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

Abstract

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.Molecular chaperones of the heat shock protein 70 (HSP70) family are involved in a variety of different tasks, like the folding of nonnative proteins to the native state (Frydman, 2001;Hartl and Hayer-Hartl, 2002), protein quality control (Bukau et al., 2006), the transport of proteins across membranes (Neupert and Brunner, 2002), the assembly and disassembly of protein complexes (Mayer, 2005), or the regulation of the stress response (Voellmy and Boellmann, 2007). HSP70s function in concert with different cochaperones, which are usually involved in one of the following processes (or a combination of them): they regulate the ATPase activity of their HSP70 partner, supply their HSP70 partner with substrates, or connect it with other proteins involved in protein folding or degradation.An important class of HSP70 cochaperones is the one of the J-domain proteins (Craig et al., 2006;Qiu et al., 2006). These interact via their J domains with HSP70s in the ATP state (Wittung-Strafshede et al., 2003), stimulate the ATPase activity of their HSP70 partner (Liberek et al., 1991), and lock the latter onto specific substrates (Han and Christen, 2003). By this, J-domain proteins mediate substrate specificity and thereby the function of...

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts

“…Interestingly, Hsp90C functions in a complex with cpHsp70 in the stroma of Chlamydomonas chloroplasts (29,45,46), raising the possibility that these two chaperones also function together at TIC complexes during protein import in vascular plants. It was previously proposed that the cpHsp70 system and the Hsp93-Tic40 systems function in parallel during protein import based on epistasis analysis of combinations of cpHsp70, Hsp93, and Tic40 mutants (9,10,47).…”

Inducible transgenic tobacco system to study the mechanisms underlying chlorosis mediated by the silencing of chloroplast heat shock protein 90

“…Moreover, we have previously suggested that PR1a induction during Tav-mediated chlorosis is independent of SA. Because Hsp90C is known to be involved in chloroplast biogenesis and retrograde signaling in A. thaliana and Chlamydomonas reinhardtti [6,30,33,34], an unknown SA-independent signaling from the affected chloroplast may have a role in the regulation of the PR1a nuclear gene. On the other hand, higher production of ROS is anticipated in malfunctioning chloroplasts of Hsp90C-silenced plants [36].…”

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