McKay Melissa M, Kahn Richard A
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd, Atlanta, GA 30322-3050, USA.
Traffic. 2004 Feb;5(2):102-16. doi: 10.1111/j.1600-0854.2004.00160.x.
GGAs comprise a family of Arf-dependent coat proteins or adaptors that regulate vesicle traffic from the trans-Golgi network (TGN). GGAs bind activated Arf, cargo, and additional components necessary for vesicle budding through interactions with their four functional domains: VHS, GAT, hinge, and GAE. We identified three sites of phosphorylation in GGA1 by tandem mass spectrometry: S268 and T270 in the GAT domain and S480 in the hinge. Expression of HA-GGA1 in mammalian cells and comparison to endogenous GGA1 confirmed their localization to late Golgi compartments. In contrast, mutations that mimic the phosphoprotein (HA-GGA1[S268D] or HA-GGA1[T270D]) at either of the sites in the GAT domain caused a decrease in the colocalization with markers of the Golgi and TGN and an increase in puncta in cytoplasm. Quantitative comparisons of the extent of colocalization of GGA1 proteins with the known components of GGA1 vesicles revealed that the composition of those markers tested in HA-GGA1[S268D] and HA-GGA1[T270D] vesicles were indistinguishable from those of HA-GGA1 vesicles. We conclude that phosphorylation of the GAT domain can stabilize the coat proteins bound and thus regulate the rate of coat protein dissociation.
GGA蛋白包含一个依赖于Arf的衣被蛋白家族或衔接蛋白，它们调节从反式高尔基体网络（TGN）的囊泡运输。GGA蛋白通过与它们的四个功能结构域：VHS、GAT、铰链区和GAE相互作用，结合活化的Arf、货物以及囊泡出芽所需的其他成分。我们通过串联质谱法在GGA1中鉴定出三个磷酸化位点：GAT结构域中的S268和T270以及铰链区中的S480。在哺乳动物细胞中表达HA-GGA1并与内源性GGA1进行比较，证实它们定位于晚期高尔基体区室。相反，在GAT结构域的任何一个位点模拟磷蛋白的突变（HA-GGA1[S268D]或HA-GGA1[T270D]）导致与高尔基体和TGN标记物的共定位减少，细胞质中的斑点增加。对GGA1蛋白与GGA1囊泡已知成分的共定位程度进行定量比较，结果显示在HA-GGA1[S268D]和HA-GGA1[T270D]囊泡中测试的那些标记物的组成与HA-GGA1囊泡的组成没有区别。我们得出结论，GAT结构域的磷酸化可以稳定结合的衣被蛋白，从而调节衣被蛋白解离的速率。
