Projection structure of a plant vacuole membrane aquaporin by electron cryo-crystallography

Abstract The water channel protein α-TIP is a member of the major intrinsic protein (MIP) membrane channel family. This aquaporin is found abundantly in vacuolar membranes of cotyledons (seed storage organs) and is synthesized during seed maturation. The water channel activity of α-TIP can be regulated by phosphorylation, and the protein may function in seed desiccation, cytoplasmic osmoregulation, and/or seed rehydration. α-TIP was purified from seed meal of the common bean ( Phaseolus vulgaris ) by membrane fractionation, solubilization in diheptanoylphosphocholine and anion-exchange chromatography. Upon detergent removal and reconstitution into lipid bilayers, α-TIP crystallized as helical tubes. Electron cryo-crystallography of flattened tubes demonstrated that the crystals exhibit plane group p 2 symmetry and c 222 pseudosymmetry. Since the 2D crystals with p 2 symmetry are derived from helical tubes, we infer that the unit of crystallization on the helical lattice is a dimer of tetramers. A projection density map at a resolution of 7.7 A revealed that α-TIP assembles as a 60 A × 60 A square tetramer. Each subunit is formed by a heart-shaped ring comprised of density peaks which we interpret as α-helices. The similarity of this structure to mammalian plasma membrane MIP-family proteins suggests that the molecular design of functionally analogous and genetically homologous aquaporins is maintained between the plant and animal kingdoms.