The most C-terminal tri-glycine segment within the polyglycine stretch of the pea Toc75 transit peptide plays a critical role for targeting the protein to the chloroplast outer envelope membrane
The most C-terminal tri-glycine segment within the polyglycine stretch of the pea Toc75 transit peptide plays a critical role for targeting the protein to the chloroplast outer envelope membrane
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DOI：
10.1111/j.1742-4658.2006.05175.x
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发表时间：
2006-04-01
期刊：
影响因子：
5.4
通讯作者：
Inoue, K
中科院分区：
文献类型：
--
作者：
Baldwin, AJ;Inoue, K
叶绿体外膜的蛋白质易位通道 (Toc75) 是作为具有 N 末端转运肽的较大前体合成的。在豌豆 Toc75 的转运肽中，含有 9 个甘氨酸残基的 10 个氨基酸长段的主要部分被证明对于在体外将蛋白质引导至叶绿体外膜是必需的 [Inoue K & Keegstra K (2003) Plant J 34, 661-669]。为了深入了解聚甘氨酸延伸介导正确靶向的机制，我们将其分为三个三甘氨酸片段，并检查每个结构域在体外靶向特异性中的重要性。用丙氨酸残基替换大部分 C 末端片段会导致蛋白质错误定位到基质，而交换其他两个三甘氨酸区域中的任何一个对正确定位没有影响。此外，用丙氨酸重复序列同时替换 N 端和中间三甘氨酸片段不会像 N 端和 C 端片段或中间和 C 端片段那样导致蛋白质的错误定位。这些结果表明，最 C 端的三甘氨酸片段对于正确靶向非常重要。用重复的亮氨酸或谷氨酸交换该部分也会导致 Toc75 错配到基质。相比之下，它被天冬酰胺、天冬氨酸、丝氨酸的重复取代。脯氨酸对正确定位影响不大。这些数据表明，该特定区域中相对紧凑且非疏水性的侧链在 Toc75 的正确分选中发挥着至关重要的作用。
The protein translocation channel at the outer envelope membrane of chloroplasts (Toc75) is synthesized as a larger precursor with an N-terminal transit peptide. Within the transit peptide of the pea Toc75, a major portion of the 10 amino acid long stretch that contains nine glycine residues was shown to be necessary for directing the protein to the chloroplast outer membrane ill vitro [Inoue K & Keegstra K (2003) Plant J 34, 661-669]. In order to get insights into the mechanism by which the polyglycine stretch mediates correct targeting, we divided it into three tri-glycine segments and examined the importance of each domain in targeting specificity ill vitro. Replacement of the most C-terminal segment with alanine residues resulted in mistargeting the protein to the stroma, while exchange of either of the other two tri-glycine regions had no effect on correct targeting. Furthermore, simultaneous replacement of the N-terminal and middle tri-glycine segments with alanine repeats did not cause mistargeting of the protein as much as those of the N- and C-terminal, or the middle and C-terminal segments. These results indicate that the most C-terminal tri-glycine segment is important for correct targeting. Exchanging this portion with a repeat of leucine or glutamic acid also caused missorting of Toc75 to the stroma. By contrast, its replacement with repeats of asparagine, aspartic acid, serine. and proline did not largely affect correct targeting. These data suggest that relatively compact and nonhydrophobic side chains in this particular region play a crucial role in correct sorting of Toc75.