Pilon M, de Kruijff B, Weisbeek P J
Institute of Molecular Biology, University of Utrecht, The Netherlands.
J Biol Chem. 1992 Feb 5;267(4):2548-56.
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.
我们研究了核编码叶绿体蛋白铁氧还蛋白的导入途径。通过在特定的体外摄取系统中使用纯化的前体蛋白和洗涤过的完整叶绿体，我们发现铁氧还蛋白自身具有完全的导入能力。此外，我们还表明，在小麦胚芽裂解物中体外合成的铁氧还蛋白并不与另一种蛋白稳定结合。导入仅依赖于ATP，不需要胞质蛋白的存在。硫醇还原剂二硫苏糖醇（DTT）可在很大程度上刺激转运。为了确定DTT作用于前体还是叶绿体，我们通过与碘乙酰胺反应修饰了前体中的5个半胱氨酸，从而防止前体中形成二硫键。添加DTT仍能刺激这种修饰前体的导入，表明DTT对叶绿体导入机制有刺激作用。对于修饰前体，导入一定是在没有铁硫簇附着于基质的情况下发生的。修饰前体的导入效率与亲本前体相似，表明导入独立于辅因子组装进行。
