Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
Nat Cell Biol. 2012 Feb 19;14(3):239-48. doi: 10.1038/ncb2427.
Clathrin-coated vesicles mediate endocytosis and transport between the trans-Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo and membranes. Two main types of clathrin adaptor act in TGN-endosome traffic: GGA proteins and the AP-1 complex. Here we characterize the relationship between GGA proteins, AP-1 and other TGN clathrin adaptors using live-cell and super-resolution microscopy in yeast. We present evidence that GGA proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PtdIns(4)P) levels at the TGN slow or uncouple AP-1 coat assembly from GGA coat assembly. Conversely, enhanced PtdIns(4)P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PtdIns(4)-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PtdIns(4)P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.
网格蛋白包被小泡介导真核细胞内高尔基体反面管网（TGN）与内体之间的内吞作用和运输。网格蛋白衔接蛋白在衣被组装中发挥核心作用，与网格蛋白、货物和膜相互作用。两种主要类型的网格蛋白衔接蛋白参与 TGN-内体运输：GGA 蛋白和 AP-1 复合物。在此，我们使用酵母活细胞和超分辨率显微镜研究了 GGA 蛋白、AP-1 和其他 TGN 网格蛋白衔接蛋白之间的关系。我们提供的证据表明，GGA 蛋白和 AP-1 被募集到 TGN 的两次网格蛋白组装波中。降低 TGN 处的磷脂酰肌醇 4-磷酸（PtdIns(4)P）水平的突变会减缓或解除 AP-1 衣被组装与 GGA 衣被组装之间的偶联。相反，增强的 PtdIns(4)P 合成会缩短衔接蛋白波之间的时间。Gga2p 直接与 TGN 的 PtdIns(4)-激酶 Pik1p 结合，并有助于 Pik1p 的募集。这些结果确定了一种基于 PtdIns(4)P 的机制，用于调节 TGN 处衔接蛋白特异性网格蛋白衣被的渐进组装。
