PEX6

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0016887 ATP hydrolysis activity	IBA GO_REF:0000033	ACCEPT	Summary: PEX6 is a well-established AAA+ ATPase. UniProt records EC 3.6.4.- with evidence from PMID:16854980. The deep research confirms ATP hydrolysis activity as a core molecular function, driving mechanical extraction of Ub-PEX5 from peroxisomal membranes. Reason: ATP hydrolysis is a core molecular function of PEX6, extensively supported by mutagenesis studies of Walker A and B motifs (PMID:16854980, PMID:21362118) and functional complementation assays (PMID:8670792). The IBA annotation is at the appropriate level of specificity for a general AAA+ ATPase activity. Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization. PMID:8670792 Substitution of an arginine for the conserved lysine residue in the ATPase domain of Pxaaa1p abolished its biological activity, suggesting that Pxaaa1p is an ATPase.
GO:0016558 protein import into peroxisome matrix	IBA GO_REF:0000033	ACCEPT	Summary: PEX6 is required for continued peroxisomal matrix protein import by recycling the PTS1 receptor PEX5. Without PEX5 recycling, import stalls. This IBA term is the parent of the more specific receptor recycling term (GO:0016562), which better describes the direct role of PEX6. Reason: While PEX6 does not directly translocate cargo into the peroxisomal matrix, it is essential for sustained matrix protein import because it recycles PEX5. The IBA annotation at this broader level is appropriate since PEX6 deficiency directly abolishes matrix protein import (PMID:16314507, PMID:8670792). The more specific child term GO:0016562 is also annotated separately, and both are valid. Supporting Evidence: PMID:16314507 In contrast, (35)S-Pex5 was imported into the peroxisome remnants of PEX1-, PEX6-, and PEX26-defective cell mutants, including those from patients with peroxisome biogenesis disorders, from which, however, (35)S-Pex5 was not exported, thereby indicating that Pex1 and Pex6 of the AAA ATPase family and their recruiter, Pex26, were essential for Pex5 export PMID:8670792 Expression of PXAAA1 restored peroxisomal protein import in fibroblasts from 16 unrelated members of complementation group 4 (CG4) of the PBD.
GO:0043335 protein unfolding	IBA GO_REF:0000033	ACCEPT	Summary: The PEX1-PEX6 complex unfolds PEX5 during ATP-dependent extraction from the peroxisomal membrane. Pedrosa et al. (PMID:29884772) demonstrated that PEX5 is globally unfolded during the dislocation event, and a folded DHFR domain fused to PEX5 arrests extraction. This is a conserved AAA+ ATPase mechanism. Reason: Protein unfolding is a mechanistically integral part of PEX6 function. The PEX1-PEX6 complex unfolds PEX5 during extraction via processive threading through its central pore (PMID:29884772, PMID:35805150). This IBA annotation is phylogenetically well supported and experimentally validated in human. Supporting Evidence: PMID:29884772 the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event PMID:35805150 Pex1 and Pex6 form a heterohexameric AAA-ATPase capable of unfolding substrate proteins via processive threading through a central pore.
GO:0005778 peroxisomal membrane	IBA GO_REF:0000033	ACCEPT	Summary: PEX6 localizes to the peroxisomal membrane via interaction with PEX26, a tail-anchored peroxisomal membrane protein. This is well established across eukaryotes. Reason: Peroxisomal membrane localization is a core feature of PEX6 function. PEX6 is recruited to peroxisomal membranes by PEX26 (PMID:16854980, PMID:21362118). UniProt also lists peroxisome membrane localization with multiple experimental references. Supporting Evidence: PMID:16854980 Pex6p and Pex26p were predominantly localized on peroxisomes. PMID:21362118 A peroxisomal C-tail-anchored type-II membrane protein, Pex26p, recruits AAA ATPase Pex1p-Pex6p complexes to peroxisomes.
GO:0005829 cytosol	IBA GO_REF:0000033	ACCEPT	Summary: PEX6 is found both in the cytosol and associated with peroxisomal membranes. A cytosolic pool is well established. Reason: Dual cytosol/peroxisome localization is a core feature of PEX6 biology. PEX6 shuttles between cytosol and peroxisomal membrane as part of the receptor recycling cycle (PMID:16854980, PMID:8670792). UniProt lists cytosol localization with experimental evidence. Supporting Evidence: PMID:16854980 endogenous Pex1p was partly localized likely as a homo-oligomer in the cytoplasm, while Pex6p and Pex26p were predominantly localized on peroxisomes. PMID:8670792 The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein.
GO:0000166 nucleotide binding	IEA GO_REF:0000043	ACCEPT	Summary: PEX6 binds ATP through its two AAA cassettes (D1 and D2). Nucleotide binding is a broader parent of ATP binding. This IEA annotation based on UniProt keywords is correct but less specific than GO:0005524 (ATP binding), which is also annotated. Reason: This is a correct but general annotation. PEX6 does bind nucleotides (specifically ATP) through its Walker A motifs. The more specific ATP binding term is also annotated. It is acceptable for IEA annotations to be broader. Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction
GO:0001750 photoreceptor outer segment	IEA GO_REF:0000044	ACCEPT	Summary: Zaki et al. (PMID:26593283) showed PEX6 expression at the base of the outer segment of photoreceptor cells using immunofluorescence. However, the localization was more specifically described as photoreceptor cilia rather than the outer segment compartment itself. Reason: The IEA mapping from UniProt subcellular location is reasonable. The study (PMID:26593283) describes localization at photoreceptor cell cilia at the junction between inner and outer segments. The more specific GO:0097733 (photoreceptor cell cilium) is also annotated with IDA evidence, and this broader IEA term is an acceptable parent-level annotation. Supporting Evidence: PMID:26593283 We show that Pex6 localizes to the apical extensions of secretory ameloblasts and differentiated odontoblasts at early stages of dentin synthesis in mice, and to cilia of retinal photoreceptor cells.
GO:0005524 ATP binding	IEA GO_REF:0000120	ACCEPT	Summary: PEX6 has two AAA cassettes with Walker A motifs (K476 and K750) that bind ATP. Mutagenesis of these lysines abolishes ATP binding and biological activity. Reason: ATP binding is a core molecular function of PEX6, directly demonstrated by mutagenesis (PMID:16854980, PMID:21362118). This IEA annotation is correct. The same term is also annotated with IMP evidence from PMID:16854980. Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization.
GO:0005778 peroxisomal membrane	IEA GO_REF:0000120	ACCEPT	Summary: Duplicate of the IBA annotation for peroxisomal membrane. Correct IEA annotation consistent with established biology. Reason: Peroxisomal membrane localization is well established for PEX6, supported by multiple experimental studies (PMID:16854980, PMID:21362118). The IEA annotation is correct and duplicates the IBA annotation, which is fine. Supporting Evidence: PMID:16854980 Pex6p and Pex26p were predominantly localized on peroxisomes.
GO:0005829 cytosol	IEA GO_REF:0000044	ACCEPT	Summary: Cytosol localization IEA annotation consistent with established PEX6 biology. PEX6 is found in both cytosol and on peroxisomal membranes. Reason: Correct IEA annotation. PEX6 has a cytosolic pool as demonstrated experimentally (PMID:16854980, PMID:8670792). Supporting Evidence: PMID:8670792 The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein.
GO:0007031 peroxisome organization	IEA GO_REF:0000120	ACCEPT	Summary: PEX6 is essential for peroxisome biogenesis and maintenance. Loss of PEX6 leads to peroxisome biogenesis disorders. This IEA annotation is correct. Reason: PEX6 is essential for peroxisome organization, as demonstrated by the peroxisome biogenesis defects in PEX6-deficient cells. Complementation restores peroxisomes (PMID:8940266, PMID:8670792). Also annotated with IMP evidence from PMID:8940266. Supporting Evidence: PMID:8940266 the full-length human PAF-2 cDNA that morphologically and biochemically restores peroxisomes of group C Zellweger fibroblasts
GO:0016787 hydrolase activity	IEA GO_REF:0000043	ACCEPT	Summary: PEX6 has ATPase (hydrolase) activity. This is a very broad parent term of ATP hydrolysis activity. While correct, it is uninformative compared to the more specific GO:0016887. Reason: This is a correct but very general IEA annotation. PEX6 is indeed a hydrolase (it hydrolyzes ATP). The more specific GO:0016887 (ATP hydrolysis activity) is also annotated. It is acceptable for IEA annotations to be at broader levels. Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction
GO:0016887 ATP hydrolysis activity	IEA GO_REF:0000002	ACCEPT	Summary: IEA annotation via InterPro mapping. PEX6 contains two AAA ATPase domains (IPR003593, IPR003959) that hydrolyze ATP. Correct and consistent with experimental evidence. Reason: Correct IEA annotation. ATP hydrolysis activity is a core function of PEX6, supported by domain structure and mutagenesis data (PMID:16854980). Duplicates the IBA and IMP annotations for the same term. Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization.
GO:0044877 protein-containing complex binding	IEA GO_REF:0000117	ACCEPT	Summary: PEX6 binds to PEX1 (forming the heterohexamer) and to PEX26. This IEA annotation from ARBA is vague but not incorrect. More specific molecular function terms would be preferred. Reason: PEX6 does bind protein-containing complexes (it interacts with the PEX1-PEX6 hexamer, PEX26, and the DTM complex). This is a general but acceptable IEA term. The same term also has IDA evidence from PMID:16854980. Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization.
GO:0005515 protein binding	IPI PMID:9588209 A cytoplasmic AAA family peroxin, Pex1p, interacts with Pex6...	MODIFY	Summary: Tamura et al. (PMID:9588209) demonstrated by co-immunoprecipitation that human PEX1 and PEX6 interact with each other. This is the foundational study establishing the PEX1-PEX6 interaction in human. However, "protein binding" is an uninformative GO term. Reason: The interaction between PEX1 and PEX6 is real and well-established, but GO:0005515 "protein binding" is too vague and uninformative per curation guidelines. The interaction reflects the formation of the PEX1-PEX6 heterohexameric AAA ATPase complex. A more informative term would describe the specific binding context. Proposed replacements: ATP hydrolysis activity Supporting Evidence: PMID:9588209 Immunoprecipitation of Pex1p using anti-Pex1p antibody resulted in concomitant recovery of 35S-Pex6p. Conversely, 35S-Pex1p was obtained in immunoprecipitate from CHO-K1 expressing human Pex6p, using anti-Pex6p antibody.
GO:0097733 photoreceptor cell cilium	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: IEA annotation transferred from experimental data via Ensembl Compara. The original experimental evidence comes from PMID:26593283, which is also annotated with IDA for this same term. This is a tissue-specific, non-core localization. Reason: Correct IEA annotation. PEX6 was shown to localize to photoreceptor cell cilia (PMID:26593283). However, this is a tissue-specific localization relevant to Heimler syndrome phenotype rather than the core peroxisomal function of PEX6. Consistent with the IDA annotation for the same term. Supporting Evidence: PMID:26593283 We show that Pex6 localizes to ... cilia of retinal photoreceptor cells.
GO:0005829 cytosol	IDA GO_REF:0000052	ACCEPT	Summary: IDA annotation based on curation of immunofluorescence data (from the Cell Atlas or similar). PEX6 cytosol localization is well established. Reason: Cytosol localization is well supported by multiple studies (PMID:16854980, PMID:8670792). The IDA from immunofluorescence curation is consistent with the broader evidence. Supporting Evidence: PMID:8670792 The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein.
GO:0005778 peroxisomal membrane	NAS PMID:35805150 Insights into the Structure and Function of the Pex1/Pex6 AA...	ACCEPT	Summary: This NAS annotation from Judy et al. (PMID:35805150) review is correct. The review extensively discusses PEX6 localization at the peroxisomal membrane via PEX26 anchoring. Reason: Peroxisomal membrane localization is a core feature of PEX6. The review (PMID:35805150) summarizes extensive evidence for PEX6 membrane localization. Duplicates other annotations for the same term, which is fine. Supporting Evidence: PMID:35805150 Pex1 and Pex6 are ATPases associated with diverse cellular activities (AAA-ATPases) and are essential for peroxisome biogenesis and maintenance
GO:0016562 protein import into peroxisome matrix, receptor recycling	NAS PMID:35805150 Insights into the Structure and Function of the Pex1/Pex6 AA...	ACCEPT	Summary: The Judy et al. review (PMID:35805150) comprehensively describes the receptor recycling function of PEX1/PEX6. This is the most specific and accurate biological process annotation for PEX6. Reason: Receptor recycling is the canonical, core biological process function of PEX6. The review (PMID:35805150) confirms: "Pex1/Pex6 is necessary to extract Pex5 from the peroxisome membrane for subsequent rounds of import. Receptor recycling remains the canonical role for Pex1/Pex6 across eukaryotes." This term is also annotated with IDA evidence from multiple publications. Supporting Evidence: PMID:35805150 Receptor recycling remains the canonical role for Pex1/Pex6 across eukaryotes.
GO:0005778 peroxisomal membrane	IDA PMID:21362118 Recruiting mechanism of the AAA peroxins, Pex1p and Pex6p, t...	ACCEPT	Summary: Nashiro et al. (PMID:21362118) used in vitro transport assays with semipermeabilized CHO cells to demonstrate that PEX6 targets to peroxisomes in an ATP-dependent manner. Reason: Peroxisomal membrane localization is a core feature. PMID:21362118 provides direct assay evidence showing PEX6 targeting to peroxisomes. This is one of multiple annotations for peroxisomal membrane with different evidence codes, which is expected and valid. Supporting Evidence: PMID:21362118 Pex6p targeting requires ATP but not its hydrolysis.
GO:0016562 protein import into peroxisome matrix, receptor recycling	IDA PMID:21362118 Recruiting mechanism of the AAA peroxins, Pex1p and Pex6p, t...	ACCEPT	Summary: Nashiro et al. (PMID:21362118) demonstrated that PEX1 and PEX6 are recruited to peroxisomes via PEX26, and that this recruitment is essential for PEX5 export/recycling. Truncation of PEX26 abolishes recruitment and fails to complement pex26 mutants. Reason: Core biological process annotation. PMID:21362118 provides direct evidence that PEX6 membrane recruitment is essential for PEX5 receptor recycling. Supporting Evidence: PMID:21362118 Pex26pDelta33-40 truncated in amino-acid residues at 33-40 abolishes the recruiting of Pex1p-Pex6p complex to peroxisomes and fails to complement the impaired phenotype of pex26 CHO cell mutant ZP167, thereby suggesting that peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins.
GO:0016562 protein import into peroxisome matrix, receptor recycling	IDA PMID:29884772 Peroxisomal monoubiquitinated PEX5 interacts with the AAA AT...	ACCEPT	Summary: Pedrosa et al. (PMID:29884772) demonstrated that PEX1-PEX6 directly interacts with monoubiquitinated PEX5 (Ub-PEX5) and extracts it from the peroxisomal DTM, with PEX5 being globally unfolded during extraction. This is the most mechanistically detailed study of receptor recycling. Reason: Core function of PEX6. PMID:29884772 provides the strongest direct evidence that Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex and is extracted (recycled) during ATP-dependent dislocation. Supporting Evidence: PMID:29884772 DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex.
GO:0043335 protein unfolding	IDA PMID:29884772 Peroxisomal monoubiquitinated PEX5 interacts with the AAA AT...	ACCEPT	Summary: Pedrosa et al. (PMID:29884772) showed using PEGylation assays that PEX5 cysteine residues located throughout the polypeptide become exposed during ATP-dependent extraction, indicating global unfolding. Furthermore, fusing a stable DHFR domain to PEX5 arrests extraction, confirming that unfolding is mechanistically required. Reason: Protein unfolding is integral to PEX6 mechanism. The PEX1-PEX6 complex unfolds PEX5 during extraction via processive threading. PMID:29884772 provides direct human experimental evidence for this. Supporting Evidence: PMID:29884772 PEX5 cysteine residues located dozens/hundreds residues apart from the pentapeptide motifs that mediate the interaction of PEX5 with the DTM (see Fig PMID:29884772 fusing the N-terminal half of PEX5 (a domain fully functional in both the import and export steps ( 50 )) to mouse DHFR results in a protein that arrests at the export step particularly when the stability of DHFR is increased by MTX
GO:0140036 ubiquitin-modified protein reader activity	IDA PMID:29884772 Peroxisomal monoubiquitinated PEX5 interacts with the AAA AT...	ACCEPT	Summary: Pedrosa et al. (PMID:29884772) demonstrated that the PEX1-PEX6 complex specifically recognizes monoubiquitinated PEX5 (Ub-PEX5) via its ubiquitin moiety. Photocross-linking showed that Ub-PEX5 interacts directly with both PEX1 and PEX6 through the ubiquitin moiety. Reason: This is a valid molecular function annotation. PEX6 (as part of the PEX1-PEX6 complex) specifically recognizes ubiquitin-modified PEX5 as its substrate. The ubiquitin moiety is the recognition signal. PMID:29884772 provides direct evidence for this activity. Supporting Evidence: PMID:29884772 DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety
GO:0140318 protein transporter activity	IDA PMID:21362118 Recruiting mechanism of the AAA peroxins, Pex1p and Pex6p, t...	ACCEPT	Summary: PEX6 as part of the PEX1-PEX6 complex transports/extracts Ub-PEX5 from the peroxisomal membrane to the cytosol. Nashiro et al. (PMID:21362118) demonstrated the requirement of PEX1-PEX6 for PEX5 export. Reason: PEX6 functions as part of a protein dislocase that transports Ub-PEX5 from the peroxisomal membrane to the cytosol. "Protein transporter activity" is appropriate for this extraction function. PMID:21362118 provides evidence for the transport/export step. Supporting Evidence: PMID:21362118 peroxisomal localization of Pex1p and Pex6p is indispensable for the transport of matrix proteins
GO:0140318 protein transporter activity	IDA PMID:29884772 Peroxisomal monoubiquitinated PEX5 interacts with the AAA AT...	ACCEPT	Summary: Pedrosa et al. (PMID:29884772) directly showed that PEX1-PEX6 extracts Ub-PEX5 from the peroxisomal DTM in an ATP-dependent manner, transporting it to the cytosol. Reason: Duplicate annotation for protein transporter activity with evidence from a different publication. PMID:29884772 provides the strongest mechanistic evidence that PEX1-PEX6 acts as a protein transporter/dislocase. Supporting Evidence: PMID:29884772 DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex
GO:0016562 protein import into peroxisome matrix, receptor recycling	IDA PMID:19208625 Properties of the ubiquitin-pex5p thiol ester conjugate.	ACCEPT	Summary: Grou et al. (PMID:19208625) studied properties of the Ub-PEX5 thiol ester conjugate. They showed that soluble Ub-PEX5 retains cargo-binding capacity and can reenter the DTM, and that monoubiquitination at Cys-11 is a requisite for ATP-dependent export/recycling by PEX1-PEX6. Reason: This annotation is appropriate. PMID:19208625 provides evidence on the PEX5 recycling mechanism, demonstrating that monoubiquitination is required for PEX5 export (mediated by PEX1-PEX6). Supporting Evidence: PMID:19208625 Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol
GO:0140036 ubiquitin-modified protein reader activity	IDA PMID:19208625 Properties of the ubiquitin-pex5p thiol ester conjugate.	ACCEPT	Summary: Grou et al. (PMID:19208625) showed that monoubiquitination of PEX5 is the signal for ATP-dependent extraction by the PEX1-PEX6 complex. The ubiquitin moiety serves as the recognition signal for the REM. Reason: This annotation is appropriate. The study demonstrates that ubiquitin conjugation to PEX5 is required for its recognition and export by the PEX1-PEX6 receptor export module, supporting ubiquitin-modified protein reader activity. Supporting Evidence: PMID:19208625 Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol PMID:19208625 Pex5(C11S)p is a very poor substrate for peroxisome-dependent monoubiquitination, it accumulates at the peroxisomal membrane
GO:0016562 protein import into peroxisome matrix, receptor recycling	IDA PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: Tamura et al. (PMID:16854980) characterized the functional assembly of PEX1-PEX6 and showed that ATP binding in both D1 and D2 domains is required for complex formation, peroxisomal localization, and peroxisome-restoring activity. Reason: Core function annotation. PMID:16854980 demonstrates the functional requirements of PEX6 for peroxisome biogenesis and receptor recycling through mutagenesis of Walker A/B motifs. Supporting Evidence: PMID:16854980 The AAA cassettes, D1 and D2, were essential for peroxisome-restoring activity of Pex1p and Pex6p.
GO:0043335 protein unfolding	IDA PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: PMID:16854980 (Tamura et al.) describes conformational changes in PEX1 upon interaction with PEX6 but does not directly demonstrate substrate protein unfolding activity. The protein unfolding evidence for PEX6 primarily comes from PMID:29884772 and structural studies. Reason: Although PMID:16854980 itself describes conformational changes in PEX1 upon binding PEX6 rather than direct substrate unfolding, protein unfolding is a well-established core function of the PEX1-PEX6 complex, confirmed by PMID:29884772 and the deep research review (PEX6-deep-research-falcon.md). The annotation is consistent with other IBA and IDA annotations for the same term and reflects an established core function. Supporting Evidence: PMID:16854980 Interaction of Pex1p with Pex6p conferred a conformational change and dissociation of the Pex1p oligomer.
GO:0140318 protein transporter activity	IDA PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: Tamura et al. (PMID:16854980) demonstrated that PEX1-PEX6 complex formation and peroxisomal localization are essential for peroxisome biogenesis, which requires PEX5 transport/recycling. Walker A mutants that cannot bind ATP fail to restore peroxisomes. Reason: The evidence from PMID:16854980 supports the protein transporter activity of PEX6 as part of the PEX1-PEX6 complex. The study shows that ATP-dependent assembly and localization of PEX1-PEX6 are required for peroxisome-restoring activity, which involves PEX5 transport/recycling. Supporting Evidence: PMID:16854980 The AAA cassettes, D1 and D2, were essential for peroxisome-restoring activity of Pex1p and Pex6p.
GO:0016562 protein import into peroxisome matrix, receptor recycling	IDA PMID:16314507 Shuttling mechanism of peroxisome targeting signal type 1 re...	ACCEPT	Summary: Miyata and Fujiki (PMID:16314507) established the cell-free PEX5 translocation system and directly demonstrated that PEX1 and PEX6 are essential for PEX5 export from peroxisomes, not for PEX5 import. ATP was required for export but not import. Reason: This is a landmark study demonstrating the specific role of PEX1/PEX6 in PEX5 receptor recycling/export. PMID:16314507 provides the clearest direct evidence that PEX6 is required for the export (recycling) step, not the import step of PEX5. Supporting Evidence: PMID:16314507 In contrast, (35)S-Pex5 was imported into the peroxisome remnants of PEX1-, PEX6-, and PEX26-defective cell mutants, including those from patients with peroxisome biogenesis disorders, from which, however, (35)S-Pex5 was not exported, thereby indicating that Pex1 and Pex6 of the AAA ATPase family and their recruiter, Pex26, were essential for Pex5 export
GO:0005829 cytosol	TAS Reactome:R-HSA-9033499	ACCEPT	Summary: Reactome pathway annotation for the PEX1-PEX6-PEX26-ZFAND6 complex dissociating Ub:PEX5L from the DTM. The complex acts in the cytosol during PEX5 extraction/recycling. Reason: Correct TAS annotation from Reactome. PEX6 has a cytosolic pool and acts at the cytosol-peroxisome interface. Consistent with experimental evidence. Supporting Evidence: PMID:16854980 endogenous Pex1p was partly localized likely as a homo-oligomer in the cytoplasm
GO:0005829 cytosol	TAS Reactome:R-HSA-9033516	ACCEPT	Summary: Reactome pathway annotation for binding of PEX1-PEX6-PEX26 and ZFAND6 to PEX2:PEX10:PEX12:Ub:PEX5L:PEX7:PEX13:PEX14 complex. Reason: Correct TAS annotation from Reactome. Consistent with other cytosol annotations. Supporting Evidence: PMID:16854980 endogenous Pex1p was partly localized likely as a homo-oligomer in the cytoplasm
GO:0005829 cytosol	TAS Reactome:R-HSA-9033533	ACCEPT	Summary: Reactome pathway annotation for binding of PEX1-PEX6-PEX26 and ZFAND6 to PEX2:PEX10:PEX12:Ub:PEX5S,L:PEX13:PEX14 complex. Reason: Correct TAS annotation from Reactome. Consistent with other cytosol annotations. Supporting Evidence: PMID:16854980 endogenous Pex1p was partly localized likely as a homo-oligomer in the cytoplasm
GO:0097733 photoreceptor cell cilium	IDA PMID:26593283 PEX6 is Expressed in Photoreceptor Cilia and Mutated in Deaf...	KEEP AS NON CORE	Summary: Zaki et al. (PMID:26593283) demonstrated PEX6 localization to cilia of retinal photoreceptor cells using immunofluorescence. This links PEX6 to ciliopathy phenotypes including the retinal degeneration and deafblindness seen in Heimler syndrome. Reason: This is a valid localization annotation supported by experimental evidence but represents a tissue-specific, non-core localization. PEX6 is primarily a peroxisomal protein, but it has additional roles in photoreceptor cells. The photoreceptor cilium localization is relevant to disease (Heimler syndrome) but is not the primary cellular context for PEX6 function. Supporting Evidence: PMID:26593283 We show that Pex6 localizes to the apical extensions of secretory ameloblasts and differentiated odontoblasts at early stages of dentin synthesis in mice, and to cilia of retinal photoreceptor cells.
GO:0005515 protein binding	IPI PMID:16257970 Mutations in the peroxin Pex26p responsible for peroxisome b...	MODIFY	Summary: Furuki et al. (PMID:16257970) demonstrated that PEX26 disease mutations impair interaction with PEX1-PEX6 complex. The study confirms the PEX6-PEX26 interaction and its functional importance, but "protein binding" is an uninformative GO term. Reason: The PEX6-PEX26 interaction is real and important for PEX6 membrane recruitment, but "protein binding" is too vague per curation guidelines. A more informative term should describe the functional context of this interaction. Proposed replacements: ATP hydrolysis activity Supporting Evidence: PMID:16257970 the instability, insufficient binding to Pex1p x Pex6p complexes, or mislocalization of patient-derived Pex26p mutants is most likely responsible for the CG8 PBDs
GO:0007031 peroxisome organization	IMP PMID:8940266 Human peroxisome assembly factor-2 (PAF-2): a gene responsib...	ACCEPT	Summary: Fukuda et al. (PMID:8940266) cloned human PAF-2 (PEX6) and showed that expression morphologically and biochemically restores peroxisomes in group C Zellweger fibroblasts. Mutations in PEX6 cause peroxisome biogenesis disorder. Reason: PEX6 is essential for peroxisome organization/biogenesis. Loss of PEX6 function leads to peroxisome biogenesis disorders and complementation restores peroxisomes. This is a well supported core biological process annotation. Supporting Evidence: PMID:8940266 the full-length human PAF-2 cDNA that morphologically and biochemically restores peroxisomes of group C Zellweger fibroblasts
GO:0005737 cytoplasm	IDA PMID:8670792 The peroxisome biogenesis disorder group 4 gene, PXAAA1, enc...	ACCEPT	Summary: Yahraus et al. (PMID:8670792) described PEX6 (then called PXAAA1) as a predominantly cytoplasmic protein. This is a broader localization term than cytosol. Reason: Cytoplasm localization is correct, though cytosol (GO:0005829) is more specific and also annotated. PEX6 is found in the cytoplasm and on peroxisomal membranes. This original characterization study correctly identified the cytoplasmic localization. Supporting Evidence: PMID:8670792 The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein.
GO:0016561 protein import into peroxisome matrix, translocation	IMP PMID:8670792 The peroxisome biogenesis disorder group 4 gene, PXAAA1, enc...	MODIFY	Summary: Yahraus et al. (PMID:8670792) showed that PEX6 expression restores peroxisomal protein import in CG4 patient cells. However, PEX6 is specifically involved in the receptor recycling step rather than the translocation step of matrix protein import. Reason: The annotation to "translocation" (GO:0016561) is inaccurate for PEX6. Subsequent work (PMID:16314507) clearly showed PEX6 is required for PEX5 export/recycling, not for the translocation of cargo across the membrane. PEX5 import into pex6 mutant peroxisomes occurs normally; it is the export that fails. The correct term is GO:0016562 (receptor recycling), which is already well annotated. Proposed replacements: protein import into peroxisome matrix, receptor recycling Supporting Evidence: PMID:16314507 In contrast, (35)S-Pex5 was imported into the peroxisome remnants of PEX1-, PEX6-, and PEX26-defective cell mutants, including those from patients with peroxisome biogenesis disorders, from which, however, (35)S-Pex5 was not exported, thereby indicating that Pex1 and Pex6 of the AAA ATPase family and their recruiter, Pex26, were essential for Pex5 export PMID:8670792 Expression of PXAAA1 restored peroxisomal protein import in fibroblasts from 16 unrelated members of complementation group 4 (CG4) of the PBD.
GO:0016887 ATP hydrolysis activity	IMP PMID:8670792 The peroxisome biogenesis disorder group 4 gene, PXAAA1, enc...	ACCEPT	Summary: Yahraus et al. (PMID:8670792) showed that mutating the conserved lysine in the ATPase domain abolished biological activity, providing indirect evidence that PEX6 acts as an ATPase. Reason: Core molecular function annotation. The Walker A lysine mutation abolishing biological activity is strong IMP evidence for ATP hydrolysis activity. This is the foundational study establishing PEX6 ATPase activity. Supporting Evidence: PMID:8670792 Substitution of an arginine for the conserved lysine residue in the ATPase domain of Pxaaa1p abolished its biological activity, suggesting that Pxaaa1p is an ATPase.
GO:0050821 protein stabilization	IMP PMID:8670792 The peroxisome biogenesis disorder group 4 gene, PXAAA1, enc...	KEEP AS NON CORE	Summary: Yahraus et al. (PMID:8670792) found that PEX6 is required for stability of the PTS1 receptor (PEX5, then called Pxr1p). In PEX6-deficient cells, PEX5 is destabilized. Reason: The observation that PEX6 is required for PEX5 stability is correct but represents an indirect consequence of PEX6 function in receptor recycling rather than a direct molecular activity. When PEX5 cannot be recycled (due to PEX6 loss), it accumulates at the membrane and is targeted for proteasomal degradation (polyubiquitination), leading to instability. This is a downstream phenotypic consequence, not a direct protein stabilization function. Supporting Evidence: PMID:8670792 Pxaaa1p is required for stability of the predominantly cytoplasmic PTS1 receptor, Pxr1p. PMID:35805150 cells deficient in Pex1 or Pex6 have fewer peroxisomes than wildtype cells, which was eventually attributed to pexophagy
GO:0005515 protein binding	IPI PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	MODIFY	Summary: Tamura et al. (PMID:16854980) demonstrated interactions between PEX6 and PEX1 and PEX26 through co-immunoprecipitation and functional assays. "Protein binding" is too vague. Reason: Per curation guidelines, "protein binding" is an uninformative term. The interactions described (PEX6-PEX1 and PEX6-PEX26) reflect the functional assembly of the AAA ATPase complex and its membrane recruitment. A more specific molecular function term is preferred. Proposed replacements: ATP hydrolysis activity Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization.
GO:0005524 ATP binding	IMP PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: Tamura et al. (PMID:16854980) showed that Walker A mutations (K476E in D1, K750E in D2) abolished ATP binding and decreased interactions with PEX1 and PEX26, providing mutant phenotype evidence for ATP binding. Reason: Core molecular function annotation. Walker A lysine mutations that abolish ATP binding also abolish PEX6 function, providing strong IMP evidence. PEX6 has two ATP-binding sites (D1 and D2 domains). Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization.
GO:0005777 peroxisome	IDA PMID:11439091 Phenotype-genotype relationships in peroxisome biogenesis di...	ACCEPT	Summary: Tamura et al. (PMID:11439091) studied PEX1-PEX6 interaction in the context of peroxisome biogenesis disorders and showed PEX6 localization at peroxisomes through interaction with PEX1. Reason: Peroxisome localization is a core feature of PEX6. The broader term "peroxisome" (GO:0005777) is a parent of "peroxisomal membrane" (GO:0005778), both of which are valid. PEX6 associates with peroxisomes via PEX26 anchoring. Supporting Evidence: PMID:11439091 Pex1p-G843D interacted with Pex6p at approx. 50% of the level of normal Pex1p, whereas Pex1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p.
GO:0005777 peroxisome	IDA PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: Tamura et al. (PMID:16854980) showed PEX6 localization to peroxisomes in HEK293 cells, with PEX6 and PEX26 predominantly on peroxisomes. Reason: Correct localization annotation. PEX6 localizes to peroxisomes. Duplicate with different reference but same term, which is valid. Supporting Evidence: PMID:16854980 Pex6p and Pex26p were predominantly localized on peroxisomes.
GO:0005829 cytosol	IDA PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: Tamura et al. (PMID:16854980) detected PEX6 in the cytosol in addition to peroxisomal membranes. Reason: Correct cytosol localization. PEX6 is found in both the cytosol and on peroxisomal membranes. This dual localization is a core feature of PEX6 biology. Supporting Evidence: PMID:16854980 endogenous Pex1p was partly localized likely as a homo-oligomer in the cytoplasm, while Pex6p and Pex26p were predominantly localized on peroxisomes.
GO:0006625 protein targeting to peroxisome	IMP PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: Tamura et al. (PMID:16854980) showed that functional PEX6 (with intact AAA cassettes) is required for peroxisome-restoring activity, which involves the targeting of matrix proteins to peroxisomes via PEX5 recycling. Reason: PEX6 is essential for protein targeting to peroxisomes by recycling the PTS1 receptor PEX5. Loss of PEX6 function abolishes matrix protein import (PMID:16854980, PMID:8670792). While receptor recycling (GO:0016562) is the more specific mechanistic annotation, protein targeting to peroxisome captures the broader biological consequence of PEX6 function. Supporting Evidence: PMID:16854980 The AAA cassettes, D1 and D2, were essential for peroxisome-restoring activity of Pex1p and Pex6p.
GO:0016887 ATP hydrolysis activity	IMP PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: Tamura et al. (PMID:16854980) performed extensive mutagenesis of Walker A (K476E, K750E) and Walker B (D532N, D803N) motifs in both D1 and D2 domains of PEX6, demonstrating that ATP binding and hydrolysis are required for biological function. Reason: Strong IMP evidence for ATP hydrolysis activity. Both ATP binding (Walker A) and hydrolysis (Walker B) mutations in PEX6 impair function. This is the most detailed mutagenesis study of PEX6 ATPase activity. Supporting Evidence: PMID:16854980 ATP binding in both AAA cassettes but not ATP hydrolysis in D2 of both Pex1p and Pex6p was prerequisite for Pex1p-Pex6p interaction and their peroxisomal localization.
GO:0044877 protein-containing complex binding	IDA PMID:16854980 Dynamic and functional assembly of the AAA peroxins, Pex1p a...	ACCEPT	Summary: Tamura et al. (PMID:16854980) demonstrated PEX6 interactions with PEX1 (forming the heterohexamer) and PEX26, and identified binding regions between these proteins. PEX6 binds to protein-containing complexes (PEX1-PEX6 hexamer, PEX26 recruitment complex). Reason: PEX6 does bind protein-containing complexes. The term is somewhat general but the IDA evidence from PMID:16854980 directly demonstrates complex binding through co-immunoprecipitation and binding assays. Supporting Evidence: PMID:16854980 We herein assigned the binding regions between human Pex1p and Pex6p and elucidated pivotal roles of the AAA cassettes, called D1 and D2 domains, in Pex1p-Pex6p interaction and peroxisome biogenesis.

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