AtJ1, a mitochondrial homologue of the Escherichia coli DnaJ protein

Barbara Kroczynska, Rengang Zhou, Clifford Wood, Jan A. Miernyk*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

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 °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.

Original languageEnglish (US)
Pages (from-to)619-629
Number of pages11
JournalPlant Molecular Biology
Volume31
Issue number3
DOIs
StatePublished - 1996

Keywords

  • ATPase activity
  • Arabidopsis thaliana
  • DnaJ
  • mitochondria
  • molecular chaperone
  • nucleotide sequence
  • precursor
  • protein import

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

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