Most chloroplast and mitochondrial proteins are synthesized in the cytosol of the plant cell and have to be imported into the organelles post-translationally. Molecular chaperones play an important role in preventing protein aggregation of freshly translated preproteins and assist in maintaining the preproteins in an import competent state. Preproteins can associate with HSP70, HSP90, and 14-3-3 proteins in the cytosol. In this study, we analyzed a large set of wheat germ-translated chloroplast preproteins with respect to their chaperone binding. Our results demonstrate that the formation of distinct 14-3-3 or HSP90 containing preprotein complexes is a common feature in post-translational protein transport in addition to preproteins that seem to interact solely with HSP70. We were able to identify a diverse and extensive class of preproteins as HSP90 substrates, thus providing a tool for the investigation of HSP90 client protein association. The analyses of chimeric HSP90 and 14-3-3 binding preproteins with exchanged transit peptides indicate an involvement of both the transit peptide and the mature part of the proteins, in HSP90 binding. We identified two partner components of the HSP90 cycle, which were present in the preprotein containing high-molecular-weight complexes, the HSP70/HSP90 organizing protein HOP, as well as the immunophilin FKBP73. The results establish chloroplast preproteins as a general class of HSP90 client proteins in plants using HOP and FKBP as novel cochaperones.

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn 4 Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in DpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electronaccepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in DpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in DpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.PSII, the oxygen-evolving pigment-protein complex of the thylakoid membrane system, is present in photosynthetic organisms from cyanobacteria to vascular plants. The proteins of the photochemically active reaction center (RC), the heterodimeric polypeptides PsbA (D1) and PsbD (D2) and the lowmolecular-weight (LMW) subunits PsbE and PsbF, and the a-and b-chains of cytochrome b 559 coordinate the redox cofactors necessary for primary PSII photochemistry. Electron flow following charge separation occurs via several electron carriers of the RC, including pheophytin a, as well as the primary and secondary quinones, Q A and Q B . The striking similarities between cyanobacterial and land plant PSII core components, including the intrinsic light-harvesting antennae, the chlorophyll-binding proteins CP43 and CP47, and their interactions during photosynthetic charge separation, suggest a high degree of structural and functional conservation. Thus, much of the information obtained from crystallization of a cyanobacterial PSII can be applied to that of higher plants (Zouni et al., 2001;Kamiya and Shen, 2003;Ferreira et al., 2004;Loll et al., 2005;Nield and Barber, 2006).Besides the RC, the PSII core contains a remarkably high number (16) of intrinsic or peripheral LMW peptides. Five of them are encoded in eukaryotes by nuclear genes (psbR, psbTn, psbW, ps...