in review: summary: Protein catabolic process is correct but too broad for the directly supported mitochondrial degradation role. action: MODIFY reason: AFG3L2 degrades mitochondrial inner membrane and matrix-facing substrates; the PN projection to mitochondrial protein catabolic process is conservative and literature-supported. proposed_replacement_terms: - id: GO:0035694 label: mitochondrial protein catabolic process supported_by: - reference_id: PMID:37917749 supporting_text: Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. - reference_id: PMID:38157846 supporting_text: mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1 - reference_id: file:projects/PROTEOSTASIS/reports/pn_projection/pn_projected_annotations.tsv supporting_text: This PN class groups mitochondrial protein-degradation pathways. GO mitochondrial protein catabolic process is the conservative shared target. - term: id: GO:0030163 label: protein catabolic process evidence_type: IDA original_reference_id: PMID:38157846 qualifier: involved_in review: summary: Protein catabolic process is correct but too broad for the directly supported mitochondrial degradation role. action: MODIFY reason: AFG3L2 degrades mitochondrial inner membrane and matrix-facing substrates; the PN projection to mitochondrial protein catabolic process is conservative and literature-supported. proposed_replacement_terms: - id: GO:0035694 label: mitochondrial protein catabolic process supported_by: - reference_id: PMID:37917749 supporting_text: Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. - reference_id: PMID:38157846 supporting_text: mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1 - reference_id: file:projects/PROTEOSTASIS/reports/pn_projection/pn_projected_annotations.tsv supporting_text: This PN class groups mitochondrial protein-degradation pathways. GO mitochondrial protein catabolic process is the conservative shared target. - term: id: GO:0072753 label: cellular response to glutathione evidence_type: IDA original_reference_id: PMID:37917749 qualifier: involved_in review: summary: AFG3L2 participates in the glutathione-response circuit by degrading SLC25A39 when mitochondrial glutathione is sufficient. action: KEEP_AS_NON_CORE reason: The annotation reflects a specific regulatory substrate and metabolite-feedback axis, but the core function remains mitochondrial proteolysis. supported_by: - reference_id: PMID:37917749 supporting_text: Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. - reference_id: PMID:38157846 supporting_text: mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1 - term: id: GO:0072753 label: cellular response to glutathione evidence_type: IDA original_reference_id: PMID:38157846 qualifier: involved_in review: summary: AFG3L2 participates in the glutathione-response circuit by degrading SLC25A39 when mitochondrial glutathione is sufficient. action: KEEP_AS_NON_CORE reason: The annotation reflects a specific regulatory substrate and metabolite-feedback axis, but the core function remains mitochondrial proteolysis. supported_by: - reference_id: PMID:37917749 supporting_text: Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. - reference_id: PMID:38157846 supporting_text: mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1 - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:19748354 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:29932645 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:31327635 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0005745 label: m-AAA complex evidence_type: IDA original_reference_id: PMID:19748354 qualifier: part_of review: summary: AFG3L2 is a constituent of the mitochondrial m-AAA protease complex, as homohexamers and as heterohexamers with SPG7. action: ACCEPT reason: The m-AAA complex is the correct cellular component context for AFG3L2 protease function. supported_by: - reference_id: PMID:31327635 supporting_text: The human m-AAA protease assembles as homohexamers of AFG3L2 subunits or heterohexamers comprising AFG3L2 subunits and subunits of the closely related homolog paraplegin (SPG7) - reference_id: PMID:14623864 supporting_text: paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane - term: id: GO:0005745 label: m-AAA complex evidence_type: IDA original_reference_id: PMID:31327635 qualifier: part_of review: summary: AFG3L2 is a constituent of the mitochondrial m-AAA protease complex, as homohexamers and as heterohexamers with SPG7. action: ACCEPT reason: The m-AAA complex is the correct cellular component context for AFG3L2 protease function. supported_by: - reference_id: PMID:31327635 supporting_text: The human m-AAA protease assembles as homohexamers of AFG3L2 subunits or heterohexamers comprising AFG3L2 subunits and subunits of the closely related homolog paraplegin (SPG7) - reference_id: PMID:14623864 supporting_text: paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane - term: id: GO:0016887 label: ATP hydrolysis activity evidence_type: IDA original_reference_id: PMID:19748354 qualifier: enables review: summary: ATP hydrolysis is a core motor activity of the AAA+ ring that powers substrate unfolding and translocation. action: ACCEPT reason: This is directly supported by m-AAA ATPase assays and AFG3L2 structural work tying ATP-dependent translocation to substrate degradation. supported_by: - reference_id: PMID:19748354 supporting_text: we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes - reference_id: PMID:31327635 supporting_text: ATP-dependent translocation to unfold and degrade targeted proteins - term: id: GO:0016887 label: ATP hydrolysis activity evidence_type: IDA original_reference_id: PMID:31327635 qualifier: enables review: summary: ATP hydrolysis is a core motor activity of the AAA+ ring that powers substrate unfolding and translocation. action: ACCEPT reason: This is directly supported by m-AAA ATPase assays and AFG3L2 structural work tying ATP-dependent translocation to substrate degradation. supported_by: - reference_id: PMID:19748354 supporting_text: we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes - reference_id: PMID:31327635 supporting_text: ATP-dependent translocation to unfold and degrade targeted proteins - term: id: GO:0051604 label: protein maturation evidence_type: IDA original_reference_id: PMID:29932645 qualifier: involved_in review: summary: Protein maturation is true for selected substrates but too generic. action: MODIFY reason: The evidence points to mitochondrial protein processing/maturation, especially MRPL32 and related mitochondrial substrates. proposed_replacement_terms: - id: GO:0034982 label: mitochondrial protein processing supported_by: - reference_id: PMID:29932645 supporting_text: conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form - reference_id: PMID:41075794 supporting_text: SLC25A45-FLAG levels only increased in cells treated with siRNA targeting AFG3L2 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:35912435 qualifier: enables supporting_entities: - UniProtKB:Q9H3K2 review: summary: The IPI row records binding to UniProtKB:Q9H3K2, but protein binding is too generic to describe AFG3L2 function. action: MARK_AS_OVER_ANNOTATED reason: Specific interactions should be interpreted through m-AAA complex membership, substrate degradation, calcium regulation, or other informative process terms rather than the generic protein binding MF term. supported_by: - reference_id: PMID:35912435 supporting_text: Besides these expected interactors, TMBIM5 (also known as GHITM or MICS1) was highly enriched in AFG3L2 precipitates - term: id: GO:0006508 label: proteolysis evidence_type: IDA original_reference_id: PMID:35912435 qualifier: involved_in review: summary: Generic proteolysis is correct but underspecified for the mitochondrial m-AAA protease role. action: MODIFY reason: The evidence supports mitochondrial substrate degradation and protein quality control, not merely generic proteolysis. proposed_replacement_terms: - id: GO:0035694 label: mitochondrial protein catabolic process supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - reference_id: file:projects/PROTEOSTASIS/reports/pn_projection/pn_projected_annotations.tsv supporting_text: This PN class groups mitochondrial protein-degradation pathways. GO mitochondrial protein catabolic process is the conservative shared target. - term: id: GO:0004222 label: metalloendopeptidase activity evidence_type: IDA original_reference_id: PMID:22354088 qualifier: enables review: summary: Metalloendopeptidase activity is the core catalytic activity of AFG3L2 as an m-AAA protease subunit. action: ACCEPT reason: Direct structural and biochemical studies support AFG3L2 as a zinc metalloprotease that cleaves substrates after ATP-driven translocation. supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0005739 label: mitochondrion evidence_type: IDA original_reference_id: PMID:22354088 qualifier: located_in review: summary: Mitochondrial localization is correct but less specific than the established inner-membrane localization. action: MODIFY reason: The earliest localization work supports mitochondria, and later biochemical/structural work resolves AFG3L2 to the mitochondrial inner membrane. proposed_replacement_terms: - id: GO:0005743 label: mitochondrial inner membrane supported_by: - reference_id: PMID:10395799 supporting_text: Immunofluorescence studies revealed that AFG3L2 and paraplegin share a similar expression pattern and the same subcellular localization, the mitochondrial compartment. - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0033619 label: membrane protein proteolysis evidence_type: IDA original_reference_id: PMID:22354088 qualifier: involved_in review: summary: AFG3L2 participates in proteolysis of mitochondrial membrane-associated proteins such as PINK1 and inner-membrane substrates. action: ACCEPT reason: The term is consistent with direct m-AAA substrate degradation, although the added mitochondrial protein catabolic process term captures the broader mitochondrial context. supported_by: - reference_id: PMID:22354088 supporting_text: we identified four mitochondrial proteases, mitochondrial processing peptidase (MPP), presenilin-associated rhomboid-like protease (PARL), m-AAA and ClpXP, involved in PINK1 degradation - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:26387735 qualifier: enables supporting_entities: - UniProtKB:Q9UQ90 review: summary: The IPI row records binding to UniProtKB:Q9UQ90, but protein binding is too generic to describe AFG3L2 function. action: MARK_AS_OVER_ANNOTATED reason: Specific interactions should be interpreted through m-AAA complex membership, substrate degradation, calcium regulation, or other informative process terms rather than the generic protein binding MF term. supported_by: - reference_id: PMID:14623864 supporting_text: To explore a potential physical interaction between paraplegin and AFG3L2, we performed coimmunoprecipitation studies in HEK293. - reference_id: PMID:26387735 supporting_text: HA antibody immunoprecipitated a known SPG7 binding partner AFG3L2 - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: located_in supporting_entities: - UniProtKB:Q8JZQ2 review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0016485 label: protein processing evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: involved_in supporting_entities: - UniProtKB:Q8JZQ2 review: summary: Protein processing is true but should be represented with the existing mitochondrial protein processing term. action: MODIFY reason: AFG3L2 processing events occur in mitochondria and are already captured more specifically by mitochondrial protein processing. proposed_replacement_terms: - id: GO:0034982 label: mitochondrial protein processing supported_by: - reference_id: PMID:29932645 supporting_text: conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form - reference_id: PMID:41075794 supporting_text: SLC25A45-FLAG levels only increased in cells treated with siRNA targeting AFG3L2 - term: id: GO:0016540 label: protein autoprocessing evidence_type: ISS original_reference_id: GO_REF:0000024 qualifier: involved_in supporting_entities: - UniProtKB:Q8JZQ2 review: summary: Autoprocessing of the imported precursor is described for AFG3L2, but it is ancillary to the mature protease role. action: KEEP_AS_NON_CORE reason: Retain as non-core because the main biological function is degradation/processing of mitochondrial substrates. supported_by: - reference_id: file:human/AFG3L2/AFG3L2-uniprot.txt supporting_text: autocatalytic proteolytic processing to generate the proteolytically active mature form - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-8949649 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-8949659 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-8949661 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-9838627 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-9839053 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-9839059 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-9839105 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-9839113 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005743 label: mitochondrial inner membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-9839149 qualifier: located_in review: summary: AFG3L2 is an integral mitochondrial inner membrane protein with catalytic domains exposed to the matrix side. action: ACCEPT reason: This is the most specific and best-supported cellular component annotation. Matrix-facing catalytic topology does not make the protein a soluble mitochondrial matrix component. supported_by: - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:27642048 qualifier: enables supporting_entities: - UniProtKB:Q8WWC4 review: summary: The IPI row records binding to UniProtKB:Q8WWC4, but protein binding is too generic to describe AFG3L2 function. action: MARK_AS_OVER_ANNOTATED reason: Specific interactions should be interpreted through m-AAA complex membership, substrate degradation, calcium regulation, or other informative process terms rather than the generic protein binding MF term. supported_by: - reference_id: PMID:27642048 supporting_text: MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE - reference_id: PMID:27642048 supporting_text: the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:27642048 qualifier: enables supporting_entities: - UniProtKB:Q9H4I9 review: summary: The IPI row records binding to UniProtKB:Q9H4I9, but protein binding is too generic to describe AFG3L2 function. action: MARK_AS_OVER_ANNOTATED reason: Specific interactions should be interpreted through m-AAA complex membership, substrate degradation, calcium regulation, or other informative process terms rather than the generic protein binding MF term. supported_by: - reference_id: PMID:27642048 supporting_text: MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE - reference_id: PMID:27642048 supporting_text: the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU - term: id: GO:0006508 label: proteolysis evidence_type: IMP original_reference_id: PMID:27642048 qualifier: involved_in review: summary: Generic proteolysis is correct but underspecified for the mitochondrial m-AAA protease role. action: MODIFY reason: The evidence supports mitochondrial substrate degradation and protein quality control, not merely generic proteolysis. proposed_replacement_terms: - id: GO:0035694 label: mitochondrial protein catabolic process supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - reference_id: file:projects/PROTEOSTASIS/reports/pn_projection/pn_projected_annotations.tsv supporting_text: This PN class groups mitochondrial protein-degradation pathways. GO mitochondrial protein catabolic process is the conservative shared target. - term: id: GO:0007409 label: axonogenesis evidence_type: IMP original_reference_id: PMID:27642048 qualifier: involved_in review: summary: Axonogenesis reflects a neuronal phenotype of impaired m-AAA protease function rather than a direct molecular role. action: KEEP_AS_NON_CORE reason: Retain as non-core/developmental consequence; do not use it to define AFG3L2 core function. supported_by: - reference_id: PMID:31327635 supporting_text: m-AAA proteases are essential for axonal development in mammals - reference_id: PMID:27642048 supporting_text: Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels - term: id: GO:0008237 label: metallopeptidase activity evidence_type: IMP original_reference_id: PMID:27642048 qualifier: enables review: summary: The annotation is directionally correct but too broad for AFG3L2. action: MODIFY reason: AFG3L2 is specifically supported as a metalloendopeptidase/m-AAA protease; use the more precise metalloendopeptidase activity term. proposed_replacement_terms: - id: GO:0004222 label: metalloendopeptidase activity supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - term: id: GO:0036444 label: calcium import into the mitochondrion evidence_type: IMP original_reference_id: PMID:27642048 qualifier: involved_in review: summary: The calcium-import phenotype is mediated through regulation of EMRE/MCU assembly rather than direct calcium transport by AFG3L2. action: MODIFY reason: Use regulation of calcium import into the mitochondrion rather than annotating AFG3L2 as directly involved in calcium import. proposed_replacement_terms: - id: GO:0110097 label: regulation of calcium import into the mitochondrion supported_by: - reference_id: PMID:27642048 supporting_text: the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU - reference_id: PMID:28396416 supporting_text: mitochondrial mAAA proteases AFG3L2 and SPG7 rapidly degrade unassembled EMRE using the energy of ATP hydrolysis - term: id: GO:0051560 label: mitochondrial calcium ion homeostasis evidence_type: IMP original_reference_id: PMID:27642048 qualifier: involved_in review: summary: AFG3L2 contributes to mitochondrial calcium homeostasis through EMRE turnover and MCU gatekeeper assembly. action: KEEP_AS_NON_CORE reason: Supported but downstream/substrate-specific relative to the m-AAA protease quality-control function. supported_by: - reference_id: PMID:27642048 supporting_text: the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU - reference_id: PMID:28396416 supporting_text: mitochondrial mAAA proteases AFG3L2 and SPG7 rapidly degrade unassembled EMRE using the energy of ATP hydrolysis - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:14623864 qualifier: enables supporting_entities: - UniProtKB:Q9UQ90 review: summary: The IPI row records binding to UniProtKB:Q9UQ90, but protein binding is too generic to describe AFG3L2 function. action: MARK_AS_OVER_ANNOTATED reason: Specific interactions should be interpreted through m-AAA complex membership, substrate degradation, calcium regulation, or other informative process terms rather than the generic protein binding MF term. supported_by: - reference_id: PMID:14623864 supporting_text: To explore a potential physical interaction between paraplegin and AFG3L2, we performed coimmunoprecipitation studies in HEK293. - reference_id: PMID:26387735 supporting_text: HA antibody immunoprecipitated a known SPG7 binding partner AFG3L2 - term: id: GO:0005739 label: mitochondrion evidence_type: TAS original_reference_id: PMID:10395799 qualifier: located_in review: summary: Mitochondrial localization is correct but less specific than the established inner-membrane localization. action: MODIFY reason: The earliest localization work supports mitochondria, and later biochemical/structural work resolves AFG3L2 to the mitochondrial inner membrane. proposed_replacement_terms: - id: GO:0005743 label: mitochondrial inner membrane supported_by: - reference_id: PMID:10395799 supporting_text: Immunofluorescence studies revealed that AFG3L2 and paraplegin share a similar expression pattern and the same subcellular localization, the mitochondrial compartment. - reference_id: PMID:31327635 supporting_text: m- and i-AAA proteases, which are tethered to the mitochondrial inner membrane (IM), but expose their enzymatic domains to the matrix and intermembrane spaces (IMS), respectively - reference_id: PMID:14623864 supporting_text: Paraplegin and AFG3L2 were recovered from the membrane fraction, indicating that both are integral proteins of the mitochondrial inner membrane - term: id: GO:0035694 label: mitochondrial protein catabolic process evidence_type: IC original_reference_id: PMID:31327635 qualifier: involved_in review: summary: Proposed new annotation from the Proteostasis Network projection. AFG3L2 directly degrades mitochondrial substrates as an inner-membrane m-AAA protease, so mitochondrial protein catabolic process is a conservative addition. action: NEW reason: The PN projection flagged GO:0035694 as more specific than the existing generic protein catabolic process annotation. Literature supports mitochondrial substrate degradation, while the parallel PN matrix localization projection is not accepted because AFG3L2 is an inner membrane protein with matrix-facing catalytic domains. additional_reference_ids: - file:human/AFG3L2/AFG3L2-notes.md - file:human/AFG3L2/AFG3L2-deep-research-falcon.md supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - reference_id: PMID:37917749 supporting_text: Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. - reference_id: file:projects/PROTEOSTASIS/reports/pn_projection/pn_projected_annotations.tsv supporting_text: This PN class groups mitochondrial protein-degradation pathways. GO mitochondrial protein catabolic process is the conservative shared target. - reference_id: file:human/AFG3L2/AFG3L2-deep-research-falcon.md supporting_text: '**m-AAA proteases** are IMM-embedded ATP-dependent protease complexes that provide **protein quality control (PQC)** by selective removal/processing of **non-assembled** or **damaged** mitochondrial proteins' references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000024 title: Manual transfer of experimentally-verified manual GO annotation data to orthologs by curator judgment of sequence similarity findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000052 title: Gene Ontology annotation based on curation of immunofluorescence data findings: [] - id: GO_REF:0000107 title: Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:10395799 title: Identification and characterization of AFG3L2, a novel paraplegin-related gene. findings: [] - id: PMID:14623864 title: Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. findings: [] - id: PMID:19748354 title: An intersubunit signaling network coordinates ATP hydrolysis by m-AAA proteases. findings: [] - id: PMID:22354088 title: Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment. findings: [] - id: PMID:26387735 title: SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore. findings: [] - id: PMID:26504172 title: Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness. findings: [] - id: PMID:27642048 title: The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria. findings: [] - id: PMID:28396416 title: Proteolytic control of the mitochondrial calcium uniporter complex. findings: [] - id: PMID:29545505 title: m-AAA and i-AAA complexes coordinate to regulate OMA1, the stress-activated supervisor of mitochondrial dynamics. findings: [] - id: PMID:29932645 title: Dissecting Substrate Specificities of the Mitochondrial AFG3L2 Protease. findings: [] - id: PMID:30683687 title: Mitochondrial stress response triggered by defects in protein synthesis quality control. findings: [] - id: PMID:31327635 title: Unique Structural Features of the Mitochondrial AAA+ Protease AFG3L2 Reveal the Molecular Basis for Activity in Health and Disease. findings: [] - id: PMID:32814053 title: Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains. findings: [] - id: PMID:34718584 title: Translation of MT-ATP6 pathogenic variants reveals distinct regulatory consequences from the co-translational quality control of mitochondrial protein synthesis. findings: [] - id: PMID:34800366 title: Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context. findings: [] - id: PMID:35912435 title: Regulation of mitochondrial proteostasis by the proton gradient. findings: [] - id: PMID:37917749 title: Autoregulatory control of mitochondrial glutathione homeostasis. findings: [] - id: PMID:38157846 title: Dual regulation of SLC25A39 by AFG3L2 and iron controls mitochondrial glutathione homeostasis. findings: [] - id: PMID:41075794 title: SLC25A45 is required for mitochondrial uptake of methylated amino acids and de novo carnitine biosynthesis. findings: [] - id: Reactome:R-HSA-8949649 title: PMPCA:PMPCB cleaves the transit peptide of proSMDT1 (proEMRE) findings: [] - id: Reactome:R-HSA-8949659 title: AFG3L2 (m-AAA protease) degrades SMDT1 that is not assembled in MCU findings: [] - id: Reactome:R-HSA-8949661 title: C2orf47:AFG3L2 binds the transit peptide of SMDT1 findings: [] - id: Reactome:R-HSA-9838627 title: AFG3L2 binds mitochondrial inner membrane proteins findings: [] - id: Reactome:R-HSA-9839053 title: AFG3L2:SPG7 binds SMDT1 (EMRE) findings: [] - id: Reactome:R-HSA-9839059 title: AFG3L2:SPG7 degrades SMDT1 (EMRE) findings: [] - id: Reactome:R-HSA-9839105 title: AFG3L2 degrades mitochondrial matrix proteins findings: [] - id: Reactome:R-HSA-9839113 title: AFG3L2 degrades mitochondrial inner membrane proteins findings: [] - id: Reactome:R-HSA-9839149 title: AFG3L2 binds mitochondrial matrix proteins findings: [] - id: file:human/AFG3L2/AFG3L2-uniprot.txt title: UniProtKB record for human AFG3L2 (Q9Y4W6) findings: [] - id: file:human/AFG3L2/AFG3L2-deep-research-falcon.md title: Falcon deep research report for human AFG3L2 findings: [] - id: file:human/AFG3L2/AFG3L2-notes.md title: AFG3L2 curator notes for Proteostasis PN review findings: [] - id: file:projects/PROTEOSTASIS/reports/pn_projection/pn_projected_annotations.tsv title: Proteostasis Network projected GO annotations report findings: [] - id: file:projects/PROTEOSTASIS/mappings/mitochondrial_proteostasis.yaml title: Proteostasis Network mitochondrial proteostasis mapping findings: [] core_functions: - description: ATP-dependent zinc metalloendopeptidase activity in the mitochondrial m-AAA protease. AFG3L2 uses its AAA+ motor to engage and unfold mitochondrial substrates and its M41 protease active sites to cleave them, supporting mitochondrial protein catabolic process, mitochondrial protein processing, and mitochondrial protein quality control. molecular_function: id: GO:0004222 label: metalloendopeptidase activity directly_involved_in: - id: GO:0035694 label: mitochondrial protein catabolic process - id: GO:0034982 label: mitochondrial protein processing - id: GO:0141164 label: mitochondrial protein quality control locations: - id: GO:0005743 label: mitochondrial inner membrane in_complex: id: GO:0005745 label: m-AAA complex supported_by: - reference_id: PMID:31327635 supporting_text: structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins - reference_id: PMID:29932645 supporting_text: Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. - reference_id: PMID:26504172 supporting_text: The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism - reference_id: PMID:29932645 supporting_text: conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form - description: AAA+ ATP hydrolysis that powers substrate unfolding, membrane-proximal engagement, and translocation into the proteolytic chamber of the m-AAA complex. molecular_function: id: GO:0016887 label: ATP hydrolysis activity directly_involved_in: - id: GO:0035694 label: mitochondrial protein catabolic process locations: - id: GO:0005743 label: mitochondrial inner membrane in_complex: id: GO:0005745 label: m-AAA complex supported_by: - reference_id: PMID:19748354 supporting_text: we demonstrate coordinated ATP hydrolysis within m-AAA protease ring complexes - reference_id: PMID:31327635 supporting_text: ATP-dependent translocation to unfold and degrade targeted proteins proposed_new_terms: [] suggested_questions: - question: Should matrix-facing but inner-membrane-anchored m-AAA proteases such as AFG3L2 be represented only with mitochondrial inner membrane/is_active_in annotations, or should GO curation add an annotation extension indicating that the catalytic domain faces the matrix? experts: - Puchades C - Glynn SE - Langer T - question: For AFG3L2 substrates such as SLC25A39 and SLC25A45, should curators represent metabolite-homeostasis consequences as non-core process annotations or restrict gene-level annotations to the proteolytic event and mitochondrial protein catabolic process? experts: - Birsoy K - Shen H - MacVicar T suggested_experiments: - hypothesis: AFG3L2 substrates can be prioritized by matrix-exposed degrons and metabolite-sensitive conformational states rather than by generic inner-membrane localization alone. description: Combine pulse-chase proteomics in AFG3L2 knockout/rescue cells with degron-mutant substrate panels for SLC25A39, SLC25A45, EMRE, and TMBIM5, measuring substrate half-life, AFG3L2 association, and mitochondrial metabolite or calcium readouts. experiment_type: proteomics and targeted substrate-turnover assays