Barone L M, Shih C, Wasserman B P
Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231, USA.
J Biol Chem. 1997 Dec 5;272(49):30672-7. doi: 10.1074/jbc.272.49.30672.
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 membrane-associated 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-X-Gly-Tyr motif of loop C and a 14 amino acid motif in apoplastic loop E, Thr-Gly-Ile/Thr-Asn-Pro-Ala-Arg-Ser-Leu/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.
水通道蛋白是存在于哺乳动物、植物和微生物中的整合膜蛋白，它们作为通道允许水双向通过细胞膜。高等植物的液泡膜和质膜中都含有丰富的水通道蛋白。水通道蛋白包含六个跨膜片段，在膜的质外体表面有三个表面环，在胞质侧有两个环。在本研究中，我们探究了质膜水通道蛋白的拓扑结构，以确定二价阳离子对水通道蛋白构象的影响，并识别区分质膜内在蛋白和液泡膜内在蛋白的结构特征。对来自甜菜贮藏组织的质膜囊泡进行有限的蛋白酶解，并使用识别31 kDa水通道蛋白的亲和纯化抗体检测蛋白酶解片段模式。在其天然的膜结合状态下，31 kDa水通道蛋白条带PMIP31对胰蛋白酶的蛋白酶解具有抗性。然而，汞化合物特异性地诱导构象变化，导致蛋白酶解切割位点暴露并形成独特的22 kDa蛋白酶解片段（p22）。对p22的N端序列分析确定其为水通道蛋白衍生片段。使用密封的外翻质膜囊泡进行的拓扑学研究表明，蛋白酶解切割位点位于表面环C，即第二个质外体环，紧接在Gly-Gly-Gly-Ala-Asn序列之前。环C的Gly-Gly-Gly-Ala-Asn-X-X-X-X-Gly-Tyr基序和质外体环E中的14个氨基酸基序Thr-Gly-Ile/Thr-Asn-Pro-Ala-Arg-Ser-Leu/Phe-Gly-Ala-Ala-Ile/Val-Ile/Val-Phe/Tyr-Asn在所有已知的源自质膜的高等植物水通道蛋白中完全保守，而在任何已知的液泡膜内在蛋白中都不存在。这些结果表明，这两个高度保守的质膜内在蛋白表面环是明显区分质膜水通道蛋白和液泡膜水通道蛋白的结构特征。
