In Vivo Competition between Plastocyanin and a Copper-Dependent Regulator of the Chlamydomonas reinhardtii Cytochrome c6 Gene

Abstract

The Chlamydomonas reinhardtii gene encoding cytochrome c₆ (Cyt c₆) is transcriptionally repressed by cupric ions. In quantitating the level of expression of this gene as a function of cupric ions available per cell, we find that transformed Chlamydomonas reinhardtii cells that accumulate high levels of plastocyanin (a type I copper protein) have a higher sensory threshold for copper-dependent repression of the Cyt c₆ gene than do untransformed, otherwise isogenic, cells that are plastocyanin-deficient. Also, in wild-type cells, the extent to which the gene is expressed at any given ratio of copper/cell is exactly correlated with the predicted deficiency (at this level of copper) in the organism's capacity to synthesize holoplastocyanin. These results support a simple model in which the sensory threshold for transcriptional repression of the Cyt c₆ gene is determined by direct competition for intracellular copper ions between a copper-binding regulator of this gene and plastocyanin. Thus, the organism is able to maintain a constant amount of Cyt c₆ plus plastocyanin per Photosystem I. With the use of in vitro-generated Cyt c₆-encoding transcripts as a standard for the quantitation of cellular Cyt c₆ mRNA levels, we estimate that whereas copper-deficient wild-type cells maintain approximately 1 × 10² to 4 × 10² Cyt c₆-specific transcripts per cell, copper-supplemented cells contain, on average, less than one Cyt c₆-encoding mRNA. Thus, repression of the Cyt c₆ gene by copper ions is essentially 100%, making it unlikely that Cyt c₆ has any essential metabolic function in copper-supplemented cells. We find also that the steady-state levels of several transcripts, including those for Cyt c₆, are influenced by cell density, so that cells harvested at low density contain several-fold as many copies of a particular message as cells harvested near stationary phase.