title: The 5A structure of heterologously expressed plant aquaporin SoPIP2;1.
abstract: SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.
authors.affiliation: Maurice E. Müller Institute for Microscopy, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: W
authors.initials: W
authors.lastname: Kukulski
authors.forename: A D
authors.initials: AD
authors.lastname: Schenk
authors.forename: U
authors.initials: U
authors.lastname: Johanson
authors.forename: T
authors.initials: T
authors.lastname: Braun
authors.forename: B L
authors.initials: BL
authors.lastname: de Groot
authors.forename: D
authors.initials: D
authors.lastname: Fotiadis
authors.forename: P
authors.initials: P
authors.lastname: Kjellbom
authors.forename: A
authors.initials: A
authors.lastname: Engel
mesh_terms: D000818:Animals; D020346:Aquaporins / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D010843:Pichia; D010940:Plant Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D018724:Spinacia oleracea; D014981:Xenopus
chemical_list: D020346:Aquaporins; D010940:Plant Proteins