ClpAP and ClpXP degrade proteins with tags located in the interior of the primary sequence.
Clp/Hsp100 ATPases comprise a large family of ATP-dependent chaperones, some of which are regulatory components of two-component proteases. Substrate specificity resides in the Clp protein and the current thinking is that Clp proteins recognize motifs located near one or the other end of the substrate. We tested whether or not ClpA and ClpX can recognize tags when they are located in the interior of the primary sequence of the substrate. A protein with an NH2-terminal ClpA recognition tag, plasmid P1 RepA, was fused to the COOH terminus of green fluorescent protein (GFP). GFP is not recognized by ClpA or ClpX and is not degraded by ClpAP or ClpXP. We found that ClpA binds and unfolds the fusion protein and ClpAP degrades the protein. Both the GFP and RepA portions of the fusion protein are degraded. A protein with a COOH-terminal ClpX tag, MuA, was fused to the NH2 terminus of GFP. ClpXP degrades MuA-GFP, however, the rate is 10-fold slower than that of GFP-MuA. The MuA portion but not the GFP portion of MuA-GFP is degraded. Thus, a substrate with an internal ClpA recognition motif can be unfolded by ClpA and degraded by ClpAP. Similarly, although less efficiently, ClpXP degrades a substrate with an internal ClpX recognition motif. We also found that ClpA recognizes the NH2-terminal 15 aa RepA tag, when it is fused to the COOH terminus of GFP. Moreover, ClpA recognizes the RepA tag in either the authentic or inverse orientation.
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Joel R
JR
Hoskins
Katsuhiko
K
Yanagihara
Kiyoshi
K
Mizuuchi
Sue
S
Wickner
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049071:Endopeptidase Clp; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D017510:Protein Folding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases