Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235.
J Cell Biol. 2013 Sep 16;202(6):875-86. doi: 10.1083/jcb.201305094. Epub 2013 Sep 9.
Vesicle-mediated protein transport between organelles of the secretory and endocytic pathways is strongly influenced by the composition and organization of membrane lipids. In budding yeast, protein transport between the trans-Golgi network (TGN) and early endosome (EE) requires Drs2, a phospholipid translocase in the type IV P-type ATPase family. However, downstream effectors of Drs2 and specific phospholipid substrate requirements for protein transport in this pathway are unknown. Here, we show that the Arf GTPase-activating protein (ArfGAP) Gcs1 is a Drs2 effector that requires a variant of the ArfGAP lipid packing sensor (+ALPS) motif for localization to TGN/EE membranes. Drs2 increases membrane curvature and anionic phospholipid composition of the cytosolic leaflet, both of which are sensed by the +ALPS motif. Using mutant forms of Drs2 and the related protein Dnf1, which alter their ability to recognize phosphatidylserine, we show that translocation of this substrate to the cytosolic leaflet is essential for +ALPS binding and vesicular transport between the EE and the TGN.
囊泡介导的分泌途径和内吞途径细胞器之间的蛋白质运输受到膜脂组成和结构的强烈影响。在出芽酵母中，跨高尔基网络（TGN）和早期内体（EE）之间的蛋白质运输需要 Drs2，它是 IV 型 P 型 ATP 酶家族中的一种磷脂转位酶。然而，Drs2 的下游效应物以及该途径中蛋白质运输的特定磷脂底物要求尚不清楚。在这里，我们表明 Arf GTP 酶激活蛋白（ArfGAP）Gcs1 是 Drs2 的一种效应物，它需要一种 ArfGAP 脂质包装传感器（+ALPS）基序的变体来定位到 TGN/EE 膜。Drs2 增加了细胞膜曲率和细胞质小叶的阴离子磷脂组成，这两者都被+ALPS 基序所感知。使用 Drs2 和相关蛋白 Dnf1 的突变形式，这些突变改变了它们识别磷脂酰丝氨酸的能力，我们表明这种底物向细胞质小叶的易位对于+ALPS 结合和 EE 与 TGN 之间的囊泡运输是必需的。
