拟南芥AtNHX5和AtNHX6调节液胞融合的功能及机理

Plant Na+,K+/H+ antiporters (NHX) are important ion transporters in plant cells and play significant roles in many aspects of plant growth and development, including salt tolerance, potassium homeostasis, cellular pH regulation and flower development. Membrane fusion is the last step of the trafficking pathways during which the transport vesicles merge with the target membrane. Membrane fusion is a central cellular process which is crucial for protein sorting, signal transduction, subcellular compartmentation, neurotansmission, cellular defence, hormone secretion, and biotic and abiotic stress. We have recently reported for the first time that ScNhx1p plays an important role in yeast vacuole fusion (Qiu and Fratti, 2010, J Cell Sci, 123:3266-3275). However, it is still not clear whether or not the plant NHX antiporters also function in membrane fusion in plants. Bioinformatic analysis has shown that Arabidopsis AtNHX5/AtNHX6 genes and yeast ScNhx1p gene share a high sequence similarity (43.3% and 42.4%, respectively), and both the AtNHX5/AtNHX6 genes and the ScNhx1p gene belong to the Endo/TGN subgroup of the Class II NHX antiporter gene family (Fig.4, Brett et al., 2005；Fig 1, Rodriguez-Rosaleset al., 2009). These bioinformatic studies strongly suggest that the plant NHX antiporters might have the same function as their yeast counterpart and play an important role in membrane fusion in plants as well. Our preliminary study found that AtNHX5 and AtNHX6 recovered the vaculoe fragmentation phenotype of the yeast nhx1p mutant cells, suggesting that AtNHX5 and AtNHX6 might function in vacuole fusion in plants. In this study, by using Arabidopsis and yeast as model systems, we plan to identify the function and underlying mechanism of AtNHX5 and AtNHX6 in vacuole fusion in plants using molecular genetic and cell biological methods and techniques. Our study will pave the way for the further understanding of the functions of plant NHX antiporters and the molecular mechanisms controlling membrane fusion in plants.

Na+,K+/H+反向交换体(NHX)是植物细胞的重要跨膜转运蛋白，在耐盐胁迫，K+平衡和pH调节以及植物生长发育中起着重要作用。生物膜融合是细胞的重要生化过程，在蛋白质分拣、信号传导、组织形态发生、植物抗病抗逆等方面起着重要作用。我们近期报道酵母ScNhx1调节液胞融合。然而植物NHX在生物膜融合中的作用还未见报道。拟南芥AtNHX5和AtNHX6与酵母ScNHX1基因序列高度相似, 同属Endo/TGN 亚类，暗示AtNHX5和AtNHX6基因可能也具有调节生物膜融合的功能。本项目将以酵母和拟南芥为模式生物，采用分子遗传学和细胞生物学技术方法及酵母异源表达体系，揭示AtNHX5和AtNHX6 调节液胞融合的生物功能及其分子机制。本项目的研究将为进一步揭示植物NHX的生物功能，阐明植物液胞融合的分子机理以及认识液胞生物发生和膜微囊运输的分子机制奠定重要的理论基础。

植物Na+,K+/H+反向交换体(NHX)在调节细胞K+和pH平衡、耐盐胁迫以及植物生长发育等过程中起着重要的作用。生物膜融合是细胞膜微囊运输的最后一个步骤，在蛋白质分拣、信号传导、组织形态发生和植物抗病抗逆等方面起着重要作用。我们近期报道酵母ScNhx1调节液胞融合。然而植物NHX在生物膜融合中的作用还未见报道。本项目以酵母和拟南芥为模式生物，采用分子遗传学、细胞生物学技术方法及酵母异源表达体系，研究了AtNHX5和AtNHX6 对液胞融合的调节作用与机理及其调控蛋白质运输和气孔运动的作用机理。我们首先研究了AtNHX5和AtNHX6在调节K+和H+稳态平衡方面的功能。我们利用酵母表达系统和拟南芥突变体，发现AtNHX5和AtNHX6在调节拟南芥K+和pH平衡中起着重要的作用。三个保守的酸性氨基酸残基对K+运输必不可少。我们以大肠杆菌EcNhaA的晶体结构为模板，采用同源建模方法，建立胡杨PeNHX3的三维结构。我们采用结构域转换(Domain-switch)技术，通过酵母异源表达和拟南芥转基因分析，验证了PeNHX3的TM11对于Na+和 Li+转运活力的必要性。通过分析AtNHX5和AtNHX6对SNARE复合物的调节作用，我们研究了AtNHX5和AtNHX6对液胞融合的调节功能。综合运用遗传、细胞和生化技术和方法，我们发现AtNHX5和AtNHX6可能通过调节SNARE复合物的亚细胞定位而调控着细胞蛋白质的运输过程。我们还发现，AtNHX5和AtNHX6可以通过其离子转运活性产生的pH调节VPS28A和VPS28B的亚细胞定位，从而影响MVB的形成，控制植物的生长发育及蛋白运输过程。我们的研究还表明，霸王ZxNHX可能通过调节气孔保卫细胞液胞的融合过程调控着叶片气孔的运动。我们的研究结果揭示了NHX调节离子平衡的功能及其结构功能关系；同时阐明了NHX调控液胞融合的功能及其调控蛋白质运输和气孔运动的作用机理。我们的研究结果为今后研究植物NHX的功能和阐明液胞融合的分子机制奠定了理论基础。