A novel Rab9 effector required for endosome-to-TGN transport.
A novel Rab9 effector required for endosome-to-TGN transport.
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DOI：
10.1083/jcb.138.2.283
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发表时间：
1997-07-28
期刊：
影响因子：
--
通讯作者：
Pfeffer SR
中科院分区：
文献类型：
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作者：
Díaz E;Schimmöller F;Pfeffer SR
Rab 9 GTdR是活细胞中甘露糖6-磷酸受体从内体转运到高尔基体网络所必需的，并且在体外系统中重建该过程。我们已经使用酵母双杂交系统来识别优先与Rab 9的活性形式相互作用的蛋白质。我们在这里报告发现的40 kD的蛋白质（p40），结合Rab 9-GTP的Rab 9-GDP的大约四倍的偏好。p40不与Rab 7或K-Ras相互作用;当它与GDI结合时，它也不能结合Rab 9。该蛋白质存在于胞质溶胶中，但有相当一部分（约30%）与细胞膜相关。在蔗糖密度梯度浮选时，膜结合的p40与含有甘露糖6-磷酸受体和Rab 9 GT3的内体共分级分离。p40是一种非常有效的转运因子，因为纯的重组蛋白可以显著地刺激体外转运测定，该测定测量甘露糖6-磷酸受体从内体转运到trans-Golgi网络。p40的功能重要性被抗p40抗体抑制体外转运的发现所证实。最后，p40在其刺激甘露糖6-磷酸受体转运的能力方面显示出与Rab 9的协同作用。这些数据与p40和Rab 9共同作用以驱动运输囊泡对接过程的模型一致。
Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9–GTP with roughly fourfold preference to Rab9–GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (∼30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.