Function Of Na~+/H~+ Antiporters In Regulating Vesicle Trafficking In Arabidopsis

Vesicle trafficking plays an important role in protein transport and signal transduction in plants. It is a complicated system that contains many organelles or membrane structures, including endoplasmic reticulum, Golgi, TGN, MVB, and vacuoles. It has been found that AtNHX5 and AtNHX6, Na+/H+ antiporters in Arabidopsis, may involve in vesicle trafficking. However, the mechanism underlying the function of AtNHX5 and AtNHX6 in regulating vesicle trafficking remains unclear.Studies have shown that the ESCRT complex and V-ATPases are key factors in vesicle trafficking. In this study, we cloned the genes of ESCRT subunits and V-ATPase subunits, and tagged them with GFP or RFP. We then transformed these genes into Col-0 or nhx5 nhx6 double mutants to generate the transgenic seedlings. By comparing the distribution of ESCRT subunits and V-ATPase subunits in Col-0 and nhx5 nhx6, we analyzed the molecular mechanism behind the regulation of vesicle trafficking by AtNHX5 and AtNHX6. The major results are listed bellows:1. We cloned the genes of the ESCRT subunits AtVPS2, AtVPS22, AtVPS28. We then constructed the expression vectors pUBC-VPS2-DN-GFP, pUBC-VPS2-GFP, pUBC-VPS22-GFP, pUBC-VPS28-GFP by the Gateway technology.2. We generated the transgenic lines containg the genes of ESCRT subunits (VPS28, VP22, VPS2) or V-ATPase subunits (VHA-al, VHA-a2, VHA-a3) using the floral dip or crossing methods.3. The subcellular distribution of ESCRT subunits and V-ATPase subunits was visualized by a confocal microscope. We found that the distribution of AtVPS2 and AtVPS28 in nhx5 nhx6 was changed compared to that in Col-0, indicating that AtNHX5 and AtNHX6 may regulate the distribution and trafficking of ESCRT subunits. However, the distribution of AtVHA-al was unchanged in nhx5 nhx6, indicating that AtNHX5 and AtNHX6 do not affect the distribution and trafficking of AtVHA-al.In a word, AtNHX5 and AtNHX6 regulate the distribution and trafficking of ESCRT subunits. AtNHX5 and AtNHX6 may control protein transport toward the vacuole through regulating the distribution and trafficking of ESCRT subunits.