ATJ1, A MITOCHONDRIAL HOMOLOG OF THE ESCHERICHIA-COLI DNAJ PROTEIN

The nucleotide sequence of a cDNA clone from Arabidopsis thaliana ecot ype Columbia was determined, and the corresponding amino sequence dedu ced. The open reading frame encodes a protein, AtJ1, of 368 residues w ith a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine -rich domains of Escherichia coli DnaJ, but the glycine/phenylalanine- rich region is not present. Based upon Southern analysis, Arabidopsis appears to have a single atJ1 structural gene. A single species of mRN A, of 1.5 kb, was detected when Arabidopsis poly(A)(+) RNA was hybridi zed with the atJ1 cDNA. The function of atJ1 was tested by complementa tion of a dnaJ deletion mutant of E. coli, allowing growth in minimal medium at 44 degrees C. The AtJ1 protein was expressed in E. coli as a fusion with the maltose binding protein. This fusion protein was puri fied by amylose affinity chromatography, then cleaved by digestion wit h the activated factor X protease. The recombinant AtJ1 protein was pu rified to electrophoretic homogeneity. In vitro, recombinant AtJ1 stim ulated the ATPase activity of both E. coli DnaK and maize endosperm cy toplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioac tive recombinant AtJ1 was synthesized in E. coli and purified. When th e labeled protein was incubated with intact pea cotyledon mitochondria , it was imported and proteolytically processed in a reaction that dep ended upon an energized mitochondrial membrane.