Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.
Genes Cells. 2012 Aug;17(8):728-36. doi: 10.1111/j.1365-2443.2012.01622.x. Epub 2012 Jul 2.
Phosphatidylserine (PS), a relatively minor constituent in the plasma membrane (PM), participates in various cellular processes such as clearance of apoptotic cells and recruitment of signaling molecules. PS also localizes in the membranes of endocytic organelles, such as recycling endosomes (REs). We recently showed that in REs, PS binds to the pleckstrin homology (PH) domain of evectin-2, thereby regulating retrograde traffic from REs to the Golgi. However, direct evidence that PS has a role in retrograde traffic is lacking. Here, we examined the contribution of PS to endosomal membrane traffic by exploiting a mutant CHO cell line (PSA-3) that is defective in PS synthesis. In PSA-3 cells, the Golgi localization of TGN38, a protein that circulates between the Golgi and the PM through endosomes by retrograde traffic, was abolished, whereas the localizations of other organelle markers remained unchanged. Increasing the cellular PS level by adding ethanolamine to the culture medium restored the Golgi localization of TGN38. Tracking the endocytic fate of cell surface TGN38 that was labeled by anti-TGN38 antibody showed that retrograde transport of TGN38 was impaired at endosomes, not at the PM. These findings provide direct evidence that intracellular PS is required for retrograde traffic through endosomes.
磷脂酰丝氨酸（PS）是质膜（PM）中相对较少的成分，参与多种细胞过程，如清除凋亡细胞和募集信号分子。PS 还定位于内吞细胞器的膜中，如再循环内体（RE）。我们最近表明，在 RE 中，PS 与 evectin-2 的 pleckstrin 同源（PH）结构域结合，从而调节从 RE 到高尔基体的逆行运输。然而，PS 在逆行运输中起作用的直接证据尚缺乏。在这里，我们通过利用一种在 PS 合成中缺陷的 CHO 细胞系（PSA-3）来研究 PS 对内体膜运输的贡献。在 PSA-3 细胞中，TGN38 的高尔基体定位被消除，TGN38 是一种通过逆行运输在高尔基体和 PM 之间循环的蛋白质，而其他细胞器标记物的定位保持不变。通过在培养基中添加乙醇胺增加细胞内 PS 水平，恢复了 TGN38 的高尔基体定位。追踪用抗 TGN38 抗体标记的细胞表面 TGN38 的内吞命运表明，TGN38 的逆行运输在内体中受损，而不是在 PM 上。这些发现提供了直接的证据，证明细胞内 PS 是通过内体进行逆行运输所必需的。
