Halperin T, Adam Z
Department of Agricultural Botany, Faculty of Agriculture, Hebrew University, Rehovot, Israel.
Plant Mol Biol. 1996 Mar;30(5):925-33. doi: 10.1007/BF00020804.
OEE33, a component of the oxygen-evolving enzyme in chloroplasts, normally resides in the thylakoid lumen. In an attempt to study the fate of mistargeted proteins in chloroplasts, we substituted the bipartite transit peptide of OEE33 with that of CAB7, an integral thylakoid-membrane protein. As a result, when imported into isolated chloroplasts, the chimeric protein protein was targeted to the stroma instead of the thylakoid lumen. Whereas the wild-type OEE33 was totally stable for at least 2 h, the chimeric protein was rapidly degraded, with a half-life of 60 min. Degradation of the chimeric protein was stimulated by ATP supplementation. Degradation could also be observed in lysed chloroplasts, in an ATP-stimulated manner. When lysates were fractionated, the proteolytic activity was found to be associated mainly with the stromal fraction. This activity was very effectively inhibited by all tested inhibitors of serine proteases. Western blot analysis demonstrated that the stromal fraction active in degrading the chimeric OEE33 contains ClpC and ClpP, homologues of the regulatory and proteolytic subunits, respectively, of the bacterial, ATP-dependent, serine-type Clp protease.
OEE33是叶绿体中放氧酶的一个组分，通常位于类囊体腔中。为了研究叶绿体中错误靶向蛋白的命运，我们将OEE33的双功能转运肽替换为CAB7（一种类囊体膜整合蛋白）的转运肽。结果，当导入分离的叶绿体时，嵌合蛋白被靶向到基质而不是类囊体腔。野生型OEE33至少2小时完全稳定，而嵌合蛋白迅速降解，半衰期为60分钟。补充ATP可刺激嵌合蛋白的降解。在裂解的叶绿体中也能观察到以ATP刺激方式进行的降解。当对裂解物进行分级分离时，发现蛋白水解活性主要与基质部分相关。所有测试的丝氨酸蛋白酶抑制剂都能非常有效地抑制这种活性。蛋白质印迹分析表明，在降解嵌合OEE33中起作用的基质部分分别含有ClpC和ClpP，它们分别是细菌ATP依赖性丝氨酸型Clp蛋白酶的调节亚基和蛋白水解亚基的同源物。
