Characterization of plastocyanin from the cyanobacterium Phormidium laminosum: copper-inducible expression and SecA-dependent targeting in Escherichia coli.
Plastocyanin from the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and the gene 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 membrane. The leader sequence consists of a positively charged N-terminal region, a hydrophobic region and a cleavage site, which are characteristic both of higher-plant chloroplast thylakoid transfer domains and of bacterial leader peptides. The petE gene and flanking regions have been cloned in Escherichia coli, and the plastocyanin protein is expressed and directed to the periplasmic space, with concomitant processing 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 transcription, 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 targeting in the cyanobacterium are discussed.
Varley
J P
JP
Department of Biochemistry, University of Cambridge, U.K.
Moehrle
J J
JJ
Manasse
R S
RS
Bendall
D S
DS
Howe
C J
CJ
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003300:Copper / Q000494:pharmacology*; D000458:Cyanobacteria / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D005798:Genes, Bacterial / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D010970:Plastocyanin / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D003300:Copper; D010970:Plastocyanin; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins