Movement Regulation Of VIPP1 Protein And Analysis Of Its GTPase Activity

VESICLE INDUCING PROTEIN IN PLASTID1?VIPP1?is conserved among oxygenic photosynthetic organisms and appears to have diverged from the bacterial PspA protein.VIPP1 localizes to the chloroplast envelope and thylakoid membrane,where it forms homooligomers of high molecular mass.Although multiple roles of VIPP1 have been inferred,including thylakoid membrane formation,envelope maintenance,membrane fusion,and regulation of photosynthetic activity,its precise role in chloroplast membrane quality control remains unknown.VIPP1 forms an oligomer through its amino-terminal domain and triggers membrane fusion in an Mg²⁺-dependent manner.We previously demonstrated that Arabidopsis?Arabidopsis thaliana?VIPP1 also exhibits dynamic complex disassembly in response to osmotic and heat stresses in vivo.The study of VIPP1dynamics would be helpful to deeply clarify the molecular mechanism of repairing chloroplast envelop.The intact chloroplasts were isolated from transgenic line VIPP1-GFP/Col of Arabidopsis.The detection of movement of VIPP1-GFP protein complex was carried out under fluorescence microscope with or without GTP and/or Ca²⁺.The results indicated that chloroplasts began to swollen up and the gap between envelop and thylakoid enlarged under hypotonic stress conditions.At the same time,VPP1-GFP protein complex started moving freely inside the chloroplast.CaCl₂ could completely inhibit the movement of VPP1-GFP protein,which was caused by Ca²⁺rather than Cl^-.GTP can effectively reverse the inhibitory effect of Ca²⁺on VPP1-GFP movement and activate protein dynamics again.The integrity of chloroplast envelop is dependent on the dynamics of VIPP1 under hypotonic stress condition.Both GTP and Ca²⁺could regulate the dynamics of VIPP1 in Arabidopsis chloroplast.But,they function in different ways:GTP promotes the movement of VIPP1 protein,while Ca²⁺inhibits the process.These results suggest that VIPP1 mediates membrane fusion/remodeling in chloroplasts.Considering that protein machines that regulate intracellular membrane fusion/remodeling events often require a capacity for GTP binding and/or hydrolysis,we questioned whether VIPP1 has similar properties.We conducted an in vitro assay using a purified VIPP1-His fusion protein expressed in Escherichia coli cells.VIPP1-His showed GTP hydrolysis activity that depends on GTP binding.Although VIPP1 does not contain a canonical G domain,the amino-terminal?-helix was found to be important for both GTP binding and GTP hydrolysis as well as for oligomer formation.