Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Nat Chem Biol. 2010 Jul;6(7):534-40. doi: 10.1038/nchembio.386. Epub 2010 May 30.
Post-translationally isoprenylated proteins represent major hubs in most membrane-connected signaling networks. GDP dissociation inhibitors (GDIs) are molecular chaperones that shuttle geranylgeranylated GTPases between membranes and the cytosol. Despite numerous studies, the mechanism of targeted membrane delivery of GTPases remains unknown. Here we have combined chemical synthesis and expressed protein ligation to generate fluorescent lipidated RabGTPase-based sensor molecules. Using these protein probes, we have demonstrated that RabGDI and the related Rab escort protein REP show a three-order-of-magnitude greater affinity for GDP-bound Rab GTPase than for the GTP-bound state. Combined with a relatively high dissociation rate of the Rab-GDI complex, this would enable guanine nucleotide exchange factors (GEFs) to efficiently dissociate the complex and promote membrane attachment of the GTPase. The findings suggest strongly that GEFs are necessary and sufficient for membrane targeting of GTPases and that the previously proposed GDI displacement factors (GDFs) are not thermodynamically required for this process.
翻译后的异戊烯化蛋白质是大多数膜连接信号网络中的主要枢纽。GDP 解离抑制剂（GDIs）是分子伴侣，可将 geranylgeranylated GTPases 在膜和细胞质之间穿梭。尽管进行了许多研究，但 GTPases 靶向膜递呈的机制仍不清楚。在这里，我们将化学合成和表达蛋白连接结合起来，生成基于荧光脂化 RabGTPase 的传感器分子。使用这些蛋白探针，我们已经证明 RabGDI 和相关的 Rab 护送蛋白 REP 对 GDP 结合的 Rab GTPase 的亲和力比对 GTP 结合状态高三个数量级。再加上 Rab-GDI 复合物的相对较高的解离速率，这将使鸟嘌呤核苷酸交换因子（GEFs）能够有效地解离复合物并促进 GTPase 的膜附着。这些发现强烈表明，GEFs 对于 GTPase 的膜靶向是必需且充分的，并且之前提出的 GDI 置换因子（GDFs）对于该过程在热力学上不是必需的。
