New insights into the import mechanism of the ferredoxin precursor into chloroplasts

We have investigated the import pathway of the nuclear-encoded chloroplast protein ferredoxin. By using purified precursor protein and washed intact chloroplasts in a defined in vitro uptake system, we show that preferredoxin is fully import-competent by itself. In addition, we show also that the in vitro, in a wheat germ lysate, synthesized preferredoxin is not stably associated with another protein. Import is dependent only on ATP and does not require the presence of cytosolic proteins. Translocation could be largely stimulated by the thiol reducing agent dithiothreitol (DTT). To determine whether DTT acts on the precursor or on the chloroplast, we modified the 5 cysteines in the precursor by a reaction with iodoacetamide, thereby preventing the formation of disulfide bridges in the precursor. The import of this modified precursor was still stimulated by the addition of DTT, indicating that DTT had a stimulating effect on the chloroplast import machinery. In the case of the modified precursor, the import must have taken place without iron-sulfur cluster attachment in the stroma. The modified precursor could be imported with a similar efficiency as the parent precursor showing that import takes place independently from cofactor assembly.