Functional reconstitution of plant plasma membrane H-ATPase into giant unilamellar vesicles.

Membrane transporters are essential for numerous biological processes by controlling the movement of ions and molecules across cell membranes. However, dissecting their molecular dynamics in complex cellular environments presents significant challenges. Reconstitution of membrane transporters in model systems offers a powerful solution. In this study, we focused on the reconstitution conditions suitable for the P3 ATPase Arabidopsis thaliana H-ATPase isoform 2 and compatible with various giant unilamellar vesicle generation techniques. Among the methods evaluated for GUV formation, including electroformation, gel-assisted formation, and charge-mediated fusion, only the gel-assisted approach successfully generated AHA2-containing giant unilamellar vesicles while preserving the pump activity. Our findings underscore the importance of carefully managing the reconstitution conditions, including the presence of ions, and selecting the appropriate lipid composition to enhance the stability and activity of AHA2 in proteoliposomes. Addressing these factors is essential for the successful formation and functional analysis of AHA2 and other P-type ATPases in experimental settings.