Plant mitochondria contain proteolytic and regulatory subunits of the ATP-dependent Clp protease

The proteolytic machinery of plant organelles is largely unknown, although indications so far point to several proteases of bacterial origin. In this study an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX, a molecular chaperone and regulatory subunit of the ATP-de pendent, serine-type Clp protease. Computer analysis of the predicted plant ClpX revealed a putative mitochondrial transit peptide at the N-terminus, as well as overall sequence similarity to other eukaryotic ClpX homologues. Specific polyclonal antibodies were made to the Arabidopsis ClpX protein a nd used to confirm its localization in plant mitochondria. In addition to C lpX, a ClpP protein located in mitochondria was also identified from the nu merous ClpP isomers in Arabidopsis. Localization of this nuclear-encoded pr otein, termed ClpP2, was determined first by its close sequence similarity to mitochondrial ClpP human, and later experimentally using ClpP2-specific antibodies with isolated plant organellar fractions. In Arabidopsis, transc ripts for both clpX and clpP2 genes were detected in various tissues and un der different growth conditions, with no significant variation in mRNA leve l (i.e. 2-fold) for each gene between samples. Using beta -casein as a subs trate, plant mitochondria were found to possess an ATP-stimulated, serine-t ype proteolytic activity that could be strongly inhibited by antibodies spe cific for ClpX or ClpP2, suggesting an active ClpXP protease. The recent di scovery of homologous mitochondrial ClpX and ClpP proteins in mammals sugge sts that this type of protease may be common to multicellular eukaryotes.