AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

Abstract

The nucleotide sequence of a cDNA clone from Arabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with 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 mRNA, of 1.5 kb, was detected when Arabidopsis poly(A)+ RNA was hybridized with the atJ1 cDNA. The function of atJ1 was tested by complementation 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 purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity. In vitro, recombinant AtJ1 stimulated the ATPase activity of both E. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized in E. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.

A Small Heat Shock Protein Cooperates with Heat Shock Protein 70 Systems to Reactivate a Heat-Denatured Protein

“…Thus, a highly functional eukaryotic refolding system can be assembled with a combination of plant, human, and yeast chaperone homologs without apparent species specificity. This result is consistent with the ability of heterologous DnaJ proteins to interact with and stimulate ATP hydrolysis by diverse Hsp70 proteins (Kroczynska et al, 1996;Takayama et al, 1999).…”

The Chloroplast DnaJ Homolog CDJ1 of Chlamydomonas reinhardtii Is Part of a Multichaperone Complex Containing HSP70B, CGE1, and HSP90C

“…Moreover, the Chlamydomonas protein within the mitochondrial clade, like its Arabidopsis homolog Sequences aligned are from E. coli DnaJ (EcDnaJ, accession AAA23693); from Arabidopsis mitochondrial and cytosolic DnaJ homologs (atDjB1 and atDjA3, accessions NP_174142 and AAB49030, respectively); from Chlamydomonas cytosolic, chloroplast, and mitochondrial DnaJ homologs (CrDNJ1, CrCDJ1, and CrMDJ1, accessions EDP04706, AAU06580, and gene model estExt_fgenesh1_pg.C_870033 at http://genome.jgi-psf.org/Chlre3/Chlre3.home.html, respectively); and from a pea chloroplast DnaJ homolog (PsPCJ1, accession CAA96305). Amino acids highlighted in black are conserved in all seven proteins; atDjB1 in that clade, lacked the G/F-rich domain normally situated between J domain and ZFD (Kroczynska et al, 1996). Hence, the Chlamydomonas protein in the cytoplasmic clade was annotated as DNJ1, that in the mitochondrial clade mitochondrial DnaJ 1 (MDJ1), and that in the chloroplast/cyanobacterial clade CDJ1 (Fig.…”

“…It was reported previously that the yeast mitochondrial DnaJ homolog Mdj1p and the Arabidopsis mitochondrial DnaJ homolog AtJ1 (now termed atDjB1) could at least partially complement the temperaturesensitive phenotype of an E. coli DdnaJ mutant strain (Kroczynska et al, 1996;Deloche et al, 1997). To test whether also CDJ1 was able to functionally replace E. coli DnaJ, we introduced the pCDF expression vector alone, or the same vector encoding CDJ1 with an N-terminal hexa-His tag into E. coli strain OD259 carrying a deletion of the dnaJ gene (Deloche et al, 1997).…”

Expression of the Arabidopsis thaliana AtJ2 Cochaperone Protein in Pichia pastoris

“…ATPase activity was quantified using the malachite green procedure, essentially as described in (9,16). A typical assay mixture contained buffer, salts, 2 mM DTT, 0.1 mM ATP, and 2 g of purified Stress70 protein in a final volume of 0.1 mL.…”

“…Protein levels were measured by the dye-binding method of Bradford (16), using BSA as the standard. The methods used for SDS-PAGE and Western blotting have been previously described (9), as has the anti-AtJ2 synthetic peptide antiserum (10). Monoclonal anti-histidine tag antibodies (18) were from Dianova GmbH (Hamburg, Germany).…”