MERCURY-INDUCED CONFORMATIONAL-CHANGES AND IDENTIFICATION OF CONSERVED SURFACE LOOPS IN PLASMA-MEMBRANE AQUAPORINS FROM HIGHER-PLANTS - TOPOLOGY OF PMIP31 FROM BETA-VULGARIS L

Aquaporins are integral membrane proteins occurring in mammals, plants , and microorganisms, which serve as channels that permit the bidirect ional passage of water through cellular membranes, Higher plants conta in abundant levels of aquaporins in both the tonoplast and plasma memb rane, 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 m embrane aquaporins to determine the effects of divalent cations on aqu aporin conformation, and to identify structural features that distingu ish plasma membrane intrinsic proteins from tonoplast intrinsic protei ns, Plasma membrane vesicles from storage tissue of Beta vulgaris L. w ere subjected to limited proteolysis, and proteolytic fragmentation pa tterns were detected using affinity-purified antibodies recognizing aq uaporins of 31-kDa, In its native membrane-associated state, the 31 aq uaporin band, PMIP31, was refractory to proteolysis by trypsin, Howeve r,mercuric compounds specifically induced a conformational change resu lting in the exposure of a proteolytic cleavage site and formation of a unique 22-kDa proteolytic fragment (p22), N-terminal sequence analys is of p22 established its identity as an aquaporin-derived fragment, T opological studies using sealed right-side-out plasma membrane vesicle s established that the proteolytic cleavage site is located at surface loop C, the second apoplastic loop, immediately preceding the sequenc e Gly-Gly-Gly-Ala-Asn, The Gly-Gly-Gly-Ala-Asn-X-X-X-X-Gly-Tyr motif o f loop C and a 14 amino acid motif in apoplastic loop E, er-Leu/Phe-Gl y-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 pr esent in any of the known tonoplast intrinsic proteins. These results demonstrate that the two highly conserved plasma membrane intrinsic pr otein surface loops are structural features that clearly distinguish p lasma membrane from tonoplast aquaporins.