The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease.
A cDNA representing the plastid-encoded homolog of the prokaryotic ATP-dependent protease ClpP was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced. ClpP and a previously isolated cDNA designated ClpC, encoding an ATPase related to proteins encoded by the ClpA/B gene family, were expressed in Escherichia coli. Antibodies directed against these recombinant proteins recognized proteins in a wide variety of organisms. N-terminal analysis of the Clp protein isolated from crude leaf extracts showed that the N-terminal methionine is absent from ClpP and that the transit peptide is cleaved from ClpC. A combination of chloroplast subfractionation and immunolocalization showed that in Arabidopsis, ClpP and ClpC localize to the stroma of the plastid. Immunoblot analyses indicated that ClpP and ClpC are constitutively expressed in all tissues of Arabidopsis at levels equivalent to those of E. coli ClpP and ClpA. ClpP, immunopurified from tobacco extracts, hydrolyzed N-succinyl-Leu-Tyr-amidomethylcoumarin, a substrate of E. coli ClpP. Purified recombinant ClpC facilitated the degradation of 3H-methylcasein by E. coli ClpP in an ATP-dependent fashion. This demonstrates that ClpC is a functional homolog of E. coli ClpA and not of ClpB or ClpX. These data represent the only in vitro demonstration of the activity of a specific ATP-dependent chloroplast protease reported to date.
Shanklin
J
J
Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.
DeWitt
N D
ND
Flanagan
J M
JM
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology; D007120:Immunochemistry; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D018087:Plastids / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases / Q000235:genetics* / Q000276:immunology
D018076:DNA, Complementary; D018744:DNA, Plant; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D049069:ATP-Dependent Proteases; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases