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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

The Chloroplast DnaJ Homolog CDJ1 of Chlamydomonas reinhardtii Is Part of a Multichaperone Complex Containing HSP70B, CGE1, and HSP90C^1,[OA]

Institute of Biology II, Plant Biochemistry, University of Freiburg, D–79104 Freiburg, Germany (F.W., K.V.D., M.S.-R., M.S.); and Max Planck Institute of Molecular Plant Physiology, D–14476 Potsdam-Golm, Germany (M.S.)

We report on the molecular and biochemical characterization of CDJ1, one of three zinc-finger-containing J-domain proteins encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that CDJ1 is a plastidic protein. In the chloroplast, CDJ1 was localized to the soluble stroma fraction, but also to thylakoids and to low density membranes. Although the CDJ1 gene was strongly heat shock inducible, CDJ1 protein levels increased only slightly during heat shock. Cellular CDJ1 concentrations were close to those of heat shock protein 70B (HSP70B), the major HSP70 in the Chlamydomonas chloroplast. CDJ1 complemented the temperature-sensitive phenotype of an Escherichia coli mutant lacking its dnaJ gene and interacted with E. coli DnaK, hence classifying it as a bona fide DnaJ protein. In soluble cell extracts, CDJ1 was found to organize into stable dimers and into complexes of high molecular mass. Immunoprecipitation experiments revealed that CDJ1 forms common complexes with plastidic HSP90C, HSP70B, and CGE1. In blue native-polyacrylamide gel electrophoresis, all four (co)chaperones migrated at 40% to 90% higher apparent than calculated molecular masses, indicating that greatest care must be taken when molecular masses of protein complexes are estimated from their migration relative to standard native marker proteins. Immunoprecipitation experiments from size-fractioned soluble cell extracts suggested that HSP90C and HSP70B exist as preformed complex that is joined by CDJ1. In summary, CDJ1 and CGE1 are novel cohort proteins of the chloroplast HSP90-HSP70 multichaperone complex. As HSP70B, CDJ1, and CGE1 are derived from the endosymbiont, whereas HSP90C is of eukaryotic origin, we observe in the chloroplast the interaction of two chaperone systems of distinct evolutionary origin.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Michael Schroda (schroda{at}mpimp-golm.mpg.de).

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www.plantphysiol.org/cgi/doi/10.1104/pp.108.127944

^* Corresponding author; e-mail schroda{at}mpimp-golm.mpg.de.

Received August 11, 2008; accepted October 14, 2008; published October 17, 2008.