研究HOPS复合体亚基VPS41的生化特性及在植物早期发育中的作用

HOPS复合体是真核生物中保守存在的多亚基栓系复合物。在酵母和动物细胞中，HOPS复合物亚基VPS41被上游小G蛋白招募到晚期内吞体上参与调控液泡融合。然而，VPS41在植物中是否具有保守的功能目前尚未知。我们前期研究表明，在模式植物拟南芥根部细胞中，VPS41定位于液泡膜和一类之前尚未报道过的无膜点状结构中。该无膜结构既能和液泡膜融合又能自我融合-分裂，与经典的相分离现象很相似。遗传学实验表明该无膜结构对植物早期发育至关重要，失去该结构导致植物苗期致死。我们前期研究也发现VPS41能与丝裂原蛋白激酶MPK6及上游激酶MKK4互作。在本项目开展过程中，我们将检测VPS41是否能在体外发生相分离并将深入研究VPS41与MKK-MPK激酶信号途径的关系及在植物早期发育中的作用。本项目研究将拓宽植物细胞生物学领域对内膜蛋白生化特征和生理学功能的理解，为后续农业生产奠定科学理论基础。

The HOPS (homotypic fusion and vacuole protein sorting) complex is a conserved, multi-subunit tethering complex present in eukaryotic cells. In yeast and mammalian cells, the HOPS subunit VPS41 is recruited to the late endosomes by upstream small GTPases and is essential for vacuole fusion. However, whether VPS41 plays the conserved roles in plant is not yet clear. Here, we demonstrate that in model plant Arabidopsis, VPS41 localizes to so-far uncharacterized membrane-less compartments in root cells in addition to its reported localization at tonoplast. The membrane-less puncta where VPS41 resides can fuse with the tonoplast and undergo self-fusion and split, similar to the classic phase separation phenomenon. Genetic experiments showed that the membrane-less puncta are essential for early plant development as the VPS41 truncated protein without forming the membrane-less structures failed to rescue the null mutant and resulted in seedling lethal. Our preliminary results also demonstrated that VPS41 specifically interacted with the mitogen activated protein kinase MPK6 and the upstream kinase MKK4. In this project, we will investigate whether VPS41 could undergo phase separation through in vitro experiments. We will further study the relationship between VPS41 and the MKK-MPK signaling pathway and its roles in plant early development. Our study will help understand the biochemical characteristics and physiological significance of the endomembrane proteins, and provide theoretical guidance for future agricultural applications.