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

The water channel protein alpha-TIP is a member of the major intrinsic prot ein (MIP) membrane channel family. This aquaporin is found abundantly in va cuolar membranes of cotyledons (seed storage organs) and is synthesized dur ing seed maturation. The water channel activity of alpha-TIP can be regulat ed by phosphorylation, and the protein may function in seed desiccation, cy toplasmic osmoregulation, and/or seed rehydration. alpha-TIP was purified f rom seed meal of the common bean (Phaseolus vulgaris) by membrane fractiona tion, solubilization in diheptanoylphosphocholine and anion-exchange chroma tography. Upon detergent removal and reconstitution into lipid bilayers, al pha-TIP crystallized as helical tubes. Electron cryo-crystallography of fla ttened tubes demonstrated that the crystals exhibit plane group p2 symmetry and c222 pseudosymmetry. Since the 2D crystals with p2 symmetry are derive d from helical tubes, we infer that the unit of crystallization on the heli cal lattice is a dimer of tetramers. A projection density map at a resoluti on of 7.7 Angstrom revealed that alpha-TIP assembles as a 60 Angstrom x 60 Angstrom square tetramer. Each subunit is formed by a heart-shaped ring com prised of density peaks which we interpret as alpha-helices. The similarity of this structure to manmalian plasma membrane MIP-family proteins suggest s that the molecular design of functionally analogous and genetically homol ogous aquaporins is maintained between the plant and animal kingdoms. (C) 1 999 Academic Press.