ClpP (CG5045) encodes the proteolytic subunit of the Drosophila mitochondrial ClpXP protease, a member of peptidase family S14. ClpP subunits form a tetradecameric barrel of two heptameric rings whose sequestered chamber contains Ser-His-Asp catalytic sites; the AAA+ partner ClpX recognizes, unfolds and translocates substrates into the chamber using ATP. In the mitochondrial matrix ClpXP degrades specific proteins, including the RNA-binding protein DmLRPPRC1, thereby regulating mitochondrial mRNA levels, transcript processing and translation, and it contributes to mitochondrial protein quality control alongside the Lon protease.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004176 ATP-dependent peptidase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: ATP-dependent peptidase activity inherited from the ClpP ancestral node (E. coli and B. subtilis ClpP). Reason: The ATP dependence of ClpXP proteolysis is supplied by the ClpX ATPase; ClpP itself is the serine peptidase. The term describes the holoenzyme activity to which ClpP contributes, so it is defensible but not the most precise description of ClpP. Supporting Evidence: PMID:28814717 ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX, which carries an ATPase associated with diverse cellular activities (AAA+) domain6, 7. PMID:28814717 we established a copper-inducible cell line expressing a catalytically inactive ClpP mutant (S124A) in which the conserved serine in the proteolytic active site was replaced with alanine. |
| GO:0004176 ATP-dependent peptidase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: ATP-dependent peptidase activity from the ClpP InterPro family signature. Reason: Same as the IBA row; the precise ClpP activity is serine-type endopeptidase. Supporting Evidence: PMID:28814717 ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX, which carries an ATPase associated with diverse cellular activities (AAA+) domain6, 7. |
| GO:0004252 serine-type endopeptidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Serine endopeptidase activity inherited from the ClpP ancestral node, which includes the fly gene among the experimentally supported descendants. Reason: Fly ClpP retains the catalytic serine whose mutation abolishes function. Supporting Evidence: PMID:28814717 we established a copper-inducible cell line expressing a catalytically inactive ClpP mutant (S124A) in which the conserved serine in the proteolytic active site was replaced with alanine. |
| GO:0004252 serine-type endopeptidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: EC 3.4.21.92 mapping. Reason: Specific family assignment consistent with fly data. Fly ClpP matches the ClpP family signatures (HAMAP MF_00444, InterPro IPR001907) including the PROSITE ClpP serine active-site motif, and in fly S2 cells ClpP is the proteolytic subunit of ClpXP, which selectively degrades DmLRPPRC1 (PMID:28814717, the source of the separate IMP row). Supporting Evidence: file:DROME/ClpP/ClpP-uniprot.txt DR PROSITE; PS00381; CLP_PROTEASE_SER; 1. file:DROME/ClpP/ClpP-uniprot.txt DR HAMAP; MF_00444; ClpP; 1. PMID:28814717 ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX PMID:28814717 Our results argue strongly that ClpXP modulates mitochondrial RNA biogenesis by the selective degradation of DmLRPPRC1. |
| GO:0004252 serine-type endopeptidase activity | IMP PMID:28814717 Drosophila protease ClpXP specifically degrades DmLRPPRC1 co... | ACCEPT | Summary: A catalytic-serine (S124A) ClpP mutant fails to degrade DmLRPPRC1 in S2 cells. Reason: Direct fly evidence that ClpP peptidase activity is required for substrate degradation. Supporting Evidence: PMID:28814717 we established a copper-inducible cell line expressing a catalytically inactive ClpP mutant (S124A) in which the conserved serine in the proteolytic active site was replaced with alanine. PMID:28814717 Collectively, our results strongly suggest that DmLRPPRC1 is a specific substrate of ClpXP in Drosophila cells. |
| GO:0005759 mitochondrial matrix | IC PMID:28814717 Drosophila protease ClpXP specifically degrades DmLRPPRC1 co... | ACCEPT | Summary: Mitochondrial matrix inferred from membership of the mitochondrial Clp complex. Reason: ClpXP is the matrix-resident Clp protease. Supporting Evidence: PMID:28814717 ClpXP and Lon localize to the matrix space whereas the m-AAA protease is anchored in the membrane with its catalytic site exposed to the matrix space. |
| GO:0006508 proteolysis | IEA GO_REF:0000002 | ACCEPT | Summary: Proteolysis from the ClpP InterPro signature. Reason: Correct, though general; the specific process is mitochondrial protein degradation, captured by the MODIFY on the protein catabolic process row. Proteolysis is kept as the generic process of the peptidase activity. Supporting Evidence: PMID:28814717 we established a copper-inducible cell line expressing a catalytically inactive ClpP mutant (S124A) in which the conserved serine in the proteolytic active site was replaced with alanine. |
| GO:0006515 protein quality control | IBA GO_REF:0000033 | MODIFY | Summary: Protein quality control inherited from the ClpP ancestral node. Reason: ClpXP degrades misfolded and regulatory matrix proteins; correct, but the evidence supports the mitochondrial process, so MODIFY to mitochondrial protein quality control, which ClpP already carries (IGI, TAS). Proposed replacements: mitochondrial protein quality control Supporting Evidence: PMID:30374414 overexpression of another matrix protease, ClpP, partially rescued the defects associated with Lon inactivation, possibly by reducing the burden of unfolded mitochondrial proteins. |
| GO:0009368 endopeptidase Clp complex | IBA GO_REF:0000033 | MODIFY | Summary: Clp protease complex membership inherited from the ancestral node. Reason: Correct parent; the fly complex is mitochondrial, so MODIFY to mitochondrial endopeptidase Clp complex, which ClpP already carries (IDA). Proposed replacements: mitochondrial endopeptidase Clp complex Supporting Evidence: PMID:28814717 ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX, which carries an ATPase associated with diverse cellular activities (AAA+) domain6, 7. file:DROME/ClpP/ClpP-uniprot.txt Tetradecamer that assembles into a two heptameric rings with a |
| GO:0009841 mitochondrial endopeptidase Clp complex | IDA PMID:28814717 Drosophila protease ClpXP specifically degrades DmLRPPRC1 co... | ACCEPT | Summary: ClpX co-immunoprecipitates with ClpP, supporting assembly of the mitochondrial ClpXP complex. Reason: Core complex membership. Supporting Evidence: PMID:28814717 FLAG-tagged ClpX is co-immunoprecipitated with ClpP and DmLRPPRC1 but not DmSLIRP1. PMID:28814717 ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX, which carries an ATPase associated with diverse cellular activities (AAA+) domain6, 7. |
| GO:0017171 serine hydrolase activity | HDA PMID:33827210 A Superfamily-wide Activity Atlas of Serine Hydrolases in &l... | MODIFY | Summary: ClpP labelled in a proteome-wide activity-based serine hydrolase atlas. Reason: High-throughput evidence that the active-site serine is reactive; correct but an uninformative parent, so MODIFY to serine-type endopeptidase activity, which ClpP already carries (IBA, IEA, IMP). Proposed replacements: serine-type endopeptidase activity Supporting Evidence: PMID:33827210 The serine hydrolase (SH) superfamily is, perhaps, one of the largest functional enzyme classes in all forms of life and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. |
| GO:0030163 protein catabolic process | IMP PMID:28814717 Drosophila protease ClpXP specifically degrades DmLRPPRC1 co... | MODIFY | Summary: ClpP depletion stabilizes DmLRPPRC1 protein without changing its mRNA. Reason: Direct evidence that ClpP degrades a specific mitochondrial matrix protein, DmLRPPRC1, so the more specific mitochondrial protein catabolic process describes this result exactly. Proposed replacements: mitochondrial protein catabolic process Supporting Evidence: PMID:28814717 Here, we showed that knockdown of ClpX or ClpP results in an increase of DmLRPPRC1 protein without an increase in the corresponding mRNA (Figs 2 and S4). PMID:28814717 Collectively, our results strongly suggest that DmLRPPRC1 is a specific substrate of ClpXP in Drosophila cells. |
| GO:0051117 ATPase binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: ATPase binding inherited from the ClpP ancestral node (E. coli ClpP-ClpX docking). Reason: ClpP docks the ClpX AAA+ ATPase to form ClpXP, and fly ClpX co-immunoprecipitates ClpP; this is a genuine interaction but a structural feature rather than the catalytic function. Supporting Evidence: PMID:28814717 FLAG-tagged ClpX is co-immunoprecipitated with ClpP and DmLRPPRC1 but not DmSLIRP1. |
| GO:0141164 mitochondrial protein quality control | IGI PMID:30374414 Lon protease inactivation in Drosophila causes unfolded prot... | ACCEPT | Summary: ClpP overexpression partially rescues Lon-knockdown flies, consistent with reducing the burden of unfolded matrix proteins. Reason: Genetic interaction with Lon supports a role in mitochondrial protein quality control. Supporting Evidence: PMID:30374414 Results of this analysis revealed that ClpP overexpression rescued the locomotor defect of Lon knockdown flies, thus supporting the hypothesis that the accumulation of unfolded proteins is responsible for the phenotypes of Lon-deficient flies (Fig. PMID:30374414 overexpression of another matrix protease, ClpP, partially rescued the defects associated with Lon inactivation, possibly by reducing the burden of unfolded mitochondrial proteins. |
| GO:0141164 mitochondrial protein quality control | TAS PMID:28814717 Drosophila protease ClpXP specifically degrades DmLRPPRC1 co... | ACCEPT | Summary: Author statement that ClpXP is the major mitochondrial matrix protease involved in quality control. Reason: Consistent with conserved ClpXP biology and the fly data. Supporting Evidence: PMID:28814717 ClpXP is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. |
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Download this section (compressed HTML)Q: Which fly mitochondrial proteins besides DmLRPPRC1 are ClpXP substrates in vivo, and do they include unfolded-protein-response triggers?
Q: Is ClpP required for the mitochondrial unfolded protein response in flies, as in C. elegans?
Experiment: Express a catalytically inactive ClpP (S124A) substrate-trap in flight muscle and identify trapped proteins by mass spectrometry, then validate candidates by cycloheximide-chase in ClpP mutant tissue.
Hypothesis: ClpXP has a restricted set of matrix substrates in fly tissues that includes respiratory-chain assembly factors.
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