Hinz G, Flügge U I
Institut für Biochemie der Pflanze, Göttingen, Federal Republic of Germany.
Eur J Biochem. 1988 Aug 15;175(3):649-59. doi: 10.1111/j.1432-1033.1988.tb14241.x.
In this report we demonstrate that a 51-kDa outer-envelope membrane protein (P51) is involved in protein translocation into chloroplasts. Furthermore it is shown that phosphorylation of P51 is functionally related to the process of binding and/or importing precursor proteins into chloroplasts. Several lines of evidence have been obtained supporting this suggestion. First, protein import into chloroplasts was inhibited by the membrane-impermeable agent pyridoxal 5'-phosphate, which has been shown to react with a component of the protein-import apparatus. Phosphorylation of envelope membrane polypeptides using [gamma-32P]ATP in the presence of pyridoxal 5'-phosphate resulted in an increased incorporation of 32P radiolabel into a 51-kDa membrane polypeptide (P51). A close correlation between the inhibition of protein import and the increase in the phosphorylation state of P51, both as a function of PLP concentration, was observed. Second, binding of purified precursor proteins to chloroplasts resulted in a specific increase in the phosphorylation state of P51. This effect was not exerted by the mature form of the precursor protein lacking the presequence. Third, internally generated ATP was able to compete specifically with externally added [gamma-32P]ATP for the phosphorylation of P51. Fourth, digestion of the outer-envelope membrane with low amounts of thermolysin resulted in a loss of protein import activity, which was associated with the removal of the phosphorylation site of P51. Phosphorylation of P51 proceeds with an apparent Km (ATP) of about 5 microM, which is much lower than the ATP concentration required for the protein translocation itself. We suggest that two different ATP-dependent processes are involved in protein translocation into chloroplasts. P51 represent presumably a regulatory component of the protein-import apparatus or the protein receptor itself.
在本报告中，我们证明了一种51 kDa的外膜蛋白（P51）参与蛋白质向叶绿体的转运。此外，研究表明P51的磷酸化与前体蛋白结合和/或导入叶绿体的过程在功能上相关。我们获得了几条证据支持这一观点。首先，蛋白质向叶绿体的导入受到膜不透性试剂磷酸吡哆醛的抑制，该试剂已被证明可与蛋白质导入装置的一个组分发生反应。在磷酸吡哆醛存在下，使用[γ-32P]ATP对包膜膜多肽进行磷酸化，导致32P放射性标记更多地掺入到一种51 kDa的膜多肽（P51）中。观察到蛋白质导入的抑制与P51磷酸化状态的增加之间存在密切相关性，二者均为PLP浓度的函数。其次，纯化的前体蛋白与叶绿体的结合导致P51磷酸化状态的特异性增加。缺乏前导序列的前体蛋白成熟形式未产生这种效应。第三，内源性产生的ATP能够与外部添加的[γ-32P]ATP特异性竞争P51的磷酸化。第四，用少量嗜热菌蛋白酶消化外膜导致蛋白质导入活性丧失，这与P51磷酸化位点的去除有关。P51的磷酸化反应的表观Km（ATP）约为5 μM，远低于蛋白质转运本身所需的ATP浓度。我们认为，蛋白质向叶绿体的转运涉及两个不同的ATP依赖性过程。P51可能代表蛋白质导入装置或蛋白质受体本身的一种调节成分。
