Clark S E, Abad M S, Lamppa G K
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
J Biol Chem. 1989 Oct 15;264(29):17544-50.
We have shown previously that during in vitro import into chloroplasts, the precursor of the major light-harvesting chlorphyll a/b-binding protein (LHCP) generated from a wheat gene gives rise to two mature forms (25 and approximately 26 kDa) which are inserted into the thylakoids. However, during incubation of the LHCP precursor with a chloroplast-soluble extract in an organelle-free processing reaction, the NH2 terminus is cleaved, yielding only a 25-kDa peptide. In the present study, mutations at the transit peptide-mature protein junction were introduced in the LHCP precursor to investigate the relationship between the two peptides and the determinants of proteolytic processing. Mutant p delta 3 lacks 3 amino acids including Met34 at the primary cleavage site thought to give rise to the 26-kDa peptide. It is still processed during import and in the organelle-free reaction yielding in both assays only a 25-kDa peptide. Mutant p + 4 has 4 amino acids inserted immediately after Met34 and a proline that disrupts the alpha-helix predicted by the Garnier-Osguthorpe-Robson method (Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120) to extend through this region. Although p + 4 is imported, it is inefficiently processed; both a 25- and 26-kDa peptide are found, but at least 60% of the imported precursor remains uncleaved. Less than 5% is processed in the organelle-free assay. Replacement of the predicted alpha-helix in the mutant p + 4 alpha restores processing upon import into the chloroplast, but this mutant, which also has a 4-amino acid insert, yields only a 26-kDa peptide. p + 4 alpha is not processed in the organelle-free reaction. These results provide evidence that the two forms of LHCP obtained during import are the result of independent processing at two cleavage sites: the first site at Met34, and a second approximately 10 amino acids downstream within what has been designated the NH2 terminus of the mature protein. Whereas p delta 3 has the first site removed but retains a functional second site, in p + 4 alpha only the first site, or one very near it, is accessible to the processing enzyme during import. The conditions of the organelle-free reaction are specific for processing at only the secondary site. We discuss the implications of these findings in terms of the heterogeneity of LHCP in vivo.
我们之前已经表明，在体外导入叶绿体的过程中，从小麦基因产生的主要捕光叶绿素a/b结合蛋白（LHCP）前体产生了两种成熟形式（25 kDa和大约26 kDa），它们被插入类囊体膜中。然而，在无细胞器加工反应中，将LHCP前体与叶绿体可溶性提取物一起温育时，其氨基末端被切割，仅产生一个25 kDa的肽段。在本研究中，在LHCP前体的转运肽-成熟蛋白连接处引入突变，以研究这两种肽段之间的关系以及蛋白水解加工的决定因素。突变体p delta 3在被认为会产生26 kDa肽段的初级切割位点缺少包括Met34在内的3个氨基酸。它在导入过程中和无细胞器反应中仍会被加工，在两种测定中都仅产生一个25 kDa的肽段。突变体p + 4在Met34之后紧接着插入了4个氨基酸以及一个脯氨酸，该脯氨酸破坏了由Garnier-Osguthorpe-Robson方法预测的延伸穿过该区域的α螺旋（Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97 - 120）。尽管p + 4被导入，但加工效率低下；发现了25 kDa和26 kDa两种肽段，但至少60%的导入前体未被切割。在无细胞器测定中，不到5%的前体被加工。在突变体p + 4 alpha中替换预测的α螺旋可恢复导入叶绿体时的加工，但该突变体（也有一个4个氨基酸的插入）仅产生一个26 kDa的肽段。p + 4 alpha在无细胞器反应中不被加工。这些结果提供了证据，表明在导入过程中获得的两种形式的LHCP是在两个切割位点独立加工的结果：第一个位点在Met34处，第二个位点在成熟蛋白氨基末端内大约下游10个氨基酸处。虽然p delta 3去除了第一个位点但保留了一个功能性的第二个位点，但在p + 4 alpha中，在导入过程中加工酶只能接近第一个位点或与其非常接近的一个位点。无细胞器反应的条件仅对在二级位点的加工具有特异性。我们根据体内LHCP的异质性讨论了这些发现的意义。
