Waegemann K, Soll J
Botanisches Institut, Universitat Kiel, Germany.
J Biol Chem. 1996 Mar 15;271(11):6545-54. doi: 10.1074/jbc.271.11.6545.
A protein kinase was located in the cytosol of pea mesophyll cells. The protein kinase phosphorylates, in an ATP-dependent manner, chloroplast-destined precursor proteins but not precursor proteins, which are located to plant mitochondria or plant peroxisomes. The phosphorylation occurs on either serine or threonine residues, depending on the precursor protein used. We demonstrate the specific phosphorylation of the precursor forms of the chloroplast stroma proteins ferredoxin (preFd), small subunit of ribulose-bisphosphate-carboxylase (preSSU), the thylakoid localized light-harvesting chlorophyll a/b-binding protein (preLHCP), and the thylakoid lumen-localized proteins of the oxygen-evolving complex of 23 kDa (preOE23) and 33 kDa (preOE33). In the case of thylakoid lumen proteins which possess bipartite transit sequences, the phosphorylation occurs within the stroma-targeting domain. By using single amino acid substitution within the presequences of preSSU, preOE23, and preOE33, we were able to tentatively identify a consensus motif for the precursor protein protein kinase. This motif is (P/G)X(n)(R/K)X(n)(S/T)X(n) (S*/T*), were n = 0-3 amino acids spacer and S*/T* represents the phosphate acceptor. The precursor protein protein kinase is present only in plant extracts, e.g. wheat germ and pea, but not in a reticulocyte lysate. Protein import experiments into chloroplasts revealed that phosphorylated preSSU binds to the organelles, but dephosphorylation seems required to complete the translocation process and to obtain complete import. These results suggest that a precursor protein protein phosphatase is involved in chloroplast import and represents a so far unidentified component of the import machinery. In contrast to sucrose synthase, a cytosolic marker protein, the precursor protein protein kinase seems to adhere partially to the chloroplast surface. A phosphorylation-dephosphorylation cycle of chloroplast-destined precursor proteins might represent one step, which could lead to a specific sorting and productive translocation in plant cells.
一种蛋白激酶定位于豌豆叶肉细胞的胞质溶胶中。该蛋白激酶以ATP依赖的方式磷酸化叶绿体靶向的前体蛋白，但不磷酸化定位于植物线粒体或植物过氧化物酶体的前体蛋白。磷酸化发生在丝氨酸或苏氨酸残基上，这取决于所使用的前体蛋白。我们证明了叶绿体基质蛋白铁氧化还原蛋白（preFd）、核酮糖-1,5-二磷酸羧化酶小亚基（preSSU）、类囊体定位的捕光叶绿素a/b结合蛋白（preLHCP）以及类囊体腔定位的23 kDa（preOE23）和33 kDa（preOE33）放氧复合体蛋白的前体形式的特异性磷酸化。对于具有双部分转运序列的类囊体腔蛋白，磷酸化发生在基质靶向结构域内。通过在preSSU、preOE23和preOE33的前序列内进行单氨基酸替换，我们能够初步确定前体蛋白蛋白激酶的共有基序。该基序为(P/G)X(n)(R/K)X(n)(S/T)X(n)(S*/T*)，其中n = 0 - 3个氨基酸间隔，S*/T*代表磷酸受体。前体蛋白蛋白激酶仅存在于植物提取物中，如小麦胚芽和豌豆，但不存在于网织红细胞裂解物中。叶绿体导入实验表明，磷酸化的preSSU与细胞器结合，但似乎需要去磷酸化才能完成转运过程并实现完全导入。这些结果表明，一种前体蛋白蛋白磷酸酶参与叶绿体导入，并且是导入机制中迄今尚未鉴定的一个组分。与胞质溶胶标记蛋白蔗糖合酶不同，前体蛋白蛋白激酶似乎部分附着于叶绿体表面。叶绿体靶向前体蛋白的磷酸化-去磷酸化循环可能代表了一个步骤，这可能导致植物细胞中的特异性分选和有效转运。
