PIK3C3 (VPS34, Q8NEB9): Pexophagy and Peroxisome Localization — Focused Curation Review

Focus type: function_assignment · Hypothesis slug: pexophagy-and-peroxisome-localization
Terms adjudicated: GO:0000425 pexophagy (BP) and GO:0005777 peroxisome (CC)
Final report (compiled across 3 iterations; provenance verified via UniProt + QuickGO, 2026-09-21)

Executive Judgment

GO:0000425 pexophagy (BP): PARTIALLY SUPPORTED (retain as a conserved role).
GO:0005777 peroxisome (CC): WEAKLY SUPPORTED / likely OVER-ANNOTATED (flag; consider removal or non-core).

Both terms on human PIK3C3 are annotated IBA (Inferred from Biological aspect of Ancestor) from GO_Central — phylogenetic propagation within the VPS34 family, not human experimental evidence (UniProt Q8NEB9, fetched 2026-09-21). They must be adjudicated separately:

• Pexophagy is a real conserved VPS34 role. Yeast Vps34 (with its PtdIns3P-binding effectors) is experimentally mandatory for regulated peroxisome degradation (PMID 21121900). Mammalian pexophagy uses the same core class III PI3K/ATG apparatus to build the engulfing autophagosome. VPS34's contribution is generating PI3P on the phagophore/omegasome, an indirect-but-required function. An IBA BP annotation here is defensible; the caveat is that it is a general-autophagy-derived role, with no human IDA/IMP.

• Peroxisome as a cellular component is the weak claim. Human VPS34's directly demonstrated locations are late endosome, autophagosome, midbody, plasma membrane, and phagophore assembly site — not the peroxisome. The CC:peroxisome IBA most plausibly propagated from the yeast observation that "Vps34 is … associated with peroxisomes during biogenesis" (PMID 21121900). This conflates the engulfing autophagic membrane with residency on the peroxisomal cargo, exactly the distinction the seed hypothesis asks curators to make. The correct location term (GO:0000407 phagophore assembly site) is already annotated, making CC:peroxisome redundant and misleading for human.

Most important caveat: absence of a human peroxisome-localization assay is not proof of absence, but the well-characterized VPS34 localization repertoire argues against stable peroxisomal residency.

Annotation lineage (QuickGO, fetched 2026-09-21)

Both human terms are PAINT propagations (GO_REF:0000033, ECO:0000318) through PANTHER family PTN000005955, with withFrom = SGD:S000004230 (yeast VPS34, UniProt P22543). Tracing to the source, each human IBA rests on a single yeast experimental annotation from one paper (PMID 21121900):

This is the crux: the process claim is grounded in a yeast mutant-phenotype assay and conserves robustly; the location claim is grounded in a yeast localization assay whose organism-specific peroxisomal association is not expected to (and empirically does not) transfer to human VPS34, whose demonstrated locations are endosome/autophagosome/midbody/plasma membrane. Note both yeast source annotations derive from the same single publication, so neither human IBA has independent corroboration.

Evidence Matrix

Artifacts: /tmp/PIK3C3_pexophagy_evidence_matrix.csv (evidence matrix); UniProt GO dump reproduced in-run.

GO Curation Implications (leads — require curator verification)

• The MF basis of the gene product (GO:0016303 PI 3-kinase activity, IDA) is solid and is the mechanistic root of any pexophagy role.
• Do not default to "protein binding"; the informative location term for VPS34's autophagy action already exists (GO:0000407).

Mechanistic Scope

• Direct molecular activity: VPS34 is the catalytic subunit of class III PI3K; it phosphorylates PtdIns to PI3P. In autophagy it operates within Complex I (VPS34–VPS15/PIK3R4–Beclin1–ATG14) on the phagophore/omegasome, and Complex II (with UVRAG) on endosomes.
• Pexophagy (indirect, required): PI3P produced on the engulfing membrane recruits WIPI/ATG effectors that build the autophagosome around a peroxisome that has been selected by cargo machinery (PEX2 ubiquitination → NBR1/p62). VPS34's role is enabling autophagosome formation, not selecting or residing on peroxisomes.
• Downstream/context effects to keep separate: peroxisome number regulation, lipid metabolism, IFN-III/MAVS antiviral signaling (PMID 41186416) are consequences of pexophagy flux, not VPS34 molecular functions.

Conflicts and Alternatives

• Paralog/ortholog carry-over: The CC:peroxisome and BP:pexophagy IBAs derive from the yeast Vps34 ancestor. Yeast Vps34 associating with peroxisomes (PMID 21121900) is an organism-specific localization not shown for human VPS34 — a classic IBA over-reach for CC.
• Machinery vs cargo confusion: Mammalian pexophagy specificity is conferred by PEX2/NBR1 (PMID 27597759); VPS34 is a shared upstream factor for all macroautophagy. Assigning a peroxisome CC to a pan-autophagy kinase risks propagating cargo terms to every selective-autophagy factor.
• No isoform-specific peroxisomal targeting is reported for PIK3C3.

Knowledge Gaps

1. Human VPS34 at peroxisomes? Checked: UniProt curated locations — none peroxisomal. Matters because it is the crux of the CC decision. Resolve: high-resolution colocalization (VPS34/PI3P biosensor vs PMP70) during induced pexophagy; APEX2 peroxisomal proximity labeling.
2. Is human pexophagy VPS34-dependent experimentally? Checked: no human IDA/IMP found; inference is by conservation. Resolve: PIK3C3 KO/inhibitor (SAR405/VPS34-IN1) block of PEX2-driven peroxisome clearance.
3. Which VPS34 complex drives pexophagy (ATG14 vs UVRAG)? Resolve: ATG14/UVRAG depletion during starvation-induced pexophagy.

Discriminating Tests

• VPS34 inhibitor (SAR405/VPS34-IN1) + PEX2 overexpression → does peroxisome clearance require VPS34 activity in human cells? (tests BP causally in human).
• PI3P biosensor (2×FYVE) live imaging during pexophagy → is PI3P generated on the nascent phagophore around the peroxisome, and is VPS34 recruited to the phagophore rather than the peroxisome membrane? (discriminates engulfing-membrane vs cargo residency → the CC question).
• Peroxisomal APEX2 proximity proteomics → is endogenous VPS34 a peroxisomal neighbor? Negative result would support removing CC:peroxisome.

Curation Leads (require curator verification)

• Reference to attach for BP pexophagy (conservation): PMID 21121900 — snippet: "The function of Vps34p-containing complex I as well as a subset of PtdIns3P-binding proteins proved to be mandatory for the regulated degradation of peroxisomes. This demonstrates that PtdIns3P-mediated signalling is required for pexophagy."
• Reference clarifying mammalian machinery (VPS34 not cargo-resident): PMID 27597759 — "the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy … degradation of peroxisomes in an NBR1-dependent autophagic process."
• Reference for correct location of VPS34 activity: PMID 19223761 — "Atg14 localizes to isolation membrane/phagophore during starvation and is essential for autophagosome formation."
• Action change candidates:
• GO:0000425 (BP): retain; annotate as conserved/non-core; keep IBA (no human experimental upgrade available).
• GO:0005777 (CC): flag for removal or reclassification as non-core; GO:0000407 (phagophore assembly site) already captures the correct autophagy location.
• Suggested curator questions: (1) Is there any human dataset placing VPS34 at the peroxisome membrane/lumen? (2) Should pan-autophagy factors inherit selective-autophagy cargo CC terms by IBA at all?
• Suggested experiments: VPS34-inhibitor pexophagy block; 2×FYVE/PI3P imaging; peroxisomal APEX2 proximity labeling (above).

Bottom line

Adjudicated independently: pexophagy (BP) is a genuine conserved VPS34 role — retain (non-core, conservation-based); peroxisome (CC) is over-annotated for human VPS34 — flag for removal/non-core, because VPS34 acts on the engulfing autophagic membrane (phagophore, already annotated as GO:0000407), not as a resident of the peroxisomal cargo. Both current annotations are IBA-only, so neither reflects human experimental evidence.