CHARACTERIZATION OF PLASTOCYANIN FROM THE CYANOBACTERIUM PHORMIDIUM-LAMINOSUM - COPPER-INDUCIBLE EXPRESSION AND SECA-DEPENDENT TARGETING INESCHERICHIA-COLI

Plastocyanin from the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and the gen e cloned and sequenced. The derived amino acid sequence indicates that the plastocyanin protein is initially synthesized with an N-terminal leader sequence of 34 amino acids to direct it across the thylakoid me mbrane. The leader sequence consists of a positively charged N-termina l region, a hydrophobic region and a cleavage site, which are characte ristic both of higher-plant chloroplast thylakoid transfer domains and of bacterial leader peptides. The petE gene and flanking regions have been cloned in Eschericha coli, and the plastocyanin protein is expre ssed and directed to the periplasmic space, with concomitant processin g to the mature form. Targeting to the periplasm and processing of the plastocyanin protein in E. coli appears to be dependent on components of the Sec apparatus, since the unprocessed precursor accumulates in the cytoplasm of a secA mutant. Expression of plastocyanin in E. coli is copper-inducible and apparently controlled at the level of transcri ption, leading to the conclusion that copper-regulated promoters exist in the regions flanking the gene and are recognized in a heterologous system. Possible implications for gene expression and protein targeti ng in the cyanobacterium are discussed.