THE STROMA OF HIGHER-PLANT PLASTIDS CONTAIN CLPP AND CLPC, FUNCTIONALHOMOLOGS OF ESCHERICHIA-COLI CLPP AND CLPA - AN ARCHETYPAL 2-COMPONENT ATP-DEPENDENT PROTEASE

A cDNA representing the plastid-encoded homolog of the prokaryotic ATP -dependent protease ClpP was amplified by reverse transcription-polyme rase chain reaction, cloned, and sequenced. ClpP and a previously isol ated cDNA designated ClpC, encoding an ATPase related to proteins enco ded by the ClpA/B gene family, were expressed in Escherichia coli. Ant ibodies directed against these recombinant proteins recognized protein s in a wide variety of organisms. N-terminal analysis of the Clp prote in isolated from crude leaf extracts showed that the N-terminal methio nine is absent from ClpP and that the transit peptide is cleaved from ClpC. A combination of chloroplast subfractionation and immunolocaliza tion showed that in Arabidopsis, ClpP and ClpC localize to the stroma of the plastid. Immunoblot analyses indicated that ClpP and ClpC are c onstitutively expressed in all tissues of Arabidopsis at levels equiva lent to those of E. coli ClpP and ClpA. ClpP, immunopurified from toba cco extracts, hydrolyzed N-succinyl-Leu-Tyr-amidomethylcoumarin, a sub strate of E. coli ClpP. Purified recombinant ClpC facilitated the degr adation of H-3-methylcasein by E. coli ClpP in an ATP-dependent fashio n. 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 demon stration of the activity of a specific ATP-dependent chloroplast prote ase reported to date.