ppgn-1 (Y38F2AR.7) encodes the Caenorhabditis elegans paraplegin-subfamily subunit of the mitochondrial m-AAA protease, an ATP-dependent proteolytic machine embedded in the inner mitochondrial membrane. The protein has the diagnostic two-module architecture of this family: an N-terminal AAA+ ATPase module that uses ATP binding and hydrolysis to unfold and translocate substrates, and a C-terminal M41 (FtsH-type) zinc-metallopeptidase module that cleaves peptide bonds; the catalytic residues of both modules (Walker A P-loop GPPGCGKT, Walker B IIYIDE, and the HExxH zinc-binding motif HEAGH) are intact in the ppgn-1 sequence. It is predicted to be an integral, multi-pass inner-membrane protein with matrix-facing catalytic domains and an N-terminal mitochondrial targeting region. Within the conserved m-AAA complex, family members carry out two coupled activities: quality-control degradation of misfolded or unassembled inner-membrane proteins, and regulated proteolytic maturation of specific substrates (the paradigm being processing of the ribosomal protein MrpL32 to control mitochondrial ribosome assembly and translation). The orthologous human subunit is paraplegin (SPG7), whose loss causes autosomal-recessive hereditary spastic paraplegia. The specific in vivo role, substrates, and complex partners of ppgn-1 in C. elegans have not been experimentally determined; C. elegans additionally carries the well-studied AFG3L2-type m-AAA subunit spg-7.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0034982
mitochondrial protein processing
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: Phylogenetic (IBA) propagation of the conserved m-AAA processing role from paraplegin/AFG3L2 orthologs. Consistent with ppgn-1's intact M41 protease domain and the family paradigm of regulated substrate maturation (e.g. MrpL32). Retained as a core function, with the caveat that no C. elegans substrate has been shown.
Reason: ppgn-1 is a clear paraplegin-subfamily m-AAA subunit (PANTHER PTHR43655:SF8) with a complete M41 zinc-peptidase module (HExxH motif HEAGH intact at residue 555), so the conserved processing activity is appropriately propagated. The direct substrate in the worm is unknown (see knowledge_gaps).
Supporting Evidence:
PMID:16239145
The mitochondrial ribosomal protein MrpL32 is processed by the m-AAA protease, allowing its association with preassembled ribosomal particles and completion of ribosome assembly in close proximity to the inner membrane.
|
|
GO:0005745
m-AAA complex
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: IBA propagation that ppgn-1 is a subunit of the inner-membrane m-AAA protease complex. Well supported by orthology to paraplegin (a conserved m-AAA subunit) and by the shared two-module (AAA+ + M41) architecture. Core.
Reason: Paraplegin is a conserved subunit of the m-AAA complex; ppgn-1 is its C. elegans ortholog. The specific composition of the worm complex (homo-oligomer vs hetero-oligomer with the AFG3L2-type subunit spg-7) is not experimentally established.
Supporting Evidence:
PMID:16647881
paraplegin, which is a conserved subunit of the ubiquitous and ATP-dependent m-AAA protease in mitochondria.
|
|
GO:0004222
metalloendopeptidase activity
|
IBA
GO_REF:0000033 |
ACCEPT |
Summary: IBA propagation of zinc-dependent endopeptidase activity from the family. Directly corroborated for the ppgn-1 sequence by the intact M41 HExxH zinc-binding catalytic motif (HEAGH at residue 555, within the Peptidase_M41 domain). Core molecular function.
Reason: The C-terminal M41 peptidase domain (InterPro IPR000642) and the intact HExxH motif support metalloendopeptidase chemistry for this specific protein, not merely by family membership.
Supporting Evidence:
PMID:9635427
Paraplegin is highly homologous to the yeast mitochondrial ATPases, AFG3, RCA1, and YME1, which have both proteolytic and chaperon-like activities at the inner mitochondrial membrane.
|
|
GO:0004176
ATP-dependent peptidase activity
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: InterPro (IEA) assignment of ATP-dependent peptidase activity. This is the most complete single molecular-function term for an m-AAA protease because it captures the coupling of the AAA+ ATPase to the M41 peptidase. Supported by the intact Walker A/B (GPPGCGKT, IIYIDE) and HExxH motifs in the ppgn-1 sequence. Core.
Reason: ppgn-1 has both an intact AAA+ ATPase module (Walker A GPPGCGKT at 325, Walker B IIYIDE at 380) and an intact M41 peptidase module (HExxH at 555), the defining combination for ATP-dependent proteolysis.
Supporting Evidence:
PMID:16239145
AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases that ensures the quality control of mitochondrial inner-membrane proteins.
|
|
GO:0004222
metalloendopeptidase activity
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: Second annotation of metalloendopeptidase activity, here from the InterPro M41 signature (IEA). Same well-supported molecular function as the IBA annotation above; kept as a corroborating electronic assignment.
Reason: Redundant with the IBA metalloendopeptidase annotation but independently supported by the InterPro Peptidase_M41 signature (IPR000642) and the intact HExxH motif.
|
|
GO:0005524
ATP binding
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: InterPro (IEA) assignment of ATP binding, an activity of the AAA+ module. Corroborated by the intact Walker A P-loop (GPPGCGKT at residue 325) in the ppgn-1 sequence. A component activity of the ATP-dependent protease.
Reason: The N-terminal AAA+ ATPase domain (SMART SM00382; InterPro IPR003959) with an intact Walker A motif provides a direct structural basis for ATP binding.
|
|
GO:0005739
mitochondrion
|
IEA
GO_REF:0000044 |
KEEP AS NON CORE |
Summary: Subcellular-location mapping (IEA) to mitochondrion. Correct but non-specific: the family resides in the inner mitochondrial membrane, captured more precisely in core_functions (GO:0005743). Retained but marked non-core because it is a generic parent of the informative location.
Reason: Consistent with the orthologous localization (paraplegin localizes to mitochondria) and with two predicted TM helices; the more informative term is mitochondrial inner membrane, so this generic term is not the core localization statement.
Supporting Evidence:
PMID:9635427
Immunofluorescence analysis and import experiments showed that Paraplegin localizes to mitochondria.
|
|
GO:0006508
proteolysis
|
IEA
GO_REF:0000002 |
KEEP AS NON CORE |
Summary: InterPro (IEA) generic proteolysis term. Correct but uninformative: the specific biological roles (protein quality-control degradation and mitochondrial protein processing) are captured in core_functions. Retained as a non-core parent term.
Reason: GO:0006508 is a high-level parent of the specific m-AAA processes; it is not wrong, but the informative annotations are mitochondrial protein processing (GO:0034982) and protein quality control (GO:0006515).
|
|
GO:0016020
membrane
|
IEA
GO_REF:0000044 |
KEEP AS NON CORE |
Summary: Subcellular-location mapping (IEA) to the generic term membrane. The protein is a multi-pass integral membrane protein, but the informative location is the mitochondrial inner membrane (GO:0005743), used in core_functions. Non-core parent.
Reason: Correct (two predicted TM helices at 111-133 and 225-245; UniProt multi-pass membrane protein) but far too generic to be a core localization statement.
|
|
GO:0016887
ATP hydrolysis activity
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: InterPro (IEA) assignment of ATP hydrolysis activity, the motor activity of the AAA+ module that drives substrate unfolding/translocation. Corroborated by the intact Walker B motif (IIYIDE at residue 380) in the ppgn-1 sequence. A component activity of the ATP-dependent protease.
Reason: The AAA+ ATPase module with intact Walker A and Walker B motifs supports ATP hydrolysis; this is the energy-providing half of the ATP-dependent protease.
|
|
GO:0005743
mitochondrial inner membrane
|
ISS | NEW |
Summary: Proposed refinement of the generic mitochondrion/membrane localizations to the specific compartment where m-AAA proteases reside. Inferred from orthology to paraplegin and from the two predicted TM helices flanking matrix-facing catalytic domains (canonical m-AAA topology).
Reason: The generic GOA terms (GO:0005739 mitochondrion, GO:0016020 membrane) understate the known compartment. The m-AAA protease is an integral protein of the mitochondrial inner membrane; this ISS annotation captures the informative location while the worm-specific topology remains to be experimentally mapped.
Supporting Evidence:
PMID:16647881
The m-AAA protease carries out protein quality control in the inner membrane of the mitochondria
|
|
GO:0006515
protein quality control for misfolded or incompletely synthesized proteins
|
ISS | NEW |
Summary: Proposed refinement of the generic proteolysis annotation to the specific quality-control process performed by the m-AAA protease: ATP-dependent degradation of misfolded/unassembled inner-membrane proteins. Inferred from orthology and the intact AAA+ + M41 catalytic modules.
Reason: GO:0006508 (proteolysis) is uninformative; the conserved biological role of the family is inner-membrane protein quality control. This ISS annotation records that specific process, complementing the mitochondrial protein processing (GO:0034982) role. The endogenous worm substrates are not yet identified.
Supporting Evidence:
PMID:16239145
AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases that ensures the quality control of mitochondrial inner-membrane proteins.
|
Q: Does ppgn-1 form a functional m-AAA protease complex on its own, or does it require the AFG3L2-type subunit spg-7, and are the two subunits functionally redundant in C. elegans?
Suggested experts: C. elegans mitochondrial biologists, m-AAA protease / AAA+ protease structural biochemists
Q: What are the endogenous substrates of ppgn-1 in C. elegans, and does it mature a MrpL32 homolog to regulate mitochondrial ribosome assembly as in yeast and mouse?
Suggested experts: Mitochondrial proteostasis / ribosome assembly researchers
Experiment: Generate a ppgn-1 null allele (CRISPR) and, in parallel with spg-7 RNAi/mutant, assay respiration, mitochondrial morphology, UPRmt (hsp-6p::GFP), fertility and lifespan to determine ppgn-1's non-redundant contribution.
Type: Reverse genetics / phenotyping
Experiment: Endogenously tag ppgn-1, purify from worm mitochondria, and identify complex partners (test for spg-7 hetero-oligomerization) and, using a catalytically dead HExxH mutant as a substrate trap, capture processed/degraded substrates.
Type: Affinity purification / mass spectrometry
Experiment: Determine ppgn-1 tissue expression and sub-mitochondrial topology with an endogenous fluorescent tag and protease-protection assays, to confirm the predicted inner-membrane, matrix-facing catalytic-domain topology.
Type: Localization
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: ppgn-1 has no experimental functional characterization in C. elegans: no loss-of-function (mutant or RNAi) phenotype, no localization, and no biochemical assay has been reported for this gene. Every GO annotation is phylogenetic (IBA) or electronic (IEA), and the only UniProt-linked reference is an unpublished nucleotide sequence submission.
OPEN BIOLOGY WHOLLY_DARK
What is known: The protein is confidently a paraplegin-subfamily m-AAA protease subunit (PANTHER PTHR43655:SF8; AAA+ + M41 architecture) with intact catalytic motifs, and the human ortholog paraplegin (SPG7) and yeast/mouse orthologs are well characterized. What is missing is any direct evidence for ppgn-1 itself in the worm.
Significance: Without any worm-level data, ppgn-1's function is entirely an orthology inference; its non-redundant contribution (if any) to C. elegans mitochondrial physiology is unknown.
What would resolve it: Generate and phenotype a ppgn-1 deletion/RNAi allele (respiration, mitochondrial morphology, UPRmt reporters, fertility, lifespan), and determine its expression and subcellular localization with an endogenous tag.
Provenance (the field's own admissions):
Gap: The endogenous substrate(s) of ppgn-1 in C. elegans are unidentified. It is unknown whether the worm protease matures a functional homolog of MrpL32 (regulated processing) and/or which misfolded inner-membrane proteins it degrades (quality control), and whether ppgn-1 and the AFG3L2-type subunit spg-7 act on the same or distinct substrate sets.
OPEN BIOLOGY MF_DARK
What is known: The conserved m-AAA protease has a defined dual activity (processing vs degradation), and MrpL32 maturation is the textbook processing substrate in yeast and mouse. ppgn-1 retains an intact HExxH metallopeptidase active site, so it is catalytically capable in principle.
Significance: Substrate identity is what distinguishes a housekeeping protease from a specific regulatory node; it is the rate-limiting unknown for placing ppgn-1 in a pathway.
What would resolve it: Substrate-trapping (catalytically inactive HExxH mutant) plus comparative proteomics of ppgn-1 vs spg-7 loss-of-function to identify processed/stabilized substrates.
Provenance (the field's own admissions):
Gap: The oligomeric composition of the C. elegans m-AAA complex containing ppgn-1 is undetermined: whether ppgn-1 forms a homo-oligomer, assembles into a hetero-oligomer with the AFG3L2-type subunit spg-7, and whether the two worm subunits are functionally redundant, is untested.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: In mammals the m-AAA protease is a hexamer of paraplegin (SPG7) and/or AFG3L2, and one catalytically dead subunit can be tolerated within an otherwise active complex. C. elegans has both a paraplegin-type (ppgn-1) and an AFG3L2-type (spg-7) subunit, but their assembly relationship is not reported.
Significance: Complex composition determines whether ppgn-1 loss can be buffered by spg-7 and how to interpret single-gene phenotypes.
What would resolve it: Co-immunoprecipitation / blue-native PAGE of endogenously tagged ppgn-1 and spg-7, and epistasis/double-mutant analysis to test redundancy.
Provenance (the field's own admissions):
Gap: Whether ppgn-1 (as opposed to its paralog spg-7) contributes to any organismal process in C. elegans - the mitochondrial unfolded-protein response, respiration, mitochondrial dynamics, fertility, longevity, or stress/pathogen resistance - is unknown. The large C. elegans mito-protease functional literature concerns spg-7 and cannot be attributed to ppgn-1.
OPEN BIOLOGY BP_DARK
What is known: Loss of m-AAA proteases across organisms causes respiratory deficiency, altered mitochondrial morphology and axonal degeneration; none of these readouts has been tested specifically for ppgn-1 in the worm.
Significance: Assigning ppgn-1 a biological process (vs treating it as a redundant paralog) requires worm phenotypes; this determines its relevance to mitochondrial surveillance and stress signaling.
What would resolve it: Test ppgn-1 loss-of-function against UPRmt (hsp-6p::GFP), respiration, mitochondrial morphology, and pathogen/stress-resistance assays, side by side with spg-7.
Provenance (the field's own admissions):
Research journal for the AI GO-annotation review. Provenance is recorded inline as
[PMID:xxxx "verbatim quote"]. This is a dark gene: the deliverable is a precise,
defensible statement of what is and is not known.
Bottom line on identity: despite the surrounding project framing, ppgn-1 is NOT an
epidermal/innate-immune/wound gene. It is the C. elegans paraplegin-subfamily
subunit of the mitochondrial m-AAA protease β an ATP-dependent, membrane-embedded
zinc metalloprotease of the inner mitochondrial membrane. All curation below is against
that identity.
The family is well characterised in yeast, mouse and human; ppgn-1's GO annotations are
IBA/IEA propagations from these orthologues.
To guard against propagating enzyme annotations onto a possible pseudoenzyme, I scanned
the ppgn-1 sequence itself (see ppgn-1-bioinformatics/, reproducible
analyze_motifs.py). All three catalytic motifs are intact and correctly ordered:
Interpretation: ppgn-1 is a catalytically competent m-AAA protease at the sequence
level, not a degenerate pseudoenzyme. This supports the enzymatic GO terms for this
specific protein, not merely by family membership. See
file:worm/ppgn-1/ppgn-1-bioinformatics/RESULTS.md.
KNOWN (high confidence, from orthology + domain/motif evidence):
- Encodes a paraplegin-subfamily m-AAA protease subunit (AAA+ ATPase + M41 Zn-peptidase).
- Retains intact ATP-binding (Walker A), ATP-hydrolysis (Walker B) and Zn-peptidase
(HExxH) catalytic motifs β catalytically competent in principle.
- Predicted mitochondrial, integral inner-membrane (two TM helices), matrix-facing
catalytic domains β the canonical m-AAA topology.
- Family function: ATP-dependent proteolytic quality control + regulated processing of
inner-membrane / matrix substrates.
NOT KNOWN (the real gaps for ppgn-1 specifically):
- No C. elegans experimental characterisation of ppgn-1: no mutant/RNAi phenotype,
no localisation, no biochemistry has been reported for this gene (all GO evidence is
IBA/IEA; the only UniProt reference is an unpublished sequence submission).
- Substrates unknown: which inner-membrane proteins ppgn-1 degrades or matures in the
worm is undetermined (no worm equivalent of the MrpL32 result attributed to ppgn-1).
- Complex composition unknown: whether ppgn-1 assembles with the AFG3L2-type subunit
spg-7 into a hetero-hexamer, forms a homo-oligomer, or is catalytically redundant with
spg-7 is untested.
- Physiological role unknown: whether ppgn-1 (as opposed to spg-7) contributes to
UPR^mt, respiration, mitochondrial morphology, fertility, longevity, stress/pathogen
responses, or is functionally dispensable, is unknown.
- Regulation unknown: expression, tissue distribution (Bgee: "Expressed in germ line
and 4 other cell types" β coarse, electronic), and conditions under which it acts are
uncharacterised.
All 10 GOA annotations are IBA/IEA and are consistent with a catalytically competent
paraplegin-subfamily m-AAA protease; none is contradicted, so none is REMOVEd.
- Core, well-supported family propagations β ACCEPT: mitochondrial protein processing
(IBA), m-AAA complex (IBA), metalloendopeptidase activity (IBA + IEA), ATP-dependent
peptidase activity (IEA), ATP binding (IEA), ATP hydrolysis activity (IEA).
- Correct but non-specific / non-core β KEEP_AS_NON_CORE with a note: proteolysis
(GO:0006508, generic parent of the specific processing/QC role), mitochondrion
(GO:0005739, generic parent of inner membrane), membrane (GO:0016020, generic parent
of inner membrane).
- The persistent uncertainty (no worm data) is captured in knowledge_gaps, not by
down-grading correct family propagations.
just deep-research-falcon worm ppgn-1 --fallback perplexity-lite was run FOREGROUND and
retried. Falcon timed out at the 600 s server limit on both attempts; the perplexity-lite
fallback returned HTTP 401 (account quota exhausted). No -deep-research-*.md file was
produced. Per repo policy I did not fabricate one. This review therefore rests on the
UniProt/InterPro record, the GOA evidence codes, the primary m-AAA/paraplegin literature
cited above (PubMed-verified, cached in publications/), and the reproducible sequence
analysis in ppgn-1-bioinformatics/.
The GO annotations for ppgn-1 (Y38F2AR.7, C. elegans paraplegin subfamily) are all
IBA/IEA β propagated from the m-AAA protease family, with no C. elegans experimental
support. A legitimate worry for any family propagation is that the target is a
pseudoenzyme: a fold-retaining paralog that has lost the catalytic residues (cf.
human AFG3L1P pseudogene, or the peptidase-dead YME1L variants). If ppgn-1 had lost its
Walker A/B or HExxH residues, the enzymatic annotations (metalloendopeptidase, ATP
binding, ATP hydrolysis, ATP-dependent peptidase) would be over-annotations for this
specific protein even though they are correct for the family.
analyze_motifs.py parses the amino-acid sequence directly from ../ppgn-1-uniprot.txt
(nothing hard-coded) and scans for the three diagnostic motifs of an m-AAA protease:
G-x(4)-G-K-[S/T]) β binds the Ξ²/Ξ³ phosphates of ATP.hhhh-D-E) β the Asp/Glu that coordinate MgΒ²βΊ and activate water for ATP hydrolysis.Run with uv run python analyze_motifs.py.
length: 747 aa
Walker A (P-loop, ATP binding): FOUND at 325 -> GPPGCGKT
Walker B (Mg-ATP hydrolysis): FOUND at 380 -> IIYIDE
HExxH (M41 zinc-binding catalytic motif): FOUND at 555 -> HEAGH
HExxH context [555..589]: HEAGHALVGWMLEHTDALLKVTIIPRTSAALGFAQ
All three catalytic elements are intact and in the expected order (AAA+ ATPase module
N-terminal, M41 peptidase module C-terminal), consistent with the domain architecture
UniProt/InterPro annotate (AAA+ core IPR003959; Peptidase M41 IPR000642). The HExxH lies
within the M41 domain and is followed downstream by an acidic residue (β¦MLEHTD),
consistent with a complete FtsH-type ZnΒ²βΊ active site.
At the sequence level ppgn-1 is a catalytically competent m-AAA protease subunit, not
a degenerate pseudoenzyme: it retains the residues required for both ATP binding/hydrolysis
and zinc-dependent peptide-bond cleavage. This supports (for this specific protein, not
merely by family membership) the molecular-function annotations ATP binding,
ATP hydrolysis activity, metalloendopeptidase activity, and ATP-dependent peptidase
activity.
id: G5EDB6
gene_symbol: ppgn-1
product_type: PROTEIN
status: DRAFT
taxon:
id: NCBITaxon:6239
label: Caenorhabditis elegans
description: >-
ppgn-1 (Y38F2AR.7) encodes the Caenorhabditis elegans paraplegin-subfamily subunit
of the mitochondrial m-AAA protease, an ATP-dependent proteolytic machine embedded in
the inner mitochondrial membrane. The protein has the diagnostic two-module
architecture of this family: an N-terminal AAA+ ATPase module that uses ATP binding and
hydrolysis to unfold and translocate substrates, and a C-terminal M41 (FtsH-type)
zinc-metallopeptidase module that cleaves peptide bonds; the catalytic residues of both
modules (Walker A P-loop GPPGCGKT, Walker B IIYIDE, and the HExxH zinc-binding motif
HEAGH) are intact in the ppgn-1 sequence. It is predicted to be an integral, multi-pass
inner-membrane protein with matrix-facing catalytic domains and an N-terminal
mitochondrial targeting region. Within the conserved m-AAA complex, family members
carry out two coupled activities: quality-control degradation of misfolded or
unassembled inner-membrane proteins, and regulated proteolytic maturation of specific
substrates (the paradigm being processing of the ribosomal protein MrpL32 to control
mitochondrial ribosome assembly and translation). The orthologous human subunit is
paraplegin (SPG7), whose loss causes autosomal-recessive hereditary spastic paraplegia.
The specific in vivo role, substrates, and complex partners of ppgn-1 in C. elegans
have not been experimentally determined; C. elegans additionally carries the
well-studied AFG3L2-type m-AAA subunit spg-7.
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000033
title: Annotation inferences using phylogenetic trees
findings: []
- id: GO_REF:0000044
title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
vocabulary mapping, accompanied by conservative changes to GO terms applied by
UniProt
findings: []
- id: PMID:9635427
title: "Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin,
a nuclear-encoded mitochondrial metalloprotease."
findings:
- statement: >-
Paraplegin (the ortholog of ppgn-1) is a nuclear-encoded mitochondrial
metalloprotease homologous to the yeast mitochondrial AAA ATPases (AFG3, RCA1,
YME1) that combine proteolytic and chaperone-like activities at the inner
mitochondrial membrane, and it localizes to mitochondria.
supporting_text: >-
Paraplegin is highly homologous to the yeast mitochondrial ATPases, AFG3, RCA1,
and YME1, which have both proteolytic and chaperon-like activities at the inner
mitochondrial membrane.
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: >-
PubMed-verified founding paper defining paraplegin as the mitochondrial m-AAA
metalloprotease subunit orthologous to ppgn-1; establishes family identity,
mitochondrial localization, and dual protease/chaperone character. Concerns human
SPG7, not C. elegans ppgn-1, so it supports the annotations by orthology only.
- id: PMID:16239145
title: "The m-AAA protease defective in hereditary spastic paraplegia controls ribosome
assembly in mitochondria."
findings:
- statement: >-
AAA proteases are a conserved family of membrane-bound ATP-dependent proteases that
ensure quality control of mitochondrial inner-membrane proteins, and the m-AAA
protease processes the ribosomal protein MrpL32 to allow completion of ribosome
assembly at the inner membrane.
supporting_text: >-
AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases
that ensures the quality control of mitochondrial inner-membrane proteins.
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: >-
PubMed-verified; defines the conserved MF/BP of the family (ATP-dependent
inner-membrane quality control and the MrpL32 processing paradigm) that the ppgn-1
IBA annotations propagate. Yeast/mouse study, not C. elegans.
- id: PMID:16647881
title: "Translating m-AAA protease function in mitochondria to hereditary spastic paraplegia."
findings:
- statement: >-
Paraplegin is a conserved subunit of the ubiquitous, ATP-dependent m-AAA protease
in mitochondria, which carries out protein quality control in the inner
mitochondrial membrane.
supporting_text: >-
paraplegin, which is a conserved subunit of the ubiquitous and ATP-dependent m-AAA
protease in mitochondria.
reference_review:
relevance: HIGH
correctness: VERIFIED
review_notes: >-
PubMed-verified review supporting the m-AAA complex membership (part_of GO:0005745)
and the inner-membrane quality-control role by orthology to paraplegin.
- id: PMID:21610694
title: "Presequence-dependent folding ensures MrpL32 processing by the m-AAA protease
in mitochondria."
findings:
- statement: >-
m-AAA proteases have dual functions in the inner mitochondrial membrane: regulated
processing of specific substrates and quality-control degradation of misfolded
polypeptides to peptides.
supporting_text: >-
m-AAA proteases exert dual functions in the mitochondrial inner membrane: they
mediate the processing of specific regulatory proteins and ensure protein quality
control degrading misfolded polypeptides to peptides.
reference_review:
relevance: MEDIUM
correctness: VERIFIED
review_notes: >-
PubMed-verified; articulates the processing-vs-degradation dual role underlying the
distinction between the two core functions. Yeast substrate mechanism, not worm.
existing_annotations:
- term:
id: GO:0034982
label: mitochondrial protein processing
evidence_type: IBA
original_reference_id: GO_REF:0000033
qualifier: involved_in
review:
summary: >-
Phylogenetic (IBA) propagation of the conserved m-AAA processing role from
paraplegin/AFG3L2 orthologs. Consistent with ppgn-1's intact M41 protease domain
and the family paradigm of regulated substrate maturation (e.g. MrpL32). Retained
as a core function, with the caveat that no C. elegans substrate has been shown.
action: ACCEPT
reason: >-
ppgn-1 is a clear paraplegin-subfamily m-AAA subunit (PANTHER PTHR43655:SF8) with a
complete M41 zinc-peptidase module (HExxH motif HEAGH intact at residue 555), so the
conserved processing activity is appropriately propagated. The direct substrate in
the worm is unknown (see knowledge_gaps).
supported_by:
- reference_id: PMID:16239145
supporting_text: >-
The mitochondrial ribosomal protein MrpL32 is processed by the m-AAA protease,
allowing its association with preassembled ribosomal particles and completion of
ribosome assembly in close proximity to the inner membrane.
- term:
id: GO:0005745
label: m-AAA complex
evidence_type: IBA
original_reference_id: GO_REF:0000033
qualifier: part_of
review:
summary: >-
IBA propagation that ppgn-1 is a subunit of the inner-membrane m-AAA protease
complex. Well supported by orthology to paraplegin (a conserved m-AAA subunit) and
by the shared two-module (AAA+ + M41) architecture. Core.
action: ACCEPT
reason: >-
Paraplegin is a conserved subunit of the m-AAA complex; ppgn-1 is its C. elegans
ortholog. The specific composition of the worm complex (homo-oligomer vs
hetero-oligomer with the AFG3L2-type subunit spg-7) is not experimentally
established.
supported_by:
- reference_id: PMID:16647881
supporting_text: >-
paraplegin, which is a conserved subunit of the ubiquitous and ATP-dependent
m-AAA protease in mitochondria.
- term:
id: GO:0004222
label: metalloendopeptidase activity
evidence_type: IBA
original_reference_id: GO_REF:0000033
qualifier: enables
review:
summary: >-
IBA propagation of zinc-dependent endopeptidase activity from the family. Directly
corroborated for the ppgn-1 sequence by the intact M41 HExxH zinc-binding catalytic
motif (HEAGH at residue 555, within the Peptidase_M41 domain). Core molecular
function.
action: ACCEPT
reason: >-
The C-terminal M41 peptidase domain (InterPro IPR000642) and the intact HExxH motif
support metalloendopeptidase chemistry for this specific protein, not merely by
family membership.
supported_by:
- reference_id: PMID:9635427
supporting_text: >-
Paraplegin is highly homologous to the yeast mitochondrial ATPases, AFG3, RCA1,
and YME1, which have both proteolytic and chaperon-like activities at the inner
mitochondrial membrane.
- term:
id: GO:0004176
label: ATP-dependent peptidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: enables
review:
summary: >-
InterPro (IEA) assignment of ATP-dependent peptidase activity. This is the most
complete single molecular-function term for an m-AAA protease because it captures
the coupling of the AAA+ ATPase to the M41 peptidase. Supported by the intact
Walker A/B (GPPGCGKT, IIYIDE) and HExxH motifs in the ppgn-1 sequence. Core.
action: ACCEPT
reason: >-
ppgn-1 has both an intact AAA+ ATPase module (Walker A GPPGCGKT at 325, Walker B
IIYIDE at 380) and an intact M41 peptidase module (HExxH at 555), the defining
combination for ATP-dependent proteolysis.
supported_by:
- reference_id: PMID:16239145
supporting_text: >-
AAA proteases comprise a conserved family of membrane bound ATP-dependent
proteases that ensures the quality control of mitochondrial inner-membrane
proteins.
- term:
id: GO:0004222
label: metalloendopeptidase activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: enables
review:
summary: >-
Second annotation of metalloendopeptidase activity, here from the InterPro M41
signature (IEA). Same well-supported molecular function as the IBA annotation
above; kept as a corroborating electronic assignment.
action: ACCEPT
reason: >-
Redundant with the IBA metalloendopeptidase annotation but independently supported
by the InterPro Peptidase_M41 signature (IPR000642) and the intact HExxH motif.
- term:
id: GO:0005524
label: ATP binding
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: enables
review:
summary: >-
InterPro (IEA) assignment of ATP binding, an activity of the AAA+ module.
Corroborated by the intact Walker A P-loop (GPPGCGKT at residue 325) in the ppgn-1
sequence. A component activity of the ATP-dependent protease.
action: ACCEPT
reason: >-
The N-terminal AAA+ ATPase domain (SMART SM00382; InterPro IPR003959) with an
intact Walker A motif provides a direct structural basis for ATP binding.
- term:
id: GO:0005739
label: mitochondrion
evidence_type: IEA
original_reference_id: GO_REF:0000044
qualifier: located_in
review:
summary: >-
Subcellular-location mapping (IEA) to mitochondrion. Correct but non-specific: the
family resides in the inner mitochondrial membrane, captured more precisely in
core_functions (GO:0005743). Retained but marked non-core because it is a generic
parent of the informative location.
action: KEEP_AS_NON_CORE
reason: >-
Consistent with the orthologous localization (paraplegin localizes to mitochondria)
and with two predicted TM helices; the more informative term is mitochondrial inner
membrane, so this generic term is not the core localization statement.
supported_by:
- reference_id: PMID:9635427
supporting_text: >-
Immunofluorescence analysis and import experiments showed that Paraplegin
localizes to mitochondria.
- term:
id: GO:0006508
label: proteolysis
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: involved_in
review:
summary: >-
InterPro (IEA) generic proteolysis term. Correct but uninformative: the specific
biological roles (protein quality-control degradation and mitochondrial protein
processing) are captured in core_functions. Retained as a non-core parent term.
action: KEEP_AS_NON_CORE
reason: >-
GO:0006508 is a high-level parent of the specific m-AAA processes; it is not wrong,
but the informative annotations are mitochondrial protein processing (GO:0034982)
and protein quality control (GO:0006515).
- term:
id: GO:0016020
label: membrane
evidence_type: IEA
original_reference_id: GO_REF:0000044
qualifier: located_in
review:
summary: >-
Subcellular-location mapping (IEA) to the generic term membrane. The protein is a
multi-pass integral membrane protein, but the informative location is the
mitochondrial inner membrane (GO:0005743), used in core_functions. Non-core parent.
action: KEEP_AS_NON_CORE
reason: >-
Correct (two predicted TM helices at 111-133 and 225-245; UniProt multi-pass
membrane protein) but far too generic to be a core localization statement.
- term:
id: GO:0016887
label: ATP hydrolysis activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
qualifier: enables
review:
summary: >-
InterPro (IEA) assignment of ATP hydrolysis activity, the motor activity of the
AAA+ module that drives substrate unfolding/translocation. Corroborated by the
intact Walker B motif (IIYIDE at residue 380) in the ppgn-1 sequence. A component
activity of the ATP-dependent protease.
action: ACCEPT
reason: >-
The AAA+ ATPase module with intact Walker A and Walker B motifs supports ATP
hydrolysis; this is the energy-providing half of the ATP-dependent protease.
- term:
id: GO:0005743
label: mitochondrial inner membrane
evidence_type: ISS
qualifier: located_in
review:
summary: >-
Proposed refinement of the generic mitochondrion/membrane localizations to the
specific compartment where m-AAA proteases reside. Inferred from orthology to
paraplegin and from the two predicted TM helices flanking matrix-facing catalytic
domains (canonical m-AAA topology).
action: NEW
reason: >-
The generic GOA terms (GO:0005739 mitochondrion, GO:0016020 membrane) understate the
known compartment. The m-AAA protease is an integral protein of the mitochondrial
inner membrane; this ISS annotation captures the informative location while the
worm-specific topology remains to be experimentally mapped.
supported_by:
- reference_id: PMID:16647881
supporting_text: >-
The m-AAA protease carries out protein quality control in the inner membrane of
the mitochondria
- term:
id: GO:0006515
label: protein quality control for misfolded or incompletely synthesized proteins
evidence_type: ISS
qualifier: involved_in
review:
summary: >-
Proposed refinement of the generic proteolysis annotation to the specific
quality-control process performed by the m-AAA protease: ATP-dependent degradation
of misfolded/unassembled inner-membrane proteins. Inferred from orthology and the
intact AAA+ + M41 catalytic modules.
action: NEW
reason: >-
GO:0006508 (proteolysis) is uninformative; the conserved biological role of the
family is inner-membrane protein quality control. This ISS annotation records that
specific process, complementing the mitochondrial protein processing (GO:0034982)
role. The endogenous worm substrates are not yet identified.
supported_by:
- reference_id: PMID:16239145
supporting_text: >-
AAA proteases comprise a conserved family of membrane bound ATP-dependent
proteases that ensures the quality control of mitochondrial inner-membrane
proteins.
core_functions:
- description: >-
ATP-dependent proteolytic quality control of the inner mitochondrial membrane. As a
subunit of the m-AAA protease, ppgn-1 couples ATP-driven substrate unfolding and
translocation (AAA+ module) to zinc-dependent peptide-bond hydrolysis (M41 module) to
degrade misfolded or unassembled inner-membrane proteins to peptides. Inferred from
orthology to paraplegin/AFG3L2 and from the intact Walker A/B and HExxH catalytic
motifs in the ppgn-1 sequence; the endogenous C. elegans substrates are not yet
identified.
molecular_function:
id: GO:0004176
label: ATP-dependent peptidase activity
directly_involved_in:
- id: GO:0006515
label: protein quality control for misfolded or incompletely synthesized proteins
locations:
- id: GO:0005743
label: mitochondrial inner membrane
in_complex:
id: GO:0005745
label: m-AAA complex
supported_by:
- reference_id: PMID:16239145
supporting_text: >-
AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases
that ensures the quality control of mitochondrial inner-membrane proteins.
- reference_id: PMID:21610694
supporting_text: >-
m-AAA proteases exert dual functions in the mitochondrial inner membrane: they
mediate the processing of specific regulatory proteins and ensure protein quality
control degrading misfolded polypeptides to peptides.
- description: >-
Regulated proteolytic maturation (processing) of specific mitochondrial substrates.
Beyond bulk degradation, m-AAA proteases perform limited, sequence-specific cleavage
that matures regulatory substrates; the conserved paradigm is processing of the
ribosomal protein MrpL32, which is required for mitochondrial ribosome assembly and
translation. ppgn-1 is inferred to contribute this metalloendopeptidase-based
processing activity within the worm m-AAA complex; the specific worm substrate(s)
remain undetermined.
molecular_function:
id: GO:0004222
label: metalloendopeptidase activity
directly_involved_in:
- id: GO:0034982
label: mitochondrial protein processing
locations:
- id: GO:0005743
label: mitochondrial inner membrane
in_complex:
id: GO:0005745
label: m-AAA complex
supported_by:
- reference_id: PMID:16239145
supporting_text: >-
The mitochondrial ribosomal protein MrpL32 is processed by the m-AAA protease,
allowing its association with preassembled ribosomal particles and completion of
ribosome assembly in close proximity to the inner membrane.
knowledge_gaps:
- gap_statement: >-
ppgn-1 has no experimental functional characterization in C. elegans: no
loss-of-function (mutant or RNAi) phenotype, no localization, and no biochemical
assay has been reported for this gene. Every GO annotation is phylogenetic (IBA) or
electronic (IEA), and the only UniProt-linked reference is an unpublished nucleotide
sequence submission.
boundary: >-
The protein is confidently a paraplegin-subfamily m-AAA protease subunit (PANTHER
PTHR43655:SF8; AAA+ + M41 architecture) with intact catalytic motifs, and the human
ortholog paraplegin (SPG7) and yeast/mouse orthologs are well characterized. What is
missing is any direct evidence for ppgn-1 itself in the worm.
gap_kind:
- BIOLOGY
dark_aspect: WHOLLY_DARK
status: OPEN
significance: >-
Without any worm-level data, ppgn-1's function is entirely an orthology inference; its
non-redundant contribution (if any) to C. elegans mitochondrial physiology is unknown.
resolution: >-
Generate and phenotype a ppgn-1 deletion/RNAi allele (respiration, mitochondrial
morphology, UPRmt reporters, fertility, lifespan), and determine its expression and
subcellular localization with an endogenous tag.
provenance:
- reference_id: PMID:9635427
supporting_text: >-
Paraplegin is highly homologous to the yeast mitochondrial ATPases, AFG3, RCA1,
and YME1, which have both proteolytic and chaperon-like activities at the inner
mitochondrial membrane.
- gap_statement: >-
The endogenous substrate(s) of ppgn-1 in C. elegans are unidentified. It is unknown
whether the worm protease matures a functional homolog of MrpL32 (regulated
processing) and/or which misfolded inner-membrane proteins it degrades (quality
control), and whether ppgn-1 and the AFG3L2-type subunit spg-7 act on the same or
distinct substrate sets.
boundary: >-
The conserved m-AAA protease has a defined dual activity (processing vs degradation),
and MrpL32 maturation is the textbook processing substrate in yeast and mouse. ppgn-1
retains an intact HExxH metallopeptidase active site, so it is catalytically capable
in principle.
gap_kind:
- BIOLOGY
dark_aspect: MF_DARK
status: OPEN
significance: >-
Substrate identity is what distinguishes a housekeeping protease from a specific
regulatory node; it is the rate-limiting unknown for placing ppgn-1 in a pathway.
resolution: >-
Substrate-trapping (catalytically inactive HExxH mutant) plus comparative proteomics
of ppgn-1 vs spg-7 loss-of-function to identify processed/stabilized substrates.
provenance:
- reference_id: PMID:21610694
supporting_text: >-
m-AAA proteases exert dual functions in the mitochondrial inner membrane: they
mediate the processing of specific regulatory proteins and ensure protein quality
control degrading misfolded polypeptides to peptides.
- gap_statement: >-
The oligomeric composition of the C. elegans m-AAA complex containing ppgn-1 is
undetermined: whether ppgn-1 forms a homo-oligomer, assembles into a hetero-oligomer
with the AFG3L2-type subunit spg-7, and whether the two worm subunits are
functionally redundant, is untested.
boundary: >-
In mammals the m-AAA protease is a hexamer of paraplegin (SPG7) and/or AFG3L2, and one
catalytically dead subunit can be tolerated within an otherwise active complex. C.
elegans has both a paraplegin-type (ppgn-1) and an AFG3L2-type (spg-7) subunit, but
their assembly relationship is not reported.
gap_kind:
- BIOLOGY
dark_aspect: RESIDUAL_SUBGAP
status: OPEN
significance: >-
Complex composition determines whether ppgn-1 loss can be buffered by spg-7 and how to
interpret single-gene phenotypes.
resolution: >-
Co-immunoprecipitation / blue-native PAGE of endogenously tagged ppgn-1 and spg-7, and
epistasis/double-mutant analysis to test redundancy.
provenance:
- reference_id: PMID:16647881
supporting_text: >-
paraplegin, which is a conserved subunit of the ubiquitous and ATP-dependent m-AAA
protease in mitochondria.
- gap_statement: >-
Whether ppgn-1 (as opposed to its paralog spg-7) contributes to any organismal process
in C. elegans - the mitochondrial unfolded-protein response, respiration, mitochondrial
dynamics, fertility, longevity, or stress/pathogen resistance - is unknown. The large
C. elegans mito-protease functional literature concerns spg-7 and cannot be attributed
to ppgn-1.
boundary: >-
Loss of m-AAA proteases across organisms causes respiratory deficiency, altered
mitochondrial morphology and axonal degeneration; none of these readouts has been
tested specifically for ppgn-1 in the worm.
gap_kind:
- BIOLOGY
dark_aspect: BP_DARK
status: OPEN
significance: >-
Assigning ppgn-1 a biological process (vs treating it as a redundant paralog) requires
worm phenotypes; this determines its relevance to mitochondrial surveillance and
stress signaling.
resolution: >-
Test ppgn-1 loss-of-function against UPRmt (hsp-6p::GFP), respiration, mitochondrial
morphology, and pathogen/stress-resistance assays, side by side with spg-7.
provenance:
- reference_id: PMID:16239145
supporting_text: >-
Inactivation of AAA proteases causes pleiotropic phenotypes in various organisms,
including respiratory deficiencies, mitochondrial morphology defects, and axonal
degeneration in hereditary spastic paraplegia (HSP).
suggested_questions:
- question: >-
Does ppgn-1 form a functional m-AAA protease complex on its own, or does it require
the AFG3L2-type subunit spg-7, and are the two subunits functionally redundant in
C. elegans?
experts:
- C. elegans mitochondrial biologists
- m-AAA protease / AAA+ protease structural biochemists
- question: >-
What are the endogenous substrates of ppgn-1 in C. elegans, and does it mature a
MrpL32 homolog to regulate mitochondrial ribosome assembly as in yeast and mouse?
experts:
- Mitochondrial proteostasis / ribosome assembly researchers
suggested_experiments:
- experiment_type: Reverse genetics / phenotyping
description: >-
Generate a ppgn-1 null allele (CRISPR) and, in parallel with spg-7 RNAi/mutant, assay
respiration, mitochondrial morphology, UPRmt (hsp-6p::GFP), fertility and lifespan to
determine ppgn-1's non-redundant contribution.
- experiment_type: Affinity purification / mass spectrometry
description: >-
Endogenously tag ppgn-1, purify from worm mitochondria, and identify complex partners
(test for spg-7 hetero-oligomerization) and, using a catalytically dead HExxH mutant
as a substrate trap, capture processed/degraded substrates.
- experiment_type: Localization
description: >-
Determine ppgn-1 tissue expression and sub-mitochondrial topology with an endogenous
fluorescent tag and protease-protection assays, to confirm the predicted inner-membrane,
matrix-facing catalytic-domain topology.