Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Institute of Plant and Food Science, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.
School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
Mol Plant. 2022 Mar 7;15(3):398-418. doi: 10.1016/j.molp.2021.11.008. Epub 2021 Nov 16.
Delivery of proteins to the plasma membrane occurs via secretion, which requires tethering, docking, priming, and fusion of vesicles. In yeast and mammalian cells, an evolutionarily conserved RAB GTPase activation cascade functions together with the exocyst and SNARE proteins to coordinate vesicle transport with fusion at the plasma membrane. However, it is unclear whether this is the case in plants. In this study, we show that the small GTPase RABA2a recruits and interacts with the VAMP721/722-SYP121-SNAP33 SNARE ternary complex for membrane fusion. Through immunoprecipitation coupled with mass spectrometry analysis followed by the validatation with a series of biochemical assays, we identified the SNARE proteins VAMP721 and SYP121 as the interactors and downstream effectors of RABA2a. Further expreiments showed that RABA2a interacts with all members of the SNARE complex in its GTP-bound form and modulates the assembly of the VAMP721/722-SYP121-SNAP33 SNARE ternary complex. Intriguingly, we did not observe the interaction of the exocyst subunits with either RABA2a or theSNARE proteins in several different experiments. Neither RABA2a inactivation affects the subcellular localization or assembly of the exocystnor the exocyst subunit mutant exo84b shows the disrupted RABA2a-SNARE association or SNARE assembly, suggesting that the RABA2a-SNARE- and exocyst-mediated secretory pathways are largely independent. Consistently, our live imaging experiments reveal that the two sets of proteins follow non-overlapping trafficking routes, and genetic and cell biologyanalyses indicate that the two pathways select different cargos. Finally, we demonstrate that the plant-specific RABA2a-SNARE pathway is essential for the maintenance of potassium homeostasis in Arabisopsis seedlings. Collectively, our findings imply that higher plants might have generated different endomembrane sorting pathways during evolution and may enable the highly conserved endomembrane proteins to participate in plant-specific trafficking mechanisms for adaptation to the changing environment.
蛋白质向质膜的输送是通过分泌来完成的，这需要囊泡的锚定、对接、引发和融合。在酵母和哺乳动物细胞中，一个进化上保守的 RAB GTP 酶激活级联与外泌体和 SNARE 蛋白一起作用，协调囊泡运输与质膜融合。然而，在植物中是否存在这种情况尚不清楚。在这项研究中，我们表明小 GTP 酶 RABA2a 招募并与 VAMP721/722-SYP121-SNAP33 SNARE 三元复合物相互作用，以进行膜融合。通过免疫沉淀结合质谱分析，并通过一系列生化实验进行验证，我们确定 SNARE 蛋白 VAMP721 和 SYP121 是 RABA2a 的相互作用蛋白和下游效应物。进一步的实验表明，RABA2a 以其 GTP 结合形式与 SNARE 复合物的所有成员相互作用，并调节 VAMP721/722-SYP121-SNAP33 SNARE 三元复合物的组装。有趣的是，在几个不同的实验中，我们没有观察到外泌体亚基与 RABA2a 或 SNARE 蛋白的相互作用。RABA2a 的失活既不影响外泌体的亚细胞定位或组装，也不影响外泌体亚基突变体 exo84b 中 RABA2a-SNARE 关联或 SNARE 组装的破坏，这表明 RABA2a-SNARE-和外泌体介导的分泌途径在很大程度上是独立的。一致地，我们的活细胞成像实验表明，这两组蛋白遵循非重叠的运输途径，遗传和细胞生物学分析表明，这两条途径选择不同的货物。最后，我们证明了植物特异性的 RABA2a-SNARE 途径对于拟南芥幼苗中钾离子稳态的维持是必不可少的。总之，我们的研究结果表明，高等植物在进化过程中可能产生了不同的内膜分选途径，并可能使高度保守的内膜蛋白参与植物特异性运输机制，以适应不断变化的环境。
