Inoshita Motoki, Mima Joji
From the Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
From the Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
J Biol Chem. 2017 Nov 10;292(45):18500-18517. doi: 10.1074/jbc.M117.811356. Epub 2017 Sep 22.
Membrane tethering is a fundamental process essential for the compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, are reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by -Rab assemblies on opposing membranes. Our findings support the novel concept that Rab-family proteins provide a membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane-tethering reactions.
膜系留是真核细胞内细胞膜运输的区室特异性所必需的基本过程。据报道，Rab家族小GTP酶和特定的Rab相互作用效应蛋白组，包括卷曲螺旋系留蛋白和多亚基系留复合物，负责膜系留。然而，这些关键成分是否以及如何直接且特异性地系留亚细胞膜仍然是个谜。我们利用用纯化的人Rab蛋白和合成脂质体膜重构的化学定义的蛋白脂质体系统来研究膜系留的分子基础，在此确定Rab家族GTP酶具有高度保守的功能，即使在没有任何类型的Rab效应器（如所谓的系留蛋白）的情况下，也能直接介导膜系留。此外，我们证明内体Rab11a介导的膜系留受到其同源Rab效应器V类肌球蛋白（Myo5A和Myo5B）的强烈且选择性的刺激，且呈GTP依赖性。值得注意的是，Myo5A和Myo5B仅识别并与其同源Rab11a的膜锚定形式协同作用，以支持由相反膜上的Rab组装介导的膜系留。我们的研究结果支持了一个新的概念，即Rab家族蛋白提供了一个膜系留，以物理方式并特异性地连接亚细胞膜的两个不同脂质双层。它们进一步表明，包括V类肌球蛋白在内的Rab相互作用效应蛋白可以调节这些Rab介导的膜系留反应。
