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...

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfidelinked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.The PM of higher plants has been the subject of extensive research focusing on the areas of recognition, water and ion transport, signal transduction, and cell wall polymer bio-

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Immunological techniques are widely used for the detection of enzymes in food systems and for elucidating protein structure and function. The generation of anti‐bodies recognizing membrane‐bound proteins is difficult due to their hydrophobic nature, and reduced antigenicity. This review summarizes strategies for the production and purification of polyclonal antibodies recognizing membrane proteins. Applications such as enzyme immunoprecipitation, use of site‐specific anti‐peptide. antibodies for determination of the topology of membrane‐embedded proteins, immunocytochemistry, and expression vector cloning are described.