Structural Characterization of Two Aquaporins Isolated from Native Spinach Leaf Plasma Membranes

Abstract

Two members of the aquaporin family, PM28A and a new one, PM28C, were isolated and shown to be the major constituents of spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a smaller one, were found by transmission electron microscopy of negatively stained solubilized proteins and by atomic force microscopy of PM28 two-dimensional crystals. The ratio of larger to smaller particles observed by transmission electron microscopy and single particle analysis correlated with the ratio of PM28A to PM28C determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are differentially expressed in spinach leaves. These findings suggest that differential expression of the various aquaporin isoforms may regulate the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.Water is the universal solvent and most important molecule for life. Immense water volumes pass across the membranes of all living cells, especially across the plasma membranes of plants (1). Simple diffusion of water through the lipid bilayer has an activation energy of Ͼ10 kcal/mol and cannot explain the rapid water flow through human red cell membranes found to exhibit an activation energy of Ͻ5 kcal/mol. This led to the hypothesis that water pores must exist (2). The discovery (3) and cloning (4) of the first known water channel, aquaporin-1 (AQP1), 1 from human erythrocytes and the demonstration of water transport in Xenopus oocytes expressing its complementary RNA (5) confirmed this hypothesis.Since then, a large number of channel-forming integral proteins homologous to AQP1 have been found in all forms of life (6). This membrane protein family was initially named the MIP family after its first sequenced member, the major intrinsic protein (MIP) of bovine lens fiber cells (7). Multiple sequence alignment and phylogenetic analysis of 164 members of the MIP family, now frequently referred to as aquaporin super family, revealed 16 subfamilies that form two distinct clusters, the aquaporin (AQP) cluster and the glycerol facilitator-like cluster (8). The AQPs are highly specific for water, whereas the glycerol facilitators allow the passage of small, nonionic molecules such as glycerol and urea (9). In addition, ovine MIP tetramers have been found to form a groove and tongue contact via their extracellular surfaces, lending support to a dual function of the protein, as a water channel and as a cell to cell adhesion molecule in the eye lens (10).Most members of the aquaporin super family...

Supported Biomimetic Membranes for Pressure-Driven Water Purification

“…The bilayer integrity is markedly improved and holes disappear for the mixed DMPC/DMTAP bilayer ( Figure 9B), apparently due to the "electrostatic stitching" effect. The lateral dimensions of the smallest protruding features magnified in the inset C closely match the ~8 nm diameter of the aquaporin tetramer (Fotiadis et al 2001). The height of protrusions, about 2 nm above the surface of the DMPC bilayer, is also in good agreement with the known height of aquaporins.…”

Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation

“…However, the characteristics of plant aquaporins at the single-molecular level have remained unclear. Although the structure of aquaporins has been clarified using x-ray crystallography and cryoelectron microscopy (Jap and Li, 1995;Fotiadis et al, 2001;Tö rnroth-Horsefield et al, 2006), no studies had reported on the oligomeric state of PIPs in the plasma membrane of living plant cells. Here, we imaged GFPlabeled PIP2;1 in the plasma membrane of root cells to determine its oligomeric state.…”

Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family

“…The structures of aquaporins have been intensively studied, revealing the functions of their conserved motifs and post-translational modification sites, in water transport [3][4][5][6][7][8][9][10][11][12][13][14][15]. The expression patterns of aquaporin genes in response to salt, drought, cold, submergence, light, and phytohormones have also been characterized in various plants [16][17][18][19][20][21][22][23][24].…”