Shashidhara L S, Lim S H, Shackleton J B, Robinson C, Smith A G
Department of Plant Sciences, University of Cambridge, United Kingdom.
J Biol Chem. 1992 Jun 25;267(18):12885-91.
A system has been developed for the import in vitro of precursor proteins into Euglena chloroplasts, which have three envelope membranes. Preparation of functional chloroplasts with intact envelope membranes has been optimized. Import of the precursor (50 kDa) for the tetrapyrrole biosynthesis enzyme porphobilinogen deaminase (PBGD), and processing to the mature size (40 kDa), occurred at 25 degrees C in the light and the presence of ATP, with an estimated efficiency of 62%. Pretreatment of the chloroplasts with proteases abolished this import, suggesting the involvement of specific protein receptors. The presequence of PBGD was found to be cleaved by Escherichia coli leader peptidase to an intermediate form (46 kDa). A construct in which the first 30 residues of the presequence (presumed to be the region removed by leader peptidase) had been deleted was no longer imported. Neither prePBGD nor the truncated precursor were imported into pea chloroplasts, although both bound to the pea chloroplast envelope. Conversely, a chimeric construct, in which the mature PBGD protein was fused downstream of the transit peptide for pea ferredoxin-NADP reductase, was efficiently imported into pea chloroplasts and processed to the mature size. However, this was not imported into Euglena chloroplasts, although again it bound to them. These results provide preliminary evidence for the possibility of two functional domains within the Euglena PBGD presequence. The implications of these findings with respect to the evolution of Euglena chloroplasts are discussed.
已经开发出一种用于将前体蛋白体外导入眼虫叶绿体的系统，眼虫叶绿体有三层包膜。完整包膜的功能性叶绿体的制备已得到优化。四吡咯生物合成酶胆色素原脱氨酶（PBGD）的前体（50 kDa）在25℃、光照和ATP存在的条件下导入，并加工成成熟大小（40 kDa），估计效率为62%。用蛋白酶预处理叶绿体消除了这种导入，表明存在特定的蛋白质受体。发现PBGD的前导序列被大肠杆菌前导肽酶切割成中间形式（46 kDa）。一种构建体，其中前导序列的前30个残基（假定为被前导肽酶去除的区域）已被删除，不再被导入。前体PBGD和截短的前体都没有导入豌豆叶绿体，尽管它们都与豌豆叶绿体包膜结合。相反，一种嵌合构建体，其中成熟的PBGD蛋白融合在豌豆铁氧还蛋白-NADP还原酶转运肽的下游，被有效地导入豌豆叶绿体并加工成成熟大小。然而，它没有导入眼虫叶绿体，尽管它再次与眼虫叶绿体结合。这些结果为眼虫PBGD前导序列中存在两个功能域的可能性提供了初步证据。讨论了这些发现对眼虫叶绿体进化的影响。
