The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing.

In eubacteria and mitochondria, Hsp70 chaperone activity is controlled by the nucleotide exchange factor GrpE. We have identified the chloroplastic GrpE homolog of Chlamydomonas, CGE1, as an approximately 26-kD protein coimmunoprecipitating with the stromal HSP70B protein. When expressed in Escherichia coli, CGE1 can functionally replace GrpE and interacts physically with DnaK. CGE1 is encoded by a single-copy gene that is induced strongly by heat shock and slightly by light. Alternative splicing generates two isoforms that differ only by two residues in the N-terminal part. The larger form is synthesized preferentially during heat shock, whereas the smaller one dominates at lower temperatures. Fractions of both HSP70B and CGE1 associate with chloroplast membranes in an ATP-sensitive manner. By colorless native PAGE and pulse labeling, CGE1 monomers were found to assemble rapidly into dimers and tetramers. In addition, CGE1 was found to form ATP-sensitive complexes with HSP70B of approximately 230 and approximately 120 kD, the latter increasing dramatically after heat shock.

Possible role for molecular chaperones in assembly and repair of photosystem II.

Genes of the HSP70 chaperone family are induced by light. In Chlamydomonas reinhardtii, the induction of HSP70 (70 kDa heat shock protein) chaperones by light results in a partial protection of photosystem II against damage by photoinhibitory conditions. Underexpression of a chloroplast-localized HSP70 protein caused an increased sensitivity of photosystem II to light. Overexpression of this protein had a protective effect. Fluorescence measurements and studies of the turnover of photosystem II core components suggest that this HSP70 might function in both the protection and the regeneration of photosystem II. This concept is supported by fractionation studies in which the plastid HSP70 was found associated with chloroplast membranes. Because the light-induced elevation of HSP70 levels provides protection for photosystem II, we examined whether the chloroplast is involved in this regulation and found that mutants defective in plastid-localized chlorophyll synthesis, i.e. the insertion of Mg(2+) into protoporphyrin IX are impaired in the induction of HSP70 by light. Exogenous addition of Mg-protoporphyrin in the dark induced the genes. The combined results support a model in which chlorophyll precursors are essential in the signalling from chloroplast to nucleus that regulates the chaperone genes.

The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas.

The Chlamydomonas reinhardtii HSP70A promoter can be induced by both heat shock and light. Several characteristics of this promoter suggest its usefulness as a tool for improved transgene expression in this alga. (i) It may by itself confer high inducibility to a transgene. Fusion of the HSP70A promoter to reporter genes HSP70B or ARS yields high levels of transgene product that, as shown for ARS, may accumulate when repeated cycles of heat shock induction are applied. (ii) It activates other promoters. Using HSP70B as a reporter gene, we show that the HSP70A promoter serves as a transcriptional activator when placed upstream of the promoters RBCS2, beta 2 TUB and HSP70B. Activation of these promoters was observed both under basal conditions and upon light induction. In addition, transformation rates obtained for the eubacterial resistance gene aadA were significantly increased, when expression of this gene was controlled by the HSP70A-RBCS2 promoter fusion as compared to the RBCS2 promoter alone.

A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition.

Dark-grown Chlamydomonas reinhardtii cultures that were illuminated at low fluence rates before exposure to high-light conditions exhibited a faster rate of recovery from photoinhibition than did dark-grown cells that were directly exposed to photoinhibitory conditions. This pretreatment has been shown to induce the expression of several nuclear heat shock protein 70 (HSP70) genes, including HSP70B, encoding a chloroplast-localized chaperone. To investigate a possible role of plastidic HSP70B in photoprotection and repair of photosystem II, which is the major target of photoinhibition, we have constructed strains overexpressing or underexpressing HSP70B. The effect of light stress on photosystem II in nuclear transformants harboring HSP70B in the sense or antisense orientation was monitored by measuring variable fluorescence, flash-induced charge separation, and relative amounts of various photosystem II polypeptides. Underexpression of HSP70B caused an increased light sensitivity of photosystem II, whereas overexpression of HSP70B had a protective effect. Furthermore, the reactivation of photosystem II after photoinhibition was enhanced in the HSP70B-overexpressing strain when compared with the wild type, both in the presence or absence of synthesis of chloroplast-encoded proteins. Therefore, HSP70B may participate in vivo both in the molecular protection of the photosystem II reaction centers during photoinhibition and in the process of photosystem II repair.

Light-inducible gene HSP70B encodes a chloroplast-localized heat shock protein in Chlamydomonas reinhardtii.

The nuclear heat shock gene HSP70B of Chlamydomonas reinhardtii is inducible by heat stress and light. Induction by either environmental cue resulted in a transient elevation in HSP70B protein. Here we describe the organization and nucleotide sequence of the HSP70B gene. The deduced protein exhibits a distinctly higher homology to prokaryotic HSP70s than to those of eukaryotes, including the cytosolic HSP70A of Chlamydomonas reinhardtii. The HSP70B protein, as previously demonstrated by in vitro translation, is synthesized with a cleavable presequence. Using an HSP70B-specific antibody, this heat shock protein was localized to the chloroplast by cell fractionation experiments. A stromal location was suggested by the presence of a conserved sequence motif used for cleavage of presequences by a signal peptidase of the stroma. Amino acid alignments of HSP70 proteins from various organisms and different cellular compartments allowed the identification of sequence motifs, which are diagnostic for HSP70s of chloroplasts and cyanobacteria.