Two metal-dependent steps in the biosynthesis of Scenedesmus obliques plastocyanin. Differential mRNA accumulation and holoprotein formation.

The accumulation of the interchangeable electron transfer catalysts plastocyanin and cytochrome c6 (cyt c6) in Scenedesmus obliquus is reciprocally regulated by the amount of copper ions in the medium. In copper-deficient cells, plastocyanin levels are severely reduced, whereas cyt c6 levels are high. Western blot analysis indicates that neither pre-apoplastocyanin nor apoplastocyanin accumulate to significant extents in copper-deficient Scenedesmus cells, and time course studies indicate that upon provision of copper salts to copper-deficient cells, the accumulation of plastocyanin to the levels maintained in copper-sufficient cells takes about 12-24 h. By 1) Northern hybridization analysis of Scenedesmus obliquus mRNA and 2) in vitro translation of polyadenylylated mRNA followed by immunoprecipitation of a 19.2-kilodalton precursor to plastocyanin, we demonstrate that the regulation of plastocyanin synthesis by copper must occur primarily at the level of mRNA accumulation. These results suggest that copper-dependent stimulation of holoplastocyanin accumulation requires de novo synthesis of the pre-apoprotein and contradict the conclusion of Bohner, H., Bohme, H., and Boger, P. (1981) FEBS Lett. 131, 386-388 that high levels of apoplastocyanin and a precursor to plastocyanin accumulate in copper-deficient Scenedesmus cells. We note also that although metal ions other than copper (e.g. silver) insert into Scenedesmus obliquus plastocyanin in vitro, synthesis of holoplastocyanin in vivo is specific for copper versus silver or mercury as it is in Chlamydomonas reinhardtii. Finally, the very different electrophoretic mobility and immunoreactivity of apoplastocyanin compared with holoplastocyanin suggests rather significant differences in structure between the copper-protein and the metal-free protein.