Brown D L, Heimann K, Lock J, Kjer-Nielsen L, van Vliet C, Stow J L, Gleeson P A
Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia.
Traffic. 2001 May;2(5):336-44. doi: 10.1034/j.1600-0854.2001.002005336.x.
Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230GRIP fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230GRIP binds to a specific population of vesicles distinct from those labelled for beta-COP or gamma-adaptin. The GFP-p230GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulo-vesicular carriers.
用于细胞内运输的囊泡载体与一组独特的辅助分子相关联，这些分子决定了在特定膜结构域上的出芽和对接。尽管这些辅助分子中的许多是外周膜蛋白，但在大多数情况下，负责其膜募集的靶向序列尚未确定。我们之前定义了一种新型的高尔基体靶向结构域（GRIP），它由一族参与膜运输的卷曲螺旋外周膜高尔基体蛋白共享。我们在此表明，p230的GRIP基序的对接位点是高尔基体膜的一个特定结构域。通过对稳定表达绿色荧光蛋白（GFP）-p230GRIP融合蛋白的HeLa细胞进行免疫电子显微镜观察，我们发现其特异性结合到反式高尔基体网络（TGN）的一部分膜上。对活HeLa细胞的实时成像显示，GFP-p230GRIP与TGN的高度动态的管状延伸相关联，这些延伸具有运输载体的外观和行为。为了进一步确定GRIP膜结合位点的性质，使用纯化的大鼠肝脏高尔基体膜和来自GFP-p230GRIP转染细胞的胞质溶胶进行了体外出芽试验。通过蔗糖梯度分级分离对高尔基体衍生的囊泡进行分析表明，GFP-p230GRIP与不同于那些标记有β-COP或γ-衔接蛋白的特定囊泡群体结合。GFP-p230GRIP融合蛋白与全长p230被募集到相同的囊泡群体中，这表明GRIP结构域单独就足以作为天然分子膜结合的靶向信号。因此，p230 GRIP是募集到反式高尔基体网络管状囊泡载体特定群体上高度选择性膜附着位点的靶向信号。
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