The role of the Clp protease in the degradation and assembly of thylakoid membrane proteins

Abstract

ClpP is the proteolytic subunit of a major chloroplast protease, Clp. In search for its in vivo substrates, we have mutated the initiation codon of the chloroplastic clpP1 gene in Chlamydomonas from AUG to AUU, thus reducing the expression level of this essential protein to 25-40% of normal. The clpP1-AUU mutation reduced the degradation rate of the cytochrome b6f complex during nitrogen starvation and in mutants of the Rieske protein (Majeran et al. 2000, Plant Cell 12,137-149). We have combined the clpP1-AUU mutation with mutations affecting assembly of the ATP synthase. In mutants lacking the a subunit of CF1, b-CF1 was stabilized, suggesting that the non-assembled b is a substrate for the protease. Non-assembled a was also stabilized by the clpP1-AUU mutation. In addition, pulse-labeling experiments indicated that attenuation of ClpP affects the translation rates of the a subunit. This effect may be exerted via the stabilization of CF1 assembly intermediates involved in the so-called epistatic control of protein synthesis. ATP synthase mutants are highly sensitive to medium intensity light. Using the mutants described above, we found that light treatment (70 µE.m-1.s-1) induces inactivation of photosystem II, followed by its proteolytic removal, and that the Clp protease is involved in this degradation process. The initial inactivation event appears to involve inactivation of QA to QB electron transfer as a result of low lumenal pH. In conclusion, our experiments implicate the Clp protease in specific degradation processes involving intrinsic and extrinsic proteins of the thylakoid membrane, shedding light on the complex substrate-enzyme relationships in chloroplast proteolysis.