Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Abstract

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.