Objective - The overall goal is to study the assembly and degradation of a key component of the photosynthetic electron transport chain, the cytochrome b-f complex. The processes involved in the assembly of a multi-subunit complex will be studied. The inter-relationship between assembly and degradation will be examined.Expected Outcome- Determination of synthesis and degradation rates or the cyt complex submits in wild-type and transgenic plants;- Analysis of assembly and degradation of Rieske mutants;- Characterisation of degradation of unassembled Rieske mutant;- Purification of proteases involved in the degradation of unassembled Rieske mutants;- The study will provide more information about the fate of unassembled proteins in the chloroplast, and the mechanisms utilised by the organelle to ensure stoichiometric levels of different components of a given complex. Insight into these processes might be useful in the future in attempts to engineer plants capable of dealing successfully with harsher growth conditions. Results so farAssembly in transgenic plants- The aim was to examine the hypothesis that the amount of the cytochrome b-f complex is determined by the amount of the Rieske FeS protein available for assembly. Transgenic tobacco plants expressing sense and antisense construct of a chloroplast Rieske Fe-S protein cDNA have been produced and analysed for effects on the assembly of the cytochrome b-f complex. A full-length cDNA encoding the tobacco chloroplast Rieske protein was inserted in both orientations in the binary vectors pROK2 and pROK8, containing the CaMV 35S promoter and tobacco RbcS promoter, respectively. These constructs were transferred to Agrobacterium tumefaciens by electroporation and into tobacco leaf discs by Agrobacterium-mediated transformation. Kanamycin-resistant transgenic plants expressing sense and antisense constructs were identified by northern blot analysis of tobacco leaf RNA using the tobacco cDNA as a probe. This identified transgenic plants expressing antisense RNA with 30-100% of wild-type levels of Rieske protein mRNA, and plants expressing sense RNA with 100-300% of wild-type levels of Rieske protein mRNA. This gave a series of transgenic plants with a 10-fold range of Rieske protein mRNA levels.- Analysis of the amounts of the Rieske protein in the leaves of these transgenic plants, by western blotting with antibodies to purified spinach Rieske protein, failed to detect differences from wild-type leaves. No differences were observed in the amounts of cytochrome f, cytochrome b-563 or the 33 kDa and 23 kDa polypeptides of the oxygen-evolving complex by western blotting with antibodies to the pea proteins. These results indicate that the amount of the chloroplast Rieske protein in tobacco leaves is regulated post-transcriptionally and that a 10-fold difference in transcript levels does not affect the synthesis of the protein or the assembly of the cytochrome b-f complex.Degradation of unassembled Rieske Fe-S protein in chloroplasts- When in vitro-synthesised wild-type Rieske Fe-S protein is imported into intact chloroplasts, it is correctly targeted to the thylakoid membrane and assembled with the cytochrome b-f complex. However, a certain proportion of the imported protein remains associated with the thylakoid membrane, but does not assemble with the complex, as indicated by its sensitivity to protease treatment. We assayed the fate of this unassembled population in organello, as well as of two mutants of the Rieske protein : a soluble mutant targeted to the stroma, due to deletion of a hydrophobic sequence, and a membrane-bound mutant containing a substitution of a single amino-acid. We found these three proteins unstable, with a half-life of ca. 60 minutes, as opposed to other imported stromal and thylakoid proteins, which are stable. The degradation of these proteins was characterised in intact and lysed chloroplasts. It was stimulated by light and was proportional to its intensity, but was independent of ATP or photosynthetic electron transport. Testing the combination of light and temperature on stability of unassembled Rieske protein suggested that the protein was damaged by light, and only then proteolysis occurred, even in the dark. The degradation activity was localised to the thylakoid membrane, was inhibited by metalloprotease inhibitors and stimulated by zinc, but not by other divalent cations. Currently, attempts are being made to purify the enzyme(s) involved in degradation of unassembled Rieske protein from detergent-solubilized thylakoid membranes.- To test the hypothesis that steady-state levels of the cytochrome b-f complex are determined by the amount of the Rieske Fe-S protein available for assembly. Identifying structural features of the Rieske protein essential for Fe-S cluster binding and for assembly with the other subunits of the complex;- Determine turn-over rates of the different components of the cytochrome b-f complex under optimal and changing environmental conditions;- To test the hypothesis that protein stability is afforded by proper assembly and that unassembled proteins are bound for rapid degradation;- To characterise the degradation of unassembled subunits of the complex;- Purification of proteases involved in degradation of unassembled Rieske protein. Programme(s) IC-ISC C - Activity (Euratom, EEC) on cooperation in science and technology, 1984- Topic(s) Data not available Call for proposal Data not available Funding Scheme CSC - Cost-sharing contracts Coordinator University of Cambridge EU contribution No data Address Downing Street CB2 3EA Cambridge United Kingdom See on map Total cost No data
