Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-enolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide.
Ribulose bisphosphate carboxylase small subunit protein is synthesized in the cytoplasm as a precursor and transported into the chloroplast where the amino-terminal portion, the transit peptide, is removed proteolytically. To obtain chloroplast delivery of the 43-kDa 5-enolpyruvyl 3-phosphoshikimate (EPSP) synthase of Salmonella typhimurium, we constructed fusion proteins between the bacterial EPSP synthase and the ribulose bisphosphate carboxylase small subunit. A fusion protein consisting of the transit peptide fused to the EPSP synthase was not transported in vitro or in vivo into chloroplasts. A second fusion protein consisting of the transit peptide and 24 amino acids of the mature small subunit fused to the EPSP synthase was transported both in vitro and in vivo into chloroplasts. It was processed into two polypeptides of 46 and 47 kDa, respectively. This heterogeneity in processing was not caused by the presence of the aroA start codon, since its removal resulted in the same pattern. Substituting 24 different amino acids for the 24 amino acids of the mature small subunit resulted in a fusion protein that was not transported into the chloroplast. It was concluded that a portion of the mature small subunit was needed for efficient chloroplast delivery.
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002678:Chimera; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D014166:Transferases / Q000235:genetics*
D046911:Macromolecular Substances; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D012273:Ribulose-Bisphosphate Carboxylase