Translocation pathway of protein substrates in ClpAP protease.
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.
Ishikawa
T
T
Laboratory 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.
Beuron
F
F
Kessel
M
M
Wickner
S
S
Maurizi
M R
MR
Steven
A C
AC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D020285:Cryoelectron Microscopy; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D049071:Endopeptidase Clp; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D015534:Trans-Activators
D004268:DNA-Binding Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D004265:DNA Helicases