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-dependent, 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 and used to confirm its localization in plant mitochondria. In addition to ClpX, a ClpP protein located in mitochondria was also identified from the numerous ClpP isomers in Arabidopsis. Localization of this nuclear-encoded protein, 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, transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions, with no significant variation in mRNA level (i.e. 2-fold) for each gene between samples. Using β-casein as a substrate, plant mitochondria were found to possess an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease. The recent discovery of homologous mitochondrial ClpX and ClpP proteins in mammals suggests that this type of protease may be common to multicellular eukaryotes.
Bibliographical noteFunding Information:
We thank E. Glaser for the antibody against SHMT, and the Arabidopsis Biological Resource Center at Ohio State University for the Arabidopsis EST clone and cDNA library. This work was supported in part by grants from BARD, the US-Israel Binational Agricultural Research and Development Fund to Z.A., and from the Swedish Agricultural and Forestry Research Council (SJFR) and the Centre for Forest Biotechnology and Chemistry to A.K.C.