Amino acid sequence motifs and mechanistic features of the membrane translocation of α-synuclein
Amino acid sequence motifs and mechanistic features of the membrane translocation of α-synuclein
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DOI：
10.1111/j.1471-4159.2006.03731.x
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发表时间：
2006-04-01
影响因子：
4.7
通讯作者：
Kim, J
中科院分区：
文献类型：
--
作者：
Ahn, KJ;Paik, SR;Kim, J
许多证据表明，α - 突触核蛋白可从细胞中分泌出来，也可进入细胞，尽管其详细机制尚不清楚。在这项研究中，我们探究了α - 突触核蛋白跨膜转运所需的氨基酸序列基序以及这一现象的机制特征。我们首先发现，不仅α - 突触核蛋白，而且β - 和γ - 突触核蛋白也能进入活细胞，这表明保守的N - 末端区域可能负责跨膜转运。通过一系列缺失突变体，我们证明了在突触核蛋白家族成员中发现的11个氨基酸的不完全重复序列在这些蛋白质的跨膜转运中起着关键作用。我们进一步证明，含有α - 突触核蛋白11个氨基酸不完全重复序列的融合肽能够穿过质膜，并且当肽包含两个重复基序时，跨膜转运效率最佳。α - 突触核蛋白似乎能够快速且高效地进入细胞，在外源处理后5分钟内细胞质中即可检测到蛋白质。有趣的是，在4℃时α - 突触核蛋白的进入情况与在37℃时观察到的进入情况相当。此外，用内吞作用抑制剂处理对α - 突触核蛋白的跨膜转运没有显著影响。这些结果表明，α - 突触核蛋白的内化对温度不敏感，并且通过一种不同于正常内吞作用的机制非常迅速地发生。
Many lines of evidence suggest that alpha-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of alpha-synuclein, and the mechanistic features of the phenomenon. We first showed that not only alpha-synuclein but also beta- and gamma-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of alpha-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. alpha-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of alpha-synuclein at 4 degrees C was comparable with the import observed at 37 degrees C. Furthermore, membrane translocation of alpha-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of alpha-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.