Effects of site-directed mutations in the chloroplast-encoded ycf4 gene on PSI complex assembly in the green alga chlamydomonas reinhardtii

Abstract

The chloroplast-encoded Ycf4 plays an essential role in PSI complex assembly in the green alga Chlamydomonas reinhardtii. To gain insight into how Ycf4 functions, we generated several mutants in which residues R120, E179 and/or E181, which are conserved among oxygenic photosynthetic organisms, were changed to A or Q. Although the single mutants R120A and R120Q accumulated 80 less Ycf4 than the wild type, they assembled a functional PSI complex and grew photosynthetically like the wild type. Thus we inferred that under laboratory growth conditions, wild-type cells accumulate a superfluous amount of Ycf4. Single mutants E179A, E179Q and E181Q assembled a functional PSI complex like the wild type, whereas the single mutant E181A and double mutant E179/181A accumulated a functional PSI complex to significantly reduced levels. Double mutant E179/181Q, in contrast, accumulated Ycf4 at the wild-type level but did not assemble any mature PSI complex, suggesting that the two glutamic acid residues play crucial roles in the functionality of Ycf4. Interestingly, sucrose density gradient centrifugation of the thylakoid extracts separated a small amount of PSI subcomplex. The apparent size of the subcomplex (150170 kDa), its composition and pulsechase protein labeling indicate that it was an unstable subcomplex consisting of a PsaAPsaB heterodimer. We inferred that the subcomplex was a PSI complex assembly intermediate that was detected because subsequent assembly steps were blocked by the E179/181Q mutation. We concluded that Ycf4 is involved in early processes of PSI complex assembly.