Reconstitution of Active Plant H⁺-ATPase AHA2 in Giant Unilamellar Vesicles

Membrane transporters mediate the selective movement of ions and molecules across biological membranes and are essential for cellular homeostasis. However, their functional characterization in living cells is often complicated by the complexity of the native membrane environment. Reconstitution into model membrane systems provides a powerful alternative by enabling precise control over lipid composition and experimental conditions. Giant unilamellar vesicles (GUVs) are particularly well suited for transporter studies, as their cell-sized dimensions allow direct microscopic observation and fluorescence-based measurements of protein activity. Here, we describe a two-step reconstitution protocol in which transport proteins are first incorporated into large unilamellar vesicles and then used to generate protein-containing giant unilamellar vesicles (proteo-GUVs) via the poly(vinyl alcohol) swelling method. This two-step approach enhances protein incorporation efficiency and preserves transporter functionality. The method is exemplified using the P3-type ATPase Arabidopsis thaliana plasma membrane H⁺-ATPase isoform 2 (AHA2). We further describe a fluorescence-based assay to assess proton transport activity in proteo-GUVs. Our approach provides a versatile and controlled platform for biochemical, biophysical, and single-molecule analysis of membrane transporters. Key features • Describes a SNAP-based labeling method. • Provides a reconstitution approach based on detergent removal using size-exclusion chromatography and bio-beads. • Introduces a gentle dehydration-rehydration strategy using gel-assisted swelling for the generation of proteo-GUVs. • Measures proton pump activity in GUVs under a confocal microscope using the pH-sensitive dye pyranine.