The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers
The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers
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DOI：
10.1034/j.1600-0854.2001.002005336.x
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发表时间：
2001-05-01
期刊：
影响因子：
4.5
通讯作者：
Gleeson, PA
中科院分区：
文献类型：
--
作者：
Brown, DL;Heimann, K;Gleeson, PA
用于细胞内运输的囊泡载体与决定特定膜结构域上的出芽和对接的独特的辅助分子组相关联。虽然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被鉴定。我们以前已经定义了一个新的高尔基体靶向结构域（GRIP）共享的一个家庭的卷曲螺旋外周膜高尔基体蛋白参与膜贩运。我们在这里表明，GRIP基序的p230的对接站点是一个特定的域的高尔基体。膜。通过免疫电子显微镜的HeLa细胞稳定表达的绿色荧光蛋白（GFP）-p230（GRIP）融合蛋白，我们显示结合特异性的一个子集的膜的trans-Golgi网络（TGN）。活HeLa细胞的实时成像显示，GFP-p230（GRIP）与TGN的高度动态管状延伸相关，其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，使用纯化的大鼠肝高尔基体膜和来自GFP-p230（GRIP）转染细胞的胞质溶胶进行体外出芽测定。通过蔗糖梯度分级分离对高尔基体衍生的囊泡的分析表明，GFP-p230（GRIP）结合到与标记为β-COP或γ-适应素的囊泡不同的特定囊泡群体。GFP-p230（GRIP）融合蛋白被募集到与全长p230相同的囊泡群体，表明GRIP结构域仅精通作为天然分子的膜结合的靶向信号。因此，p230 GRIP是一种靶向信号，用于招募到特定群体的trans-Golgi网络管泡载体上的高选择性膜附着位点。
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 immunoelectron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230(GRIP) 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-p230(GRIP) 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-p230(GRIP) transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230(GRIP) binds to a specific population of vesicles distinct from those labelled for beta -COP or gamma -adaptin. The GFP-p230(GRIP) 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 tubulovesicular carriers.