HEAT SHOCK PROTEIN 90C Is a Bona Fide Hsp90 That Interacts with Plastidic HSP70B in Chlamydomonas reinhardtii

Abstract

We report on the molecular and biochemical characterization of HEAT SHOCK PROTEIN 90C (HSP90C), one of the three Hsp90 chaperones encoded by the Chlamydomonas reinhardtii genome. Fractionation experiments indicate that HSP90C is a plastidic protein. In the chloroplast, HSP90C was localized to the soluble stroma fraction, but also to thylakoids and lowdensity membranes containing inner envelopes. HSP90C is expressed under basal conditions and is strongly induced by heat shock and moderately by light. In soluble cell extracts, HSP90C was mainly found to organize into dimers, but also into complexes of high molecular mass. Also, heterologously expressed HSP90C was mainly found in dimers, but tetramers and fewer monomers were detected, as well. HSP90C exhibits a weak ATPase activity with a K m for ATP of approximately 48 mM and a k cat of approximately 0.71 min 21 . This activity was inhibited by the Hsp90-specific inhibitor radicicol. In coimmunoprecipitation experiments, we found that HSP90C interacts with several proteins, among them plastidic HSP70B. The cellular concentration of HSP70B was found to be 2.9 times higher than that of HSP90C, giving a 4.8:1 stoichiometry of HSP70B monomers to HSP90C dimers. The strong inducibility of HSP90C by heat shock implies a role of the chaperone in stress management. Furthermore, its interaction with HSP70B suggests that, similar to their relatives in cytosol and the endoplasmic reticulum, both chaperones might constitute the core of a multichaperone complex involved in the maturation of specific client proteins, e.g. components of signal transduction pathways.

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).…”

“…Consequently, we tested the effects of radicicol, a specific inhibitor of the Hsp90 N-terminal ATP binding site (28), in in vitro import reactions with isolated chloroplasts. A previous study demonstrated that radicicol inhibited the ATPase activity of Hsp90C isolated from the green algae, Chlamydomonas reinhardtii (29). Radicicol also inhibited purified recombinant Arabidopsis Hsp90C ATPase activity, with 95% inhibition observed at 100 μM radicicol (Fig.…”

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].…”

Assistance for a Chaperone

“…Stromal Hsp70 is involved in the maturation of chloroplast proteins (19 -22), in the protection of photosystem II from photodamage (23), and in the assembly/disassembly of VIPP1 oligomers (24,25). Furthermore, stromal HSP70B was found to interact with plastidic HSP90C (26).…”

“…Next, pMS408 was cleaved with SapI-XhoI, and the resulting ϳ490-bp fragment was ligated into SapI-XhoI-digested pTYB11 (New England Biolabs), generating pMS410. pMS410 was transformed into strain ER2566 and, like CGE1b (pMS301) and HSP70B (pMS307), HEP2 was expressed with an intein/chitin binding domain fused to its N terminus, purified by chitin affinity chromatography, and eluted after thiol-induced cleavage as described earlier (26,27). 1 mg of purified HEP2 was used for the immunization of a rabbit.…”