Hourglass pore-forming domains restrict aquaporin-1 tetramer assembly.
The AQP1 water channel protein is a homotetramer with 28 kDa subunits containing six transmembrane domains. The sequence-related loops B (cytoplasmic) and E (extracellular) were predicted to overlap within the membrane, forming an aqueous pore ("the hourglass") flanked by the corresponding B and E residues 73 and 189. Cryoelectron microscopy of AQP1 previously revealed the central hourglass structure surrounded by six transmembrane helices which provide contact points between subunits. Several mutants in loop B and E residues were nonfunctional when expressed in X. laevis oocytes, but their ability to form tetramers is unknown. To explore the possible functional dependence of hourglass domains in adjacent subunits, we prepared a series of tandem dimers as single 55 kDa polypeptides containing different combinations of wild-type (AQP1) or mutant subunits (A73M or C189M). In oocytes, AQP1-AQP1 exhibited high osmotic water permeability, and AQP1-C189M exhibited half activity. Dimer polypeptides with A73M were nonfunctional or not expressed. In yeast secretory vesicles, AQP1-AQP1 exhibited high water permeability, AQP1-C189M exhibited half activity, and both were inhibited by pCMBS. Although expressed, the dimer polypeptides with A73M were all nonfunctional. Tetramer formation was investigated by detergent solubilization and velocity sedimentation through sucrose gradients. Dimer polypeptides containing one A73M subunit or two C189M subunits migrated with slower velocity (s < 3.5 S). In contrast, dimer polypeptides with one C189M subunit migrated with velocity similar to native AQP1 tetramers (s approximately 6 S). Thus, although hourglass pore-forming domains are not points of subunit-subunit contact, the structure of loop B is important to normal tetramer assembly.
Mathai
J C
JC
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
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D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001692:Biological Transport / Q000235:genetics; D002499:Centrifugation, Density Gradient; D003902:Detergents; D005822:Genetic Vectors; D009865:Oocytes / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014867:Water / Q000378:metabolism; D014982:Xenopus laevis
D020346:Aquaporins; D003902:Detergents; D011994:Recombinant Proteins; D014867:Water; D051398:Aquaporin 1