Molecular chaperones are present in the thylakoid lumen of pea chloroplasts.

A soluble protein fraction was obtained from pea chloroplast thylakoids, which represents highly enriched lumenal components. Using antisera against chaperonin 60 (cpn60), chaperonin 10 (cpn10) and the heat shock cognate protein of 70 kDa (hsc70) we are able to demonstrate, that the thylakoid lumen contains a separate set of molecular chaperones, which is distinct from the stromal one. In contrast to the alpha and beta subunits of cpn60 present in the stroma the lumen contains only one cpn60 isoform of distinct isoelectric point. Furthermore the lumenal cpn10 is of 'normal' size and not like its stromal counterpart of a double-domain tandem architecture. The immunoreactive hsc70 isoforms in the lumen seem also to be different from the stromal ones. Thus, chloroplasts seem to contain the largest number of molecular chaperone isoforms present in one organelle.

Chloroplastic isoforms of DnaJ and GrpE in pea.

The folding and assembly of proteins in cells often requires the assistance of molecular chaperones such as the Hsp70 and Hsp60 heat shock proteins. Hsp70 chaperones cooperate with DnaJ and GrpE homologues to ensure a productive folding cycle. In this study we describe the gene for the first chloroplast localized DnaJ homologue and present evidence that the gene product is at least partially associated with the inner envelope membrane. Immunoblot analysis also provides evidence for the presence of a GrpE homologue in plastids.

Reconstitution of a chloroplast protein import channel.

The chloroplastic outer envelope protein OEP75 with a molecular weight of 75 kDa probably forms the central pore of the protein import machinery of the outer chloroplastic membrane. Patch-clamp analysis shows that heterologously expressed, purified and reconstituted OEP75 constitutes a voltage-gated ion channel with a unit conductance of Lambda = 145pS. Activation of the OEP75 channel in vitro is completely dependent on the magnitude and direction of the voltage gradient. Therefore, movements of protein charges of parts of OEP75 in the membrane electric field are required either for pore formation or its opening. In the presence of precursor protein from only one side of the bilayer, strong flickering and partial closing of the channel was observed, indicating a specific interaction of the precursor with OEP75. The comparatively low ionic conductance of OEP75 is compatible with a rather narrow aqueous pore (dporeapproximately equal to 8-9 A). Provided that protein and ion translocation occur through the same pore, this implies that the environment of the polypeptide during the transit is mainly hydrophilic and that protein translocation requires almost complete unfolding of the precursor.