Volume 271, Number 49, Issue of December 6, 1996 pp. 31283-31289
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Molecular Genetic Analysis of Plastocyanin Biosynthesis in Chlamydomonas reinhardtii

(Received for publication, June 12, 1996, and in revised form, September 14, 1996)

Hong Hua Li , Jeanette Quinn , Duane Culler , Jacqueline Girard-Bascou ^§ and Sabeeha Merchant

From the  Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569 and ^§ Service de Photosynthese, Institut de Biologie Physico-Chemique, 13 rue Pierre et Marie Curie, Paris 75005, France

Five plastocyanin-deficient mutants were identified from a population of UV-mutagenized Chlamydomonas reinhardtii cells. Genetic complementation experiments indicated that four mutants represented alleles at the PCY1 locus (pcy1-2, pcy1-3, pcy1-4, and pcy1-5). Sequence analysis confirmed that two strains, pcy1-2 and pcy1-3, carry a frameshift (1) and a nonsense mutation, respectively, while strains pcy1-4 and pcy1-5 synthesize an extended protein as a result of read-through mutations at the stop codon. The C-terminal extension does not affect synthesis or processing of the pre-proteins, but the polypeptides are rapidly degraded after the second (lumenal) processing event. The frameshift mutation in pcy1-2 results in loss of Pcy1 mRNA, as noted previously for strain ac208 (pcy1-1), but the abundance of Pcy1 mRNA in strain pcy1-3, which carries a nonsense mutation at codon 26, is unaffected relative to wild-type cells. The decreased abundance of frameshifted Pcy1 mRNA is attributed to increased degradation rather than decreased synthesis, since the mRNAs can be stabilized by treatment of cells with cycloheximide or anisomycin. The fifth strain has a wild-type plastocyanin-encoding gene, but the strain accumulates apoplastocyanin at the expense of holoplastocyanin. We suggest that the mutation identifies a new locus (PCY2) whose function is required for normal holoplastocyanin accumulation. Like ac208 (pcy1-1), several of the new mutants were suppressed spontaneously owing to accumulation of cytochrome c₆ (a functional substitute for plastocyanin). The suppressor mutation(s) displayed Mendelian inheritance and segregated independently from the PCY1 locus, which confirms that regulation of Cyc6 expression is not tightly linked to plastocyanin function.

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