Graduate Program in Functional and Molecular Biology, Institute of Biology, University of Campinas, Campinas, SP, Brazil; Brazilian Biosciences National Laboratory (LNBio), Brazilian Center for Research in Energy and Materials (CNPEM), Zip Code 13083-100 Campinas, Sao Paulo, Brazil.
Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan; Department of Physiological Chemistry, Faculty of Medicine and Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
J Proteomics. 2020 Feb 10;212:103549. doi: 10.1016/j.jprot.2019.103549. Epub 2019 Nov 5.
Vertebrates usually have three class V myosin paralogues (MyoV) to control membrane trafficking in the actin-rich cell cortex, but their functional overlapping or differentiation through cargoes selectivity is yet only partially understood. In this work, we reveal that the globular tail domain of MyoVc binds to the active form of small GTPase Rab3A with nanomolar affinity, a feature shared with MyoVa but not with MyoVb. Using molecular docking analyses guided by chemical cross-linking restraints, we propose a model to explain how Rab3A selectively recognizes MyoVa and MyoVc via a distinct binding site from that used by Rab11A. The MyoVa/c binding interface involves multiple residues from both lobules (I and II) and the short helix at the α2-α3 link region, which is conserved between MyoVa and MyoVc, but not in MyoVb. This motif is also responsible for the selective binding of RILPL2 by MyoVa and potentially MyoVc. Together, these findings support the selective recruitment of MyoVa and MyoVc to exocytic pathways via Rab3A and expand our knowledge about the functional evolution of class V myosins. SIGNIFICANCE: Hormone secretion, neurotransmitter release, and cytoplasm membrane recycling are examples of processes that rely on the interaction of molecular motors and Rab GTPases to regulate the intracellular trafficking and tethering of vesicles. Defects in these proteins may cause neurological impairment, immunodeficiency, and other severe disorders, being fatal in some cases. Despite their crucial roles, little is known about how these molecular motors are selectively recruited by specific members of the large family of Rab GTPases. In this study, we unveil the interaction between the actin-based molecular motor Myosin Vc and the small GTPase Rab3A, a key coordinator of vesicle trafficking and exocytosis in mammalian cells. Moreover, we propose a model for their recognition and demonstrate that Rab3A specifically binds to the globular tail of Myosins Va and Vc, but not of Myosin Vb, advancing our knowledge about the molecular basis for the selective recruitment of class V myosins by Rab GTPases.
脊椎动物通常有三个类 V 肌球蛋白同工酶（MyoV）来控制富含肌动蛋白的细胞皮质中的膜运输，但它们通过货物选择性的功能重叠或分化仅部分理解。在这项工作中，我们揭示了 MyoVc 的球状尾部结构域以纳摩尔亲和力与小 GTPase Rab3A 的活性形式结合，这一特征与 MyoVa 共享，但与 MyoVb 不共享。使用化学交联限制引导的分子对接分析，我们提出了一个模型来解释 Rab3A 如何通过与 Rab11A 不同的结合位点选择性识别 MyoVa 和 MyoVc。MyoVa/c 结合界面涉及来自两个叶（I 和 II）和α2-α3 连接区短螺旋的多个残基，该残基在 MyoVa 和 MyoVc 之间保守，但在 MyoVb 中不保守。该模体还负责 RILPL2 被 MyoVa 并可能被 MyoVc 的选择性结合。总之，这些发现支持 MyoVa 和 MyoVc 通过 Rab3A 选择性募集到胞吐途径，并扩展了我们对类 V 肌球蛋白功能进化的认识。
激素分泌、神经递质释放和细胞质膜回收是依赖于分子马达和 Rab GTPases 相互作用来调节囊泡的细胞内运输和固定的过程的例子。这些蛋白质的缺陷可能导致神经损伤、免疫缺陷和其他严重疾病，在某些情况下甚至是致命的。尽管它们具有重要作用，但对于这些分子马达如何被 Rab GTPase 大家族的特定成员选择性募集知之甚少。在这项研究中，我们揭示了肌球蛋白 Vc 与小 GTPase Rab3A 之间的相互作用，Rab3A 是哺乳动物细胞中囊泡运输和胞吐作用的关键协调因子。此外，我们提出了它们的识别模型，并证明 Rab3A 特异性结合肌球蛋白 Va 和 Vc 的球状尾部，而不是肌球蛋白 Vb 的，这提高了我们对 Rab GTPases 选择性募集类 V 肌球蛋白的分子基础的认识。
