Bai Hongdong, Doray Balraj, Kornfeld Stuart
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2004 Apr 23;279(17):17411-7. doi: 10.1074/jbc.M401158200. Epub 2004 Feb 18.
The Golgi-associated gamma-adaptin-related ADP-ribosylation factor-binding proteins (GGAs) are critical components of the transport machinery that mediates the trafficking of the mannose 6-phosphate receptors and associated cargo from the trans-Golgi network to the endosomes. The GGAs colocalize in vivo with the clathrin adaptor protein AP-1 and bind to AP-1 in vitro, suggesting that the two proteins may cooperate in packaging the mannose 6-phosphate receptors into clathrin-coated vesicles at the trans-Golgi network. Here, we demonstrate that the sequence, (382)WNSF(385), in the hinge region of GGA1 mediates its interaction with the AP-1 gamma-ear. The Trp and Phe constitute critical amino acids in this interaction. The binding of Rabaptin5 to the AP-1 gamma-ear, which occurs through a FXXPhi motif, is inhibited by a peptide encoding the GGA1 (382)WNSF(385) sequence. Moreover, mutations in the AP-1 gamma-ear that abolish its interaction with Rabaptin5 also preclude its association with GGA1. These results suggest that the GGA1 WXXF-type and Rabaptin5 FXXPhi-type motifs bind to the same or highly overlapping sites in the AP-1 gamma-ear. This binding is modulated by residues adjacent to the core motifs.
高尔基体相关的γ-衔接蛋白相关ADP核糖基化因子结合蛋白（GGAs）是转运机制的关键组成部分，介导甘露糖6-磷酸受体及相关货物从反式高尔基体网络向内体的运输。GGAs在体内与网格蛋白衔接蛋白AP-1共定位，且在体外与AP-1结合，这表明这两种蛋白可能在反式高尔基体网络中将甘露糖6-磷酸受体包装到网格蛋白包被小泡的过程中发挥协同作用。在此，我们证明GGA1铰链区的序列（382）WNSF（385）介导其与AP-1γ耳的相互作用。色氨酸和苯丙氨酸在这种相互作用中构成关键氨基酸。Rabaptin5通过一个FXXPhi基序与AP-1γ耳结合，而编码GGA1（382）WNSF（385）序列的肽段可抑制这种结合。此外，AP-1γ耳中消除其与Rabaptin5相互作用的突变也会阻止其与GGA1的结合。这些结果表明，GGA1的WXXF型基序和Rabaptin5的FXXPhi型基序与AP-1γ耳中的相同或高度重叠位点结合。这种结合受核心基序附近残基的调节。
