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Updated: Jul 24, 2026
In vivo and in vitro Studies of Adaptor-clathrin Interaction
Published on: January 26, 2011
Translocation pathway of protein substrates in ClpAP protease
T Ishikawa1, F Beuron, M Kessel
1Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, and Laboratories of Cell Biology and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATPgammaS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through approximately 150 A into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.
Insights
The ClpAP protease complex unfolds and translocates protein substrates like RepA into its digestion chamber. Cryo-EM reveals RepA accumulates at intermediate sites within ClpP during translocation.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Intracellular protein degradation is crucial for cellular function and tightly regulated by ATP-dependent proteases.
- Multicomponent enzymes like ClpAP utilize chaperone-like ATPases to unfold and deliver protein substrates for degradation.
Purpose of the Study:
- To characterize the structural mechanism of substrate translocation by the ClpAP protease complex using cryo-electron microscopy.
- To visualize the interaction of the ClpAP complex with its model substrate, bacteriophage P1 protein RepA.
Main Methods:
- Cryo-electron microscopy (cryo-EM) was employed to study ClpAP complexes.
- Interactions with the model substrate RepA were analyzed in the presence of ATPgammaS and ATP.
- A ClpP mutant with an occupied digestion chamber was used to trap translocation intermediates.
Main Results:
- RepA dimers bind to ClpA in ATPgammaS-stabilized complexes.
- Upon ATP addition, RepA translocates into the ClpP digestion chamber with minimal ClpAP structural changes.
- Cryo-EM of a ClpP mutant revealed intermediate binding sites for RepA within ClpP, suggesting a stepwise translocation pathway.
Conclusions:
- The ClpAP complex translocates unfolded RepA substrates through a defined pathway.
- Substrate translocation occurs without major conformational changes in the ClpA hexamer.
- Intermediate accumulation sites within ClpP highlight the dynamic nature of protein translocation and degradation.
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