Viitanen P V, Doran E R, Dunsmuir P
Central Research and Development, Du Pont Experimental Station, Wilmington, Delaware 19898.
J Biol Chem. 1988 Oct 15;263(29):15000-7.
Whereas it is widely accepted that the transit peptide of the precursor for the light-harvesting chlorophyll a/b protein (preLHCP) is responsible for targeting this polypeptide to chloroplasts, the signals which govern its intraorganellar targeting appears to be transit peptide-mediated for plastocyanin (Smeekins, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375) and several other nuclear-encoded, thylakoid luminal proteins. To determine whether a similar mechanism operates for LHCP (an integral thylakoid protein), we have used oligonucleotide-directed mutagenesis to delete the proposed transit sequence from a petunia precursor of this polypeptide. Intact preLHCP and the deletion mutant product have been expressed in vitro, and their abilities to integrate into purified thylakoids have been compared. We have found that both polypeptides insert into thylakoids correctly, provided the latter are supplemented with a membrane-free stromal extract and Mg.ATP. Our results clearly demonstrate that whereas the transit peptide is required for transport into chloroplasts, thylakoid integration of preLHCP is determined by mature portions of the polypeptide. In addition, we note that transit peptide removal has little effect on the apparent solubility of the in vitro translation products.
尽管人们普遍认为，捕光叶绿素a/b蛋白前体（preLHCP）的转运肽负责将该多肽靶向叶绿体，但对于质体蓝素（Smeekins, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365 - 375）以及其他几种核编码的类囊体腔蛋白而言，控制其细胞器内靶向的信号似乎是由转运肽介导的。为了确定类似的机制是否也适用于LHCP（一种类囊体整合蛋白），我们利用寡核苷酸定向诱变技术从该多肽的矮牵牛前体中删除了推测的转运序列。完整的preLHCP和缺失突变体产物已在体外表达，并比较了它们整合到纯化类囊体中的能力。我们发现，只要向类囊体补充无膜的基质提取物和Mg.ATP，这两种多肽都能正确插入类囊体。我们的结果清楚地表明，虽然转运肽是进入叶绿体所必需的，但preLHCP的类囊体整合是由多肽的成熟部分决定的。此外，我们注意到去除转运肽对体外翻译产物的表观溶解度影响很小。
