Illumination changes elicit modifications of thylakoid proteins and reorganization of the photosynthetic machinery. This involves, in the short term, phosphorylation of photosystem II (PSII) and light-harvesting (LHCII) proteins. PSII phosphorylation is thought to be relevant for PSII turnover 1,2 , whereas LHCII phosphorylation is associated with the relocation of LHCII and the redistribution of excitation energy (state transitions) between photosystems 3,4 . In the long term, imbalances in energy distribution between photosystems are counteracted by adjusting photosystem stoichiometry 5,6 . In the green alga Chlamydomonas and the plant Arabidopsis, state transitions require the orthologous protein kinases STT7 and STN7, respectively 7,8 . Here we show that in Arabidopsis a second protein kinase, STN8, is required for the quantitative phosphorylation of PSII core proteins. However, PSII activity under high-intensity light is affected only slightly in stn8 mutants, and D1 turnover is indistinguishable from the wild type, implying that reversible protein phosphorylation is not essential for PSII repair. Acclimation to changes in light quality is defective in stn7 but not in stn8 mutants, indicating that short-term and long-term photosynthetic adaptations are coupled. Therefore the phosphorylation of LHCII, or of an unknown substrate of STN7, is also crucial for the control of photosynthetic gene expression.STT7 and STN7 are orthologous protein kinases required for LHCII phosphorylation and for state transitions in Chlamydomonas and Arabidopsis, respectively 7,8 . In Arabidopsis, another STT7/STN7-like protein (STN8) exists that is not required for state transitions 8 . STN8 is located in the chloroplast, as shown by in vivo subcellular localization of its amino-terminal region fused to the dsRED protein and by the import of, and transit peptide removal from, STN8 translated in vitro (Fig. 1a, b). Chloroplast subfractionation after import revealed that the protein is associated, like STT7 and STN7, with thylakoids ( Fig. 1c) (refs 7, 8).Insertion mutants for STN8 and STN7 were obtained from the Salk collection 9 , and for each gene two independent mutant alleles lacking the respective transcript were identified (Supplementary Fig. S1). The stn7 stn8 double mutant was generated by crossing stn7 and stn8 single knockouts and screening the resulting F 2 generation for homozygous double mutants. All mutants were indistinguishable from the wild type with regard to the timing of seed germination and growth rate in the greenhouse ( Supplementary Fig. S1). In stn7 and stn7 stn8 mutants, a slight decrease in the levels of neoxanthin, lutein and total chlorophyll was found (Supplementary Table S1). These subtle changes can be attributed to a minor decrease in LHCII content, not detectable by polyacrylamide-gel electrophoresis (PAGE) analysis ( Supplementary Fig. S2).Photosynthetic electron flow, measured on the basis of chlorophyll fluorescence, was not altered in the mutants (Supplementary Table S2). State transitions w...

Heat stress transcription factors (Hsfs) regulate gene expression in response to environmental stress. The Hsf network in plants is controlled at the transcriptional level by cooperation of distinct Hsf members and by interaction with chaperones. We found two general mechanisms of Hsf regulation by chaperones while analyzing the three major Hsfs, A1, A2, and B1, in tomato (Solanum lycopersicum). First, Hsp70 and Hsp90 regulate Hsf function by direct interactions. Hsp70 represses the activity of HsfA1, including its DNA binding, and the coactivator function of HsfB1 in the complex with HsfA2, while the DNA binding activity of HsfB1 is stimulated by Hsp90. Second, Hsp90 affects the abundance of HsfA2 and HsfB1 by modulating hsfA2 transcript degradation involved in regulation of the timing of HsfA2 synthesis. By contrast, HsfB1 binding to Hsp90 and to DNA are prerequisites for targeting this Hsf for proteasomal degradation, which also depends on a sequence element in its carboxyl-terminal domain. Thus, HsfB1 represents an Hsp90 client protein that, by interacting with the chaperone, is targeted for, rather than protected from, degradation. Based on these findings, we propose a versatile regulatory regime involving Hsp90, Hsp70, and the three Hsfs in the control of heat stress response.

Mitochondria and chloroplasts import the vast majority of their proteins across two membranes, and use translocases of the outer membrane as an entry gate. These translocases interact with the incoming precursor protein and guiding chaperone factors. Within the translocon, precursor-protein receptors dock to a central component that mediates both transfer through a cation-selective channel and initial sorting towards internal subcompartments. Despite these similarities, the mode of translocation differs between the two organelles: in chloroplasts, GTP-binding and hydrolysis by the receptors is required for transport, whereas in mitochondria passage of the preprotein is driven by its increasing affinity for the translocase subunits.

SummaryYeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3′ cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.

Precursor protein targeting toward organellar surfaces is assisted by different cytosolic chaperones. We demonstrate that the chloroplast protein translocon subunit Toc64 is the docking site for Hsp90 affiliated preproteins. Thereby, Hsp90 is recognised by the clamp type TPR domain of Toc64. The subsequent transfer of the preprotein from Toc64 to the major receptor of the Toc complex, namely Toc34, is affinity driven and nucleotide dependent. We propose that Toc64 acts as an initial docking site for Hsp90 associated precursor proteins. We outline a mechanism in which chaperones are recruited for a specific targeting event by a membrane-inserted receptor.