Lundberg Mathias, Wikström Sara, Johansson Magnus
Division of Clinical Virology F68, Karolinska Institute, Huddinge University Hospital, S-14186, Stockholm, Sweden.
Mol Ther. 2003 Jul;8(1):143-50. doi: 10.1016/s1525-0016(03)00135-7.
Protein transduction domains (PTD), such as the HIV TAT and the herpes simplex virus VP22 proteins, are reported to translocate across the membranes of mammalian cells. The mechanism of PTD membrane translocation has largely remained elusive, but recent studies suggest that the reported PTD translocation is due to a fixation artifact. We have constructed and expressed the PTDs VP22, TAT, polyarginine, and polylysine fused to the green fluorescent protein to visualize these proteins in both living and fixed cells. The investigated PTDs strongly adhered to the surface of living cells and were internalized by constitutive endocytosis. No cytosolic or nuclear import of the proteins was detected. In contrast, the PTD-GFP fusion proteins were redistributed to the cytosol and nucleus directly after fixation. Our findings suggest that the PTDs only mediate cell surface adherence, a property shared with many other positively charged macromolecules. The cell surface adherence results in endocytosis and accumulation of proteins in endosomes. We suggest that the biological effects observed for PTD fusion proteins are due to cell surface interactions and internalization of the proteins into cells by classical endocytosis.
据报道，蛋白质转导结构域（PTD），如HIV TAT和单纯疱疹病毒VP22蛋白，可穿过哺乳动物细胞的膜。PTD跨膜转运的机制在很大程度上仍然难以捉摸，但最近的研究表明，所报道的PTD转运是由于固定假象。我们构建并表达了与绿色荧光蛋白融合的PTDs VP22、TAT、聚精氨酸和聚赖氨酸，以便在活细胞和固定细胞中可视化这些蛋白质。所研究的PTDs强烈粘附于活细胞表面，并通过组成型内吞作用被内化。未检测到蛋白质的胞质或核内导入。相反，PTD-GFP融合蛋白在固定后直接重新分布到细胞质和细胞核中。我们的研究结果表明，PTDs仅介导细胞表面粘附，这是许多其他带正电荷的大分子共有的特性。细胞表面粘附导致蛋白质的内吞作用和在内涵体中的积累。我们认为，观察到的PTD融合蛋白的生物学效应是由于细胞表面相互作用以及蛋白质通过经典内吞作用进入细胞。
