Mercury-induced Conformational Changes and Identification of Conserved Surface Loops in Plasma Membrane Aquaporins from Higher Plants

Abstract

Aquaporins are integral membrane proteins occurring in mammals, plants, and microorganisms, which serve as channels that permit the bidirectional passage of water through cellular membranes. Higher plants contain abundant levels of aquaporins in both the tonoplast and plasma membrane. Aquaporins contain six transmembrane segments with three surface loops located at the apoplastic face of the membrane and two loops at the cytosolic side. In this study, we probed the topology of plasma membrane aquaporins to determine the effects of divalent cations on aquaporin conformation, and to identify structural features that distinguish plasma membrane intrinsic proteins from tonoplast intrinsic proteins. Plasma membrane vesicles from storage tissue of Beta vulgaris L. were subjected to limited proteolysis, and proteolytic fragmentation patterns were detected using affinity-purified antibodies recognizing aquaporins of 31-kDa. In its native membraneassociated state, the 31-aquaporin band, PMIP31, was refractory to proteolysis by trypsin. However, mercuric compounds specifically induced a conformational change resulting in the exposure of a proteolytic cleavage site and formation of a unique 22-kDa proteolytic fragment (p22). N-terminal sequence analysis of p22 established its identity as an aquaporin-derived fragment. Topological studies using sealed right-side-out plasma membrane vesicles established that the proteolytic cleavage site is located at surface loop C, the second apoplastic loop, immediately preceding the sequence Gly-Gly-Gly-Ala-Asn. The Gly-Gly-Gly-Ala-Asn-X-X-X-XGly-Tyr motif of loop C and a 14 amino acid motif in apoplastic loop E, Thr-Gly-Ile/Thr-Asn-Pro-Ala-Arg-SerLeu/Phe-Gly-Ala-Ala-Ile/Val-Ile/Val-Phe/Tyr-Asn are completely conserved in all known higher plant aquaporins of plasma membrane origin and are not present in any of the known tonoplast intrinsic proteins. These results demonstrate that the two highly conserved plasma membrane intrinsic protein surface loops are structural features that clearly distinguish plasma membrane from tonoplast aquaporins.Aquaporins are members of the MIP 1 superfamily that permit the entry and exit of water through biological membranes. MIPs are hydrophobic integral membrane proteins ranging in apparent size from 31 to 23 kDa (1-6). Each monomer contains six transmembrane segments arranged in the form of righttilted ␣-helices with both the N and C termini cytosolically oriented (7,8). In higher plants, MIPs are believed to be responsible for the maintenance of structural integrity, osmoregulation, and responses to water and salt stress (9 -12). Plant aquaporin isoforms have been localized within various membrane systems including the tonoplast (13-18) and the PM (11, 19 -25) with over a dozen differentially expressed aquaporin genes documented in Arabidopsis.Based upon N-terminal and internal sequence analysis of polypeptides electroeluted from SDS gels, our laboratory has identified aquaporins of 31, 29, and 27 kDa in PM vesicles obtained from storage tissue of Bet...

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].…”

Plant aquaporins: multifunctional water and solute channels with expanding roles

“…there may be higher energy-barriers to overcome. For this reason also slight conformational changes induced by low concentrations of HgCl 2 (see Barone et al 1997) may have a bigger effect on NH 3 permeation.…”

The Grapevine Root-Specific Aquaporin VvPIP2;4N Controls Root Hydraulic Conductance and Leaf Gas Exchange under Well-Watered Conditions But Not under Water Stress

“…Analysis of the deduced protein sequence of VvPIP2;4N allowed the prediction of all residues characteristic of the PIP family: six transmembrane domains connected by five loops, two NPA (Asn-Pro-Ala) motifs, which are conserved features of all aquaporins (Chrispeels and Maurel, 1994), and the sequences G-G-G-A-N-X-X-X-X-G-Y in loop C and T-G-I/T-N-P-A-R-S-L/F-G-A-A-I/V-I/V-F/Y-N in loop E (Barone et al, 1997). VvPIP2;4N also shows a shorter N-terminal region and a longer Cterminal region when compared with PIP1 proteins, as described in the literature (Chaumont et al, 2000).…”