Single-molecule Analysis Of AtHIR3 And Its Interaction With AtVHA-A

Hypersensitive Induced Reaction proteins(AtHIRs)are plant-specific proteins with SPFH(stomatin/prohibitin/flotillin/Hflk/C)domain,which have closly association with membrane microdomain formation and play an important role in plant immune responses.In this study,we investigated AtHIR3 using laser scanning confocal microscopy(LSCM)and variable-angle total internal reflection fluorescence microscopy(VA-TIRFM).The result showed that AtHIR3-EGFP was expressed in all tissues of Arabidopsis seedlings and unevenly distributed on the tonoplast.AtHIR3-EGFP presented four types of diffusion,including Brownian motion,restricted motion,directional motion and mixed motion.Using mass spectroscopy,this study revealed that AtHIR3 and AtVHA-A subunit of V-ATPase(vacuolar H+-ATPase)are located in the same protein complexFurther studies by Bimolecular Fluorescence Complementation(BiFC)and yeast two hybrid experiments confirmed the interaction between AtHIR3 and AtVHA-A.Moreover,confocal microscopy revealed that AtHER3 and AtVHA-A were colocalizated on the tonoplast in Arabidopsis.In Athir3 mutant,the diffusion coefficient,motion range and recovery rate after fluorescence bleaching of AtVHA-A-EGFP were higher than those of wild type.After NaCl treatment for 1 h,the vacuole pH of root epidermal cells in Athir3 increased more than that in WT.In addition,NaCl affected the diffusion of AtHIR3-EGFP on the tonoplast,slowed down its diffusion rate and reduced its motion range.In summary,AtHIR3 was mainly located on the vacuolar membrane microdomains of Arabidopsis thaliana and interacted with VHA-A subunit of V-ATPase,which limited the movement of VHA-A on the tonoplast.It is presumed that AtHIR3 promotes the H+transport of V-ATPase into vacuoles by recruiting VHA-A to vacuolar membrane microdomains.The above results provide new insights into the regulation mechanism of H+transport of V-ATPase.