Oligomerization State of Water Channels and Glycerol Facilitators

Abstract

The major intrinsic protein (MIP) family includes water channels aquaporins (AQPs) and facilitators for small solutes such as glycerol (GlpFs). Velocity sedimentation on sucrose gradients demonstrates that heterologous AQPcic expressed in yeast orXenopus oocytes behaves as an homotetramer when extracted by n-octyl β-d-glucopyranoside (OG) and as a monomer when extracted by SDS. We performed an analysis of GlpF solubilized from membranes of Escherichia coli or of mRNA-injected Xenopus oocytes. The GlpF protein extracted either by SDS or by nondenaturing detergents, OG and Triton X-100, exhibits sedimentation coefficients only compatible with a monomeric form of the protein in micelles. We then substituted in loop E of AQPcic two amino acids predicted to play a role in the functional/structural properties of the MIPs. In two expression systems, yeast and oocytes, the mutant AQPcic-S205D is monomeric in OG and in SDS. The A209K mutation does not modify the tetrameric form of the heterologous protein in OG. This study shows that the serine residue at position 205 is essential for AQPcic tetramerization. Because the serine in this position is highly conserved among aquaporins and systematically replaced by an acid aspartic in GlpFs, we postulate that glycerol facilitators are monomers whereas aquaporins are organized in tetramers. Our data suggest that the role of loop E in MIP properties partly occurs through its ability to allow oligomerization of the proteins. The major intrinsic protein (MIP) family includes water channels aquaporins (AQPs) and facilitators for small solutes such as glycerol (GlpFs). Velocity sedimentation on sucrose gradients demonstrates that heterologous AQPcic expressed in yeast orXenopus oocytes behaves as an homotetramer when extracted by n-octyl β-d-glucopyranoside (OG) and as a monomer when extracted by SDS. We performed an analysis of GlpF solubilized from membranes of Escherichia coli or of mRNA-injected Xenopus oocytes. The GlpF protein extracted either by SDS or by nondenaturing detergents, OG and Triton X-100, exhibits sedimentation coefficients only compatible with a monomeric form of the protein in micelles. We then substituted in loop E of AQPcic two amino acids predicted to play a role in the functional/structural properties of the MIPs. In two expression systems, yeast and oocytes, the mutant AQPcic-S205D is monomeric in OG and in SDS. The A209K mutation does not modify the tetrameric form of the heterologous protein in OG. This study shows that the serine residue at position 205 is essential for AQPcic tetramerization. Because the serine in this position is highly conserved among aquaporins and systematically replaced by an acid aspartic in GlpFs, we postulate that glycerol facilitators are monomers whereas aquaporins are organized in tetramers. Our data suggest that the role of loop E in MIP properties partly occurs through its ability to allow oligomerization of the proteins. major intrinsic protein aquaporin glycerol facilitator n-octyl β-d-glucopyranoside. The MIP1 family is a rapidly growing group of currently 150 proteins that have been widely identified from bacteria to man. Functional characterization data have distinguished two major subgroups of specific channels: aquaporins (AQPs), which transfer water, and glycerol facilitators (GlpFs), which transfer small neutral solutes. In the aquaporin subgroup, the eukaryotic AQP0 (1, 2), AQP1 (3Smith B.L. Agre P. J. Biol. Chem. 1991; 266: 6407-6415Abstract Full Text PDF PubMed Google Scholar, 4Verbavatz J.M. Brown D. Sabolic I. Valenti G. Austiello D. van Hoek A. Ma T. Verkman A.S. J. Cell Biol. 1993; 123: 605-618Crossref PubMed Scopus (214) Google Scholar), AQPcic (initially called P25 in Ref. 5Beuron F. Le Cahérec F. Guillam M-T. Cavalier A. Garret A. Tassan J-P. Delamarche C. Schultz P. Mallouh V. Rolland J-P. Hubert J-F. Gouranton J. Thomas D. J. Biol. Chem. 1995; 270: 17414-17422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and AQP4 (6Verbavatz J-M. Ma T. Gobin R. Verkman A.S. J. Cell Sci. 1997; 110: 2855-2860Crossref PubMed Google Scholar) have been demonstrated to exist as homotetramers. The procaryotic AqpZ was also recently shown to be tetrameric (7Ringler P. Borgnia M. Scheuring S. Agre P. Engel A. Biol. Cell. 1998; 90: 33-34Crossref Google Scholar). The role for assembly of AQPs in tetramers is unclear but may be required for stability and function (8van Hoek A.N. Hom M.L. Luthjens L.H. de Jong M.D. Dempster J.A. van Os C.H. J. Biol. Chem. 1991; 266: 16633-16635Abstract Full Text PDF PubMed Google Scholar, 9Preston G. Jung J.S. Guggino W.B. Agre P. J. Biol. Chem. 1993; 268: 17-20Abstract Full Text PDF PubMed Google Scholar, 10Zhang R. van Hoek A.N. Biwersi J. Verkman A.S. Biochemistry. 1993; 32: 2938-2941Crossref PubMed Scopus (124) Google Scholar, 11Shi L.B. Skach W.R. Verkman A.S. J. Biol. Chem. 1994; 269: 10417-10422Abstract Full Text PDF PubMed Google Scholar). According to the projection map of AQP1 obtained by electron crystallography, the protein in its tetrameric native form possesses six transmembrane domains surrounding a high density zone (12Walz T. Hirai T. Murata K. Heyman J.B. Mitsouka K. Fujiyosi Y. Smith B.L. Agre P. Engel A. Nature. 1997; 387: 624-627Crossref PubMed Scopus (377) Google Scholar, 13Heymann J. Agre P. Engel A. J. Struct. Biol. 1998; 121: 191-206Crossref PubMed Scopus (76) Google Scholar). This density area is likely constituted by loops B, C, and E that fold into the membrane through hydrophobic interactions with the transmembrane helical domains according to the “hourglass model” proposed for AQP1 (14Jung J.S. Preston G.M. Smith B.L. Guggino W.B. Agre P. J. Biol. Chem. 1994; 269: 14648-14654Abstract Full Text PDF PubMed Google Scholar). However, the domains involved in aquaporin tetramerization have not yet been identified. It has been shown that some mutations introduced in aquaporin loops affect water channel function (14Jung J.S. Preston G.M. Smith B.L. Guggino W.B. Agre P. J. Biol. Chem. 1994; 269: 14648-14654Abstract Full Text PDF PubMed Google Scholar, 15Bai L. Fushimi K. Sasaki S. Marumo F. J. Biol. Chem. 1996; 271: 5171-5176Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), whereas other mutations affect normal routing (16Deen P.M. Croes H. van Aubel R.A. Ginsel L.A. van Os C.H. J. Clin. Invest. 1995; 95: 2291-2296Crossref PubMed Scopus (215) Google Scholar, 17Mulders S.M. Knoers N.V. Van Lieburg A.F. Monnens L.A. Leumann E. Wuhl E. Schober E. Rijss J.P. Van Os C.H. Deen P.M. J. Am. Soc. Nephrol. 1997; 8: 242-248Crossref PubMed Google Scholar, 18Mulders S.M. Rijss J.P. Hartog A. Bindels R.J. van Os C.H. Deen P.M. Am. J. Physiol. 1997; 273: F451-F456PubMed Google Scholar) or expression in Xenopus oocytes (19Lagrée V. Pellerin I. Hubert J.F. Tacnet F. Le Cahérec F. Roudier N. Thomas D. Gouranton J. Deschamps S. J. Biol. Chem. 1998; 273: 12422-12426Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Few data exist about the consequences of mutations on oligomerization of aquaporins. Jung et al. (14Jung J.S. Preston G.M. Smith B.L. Guggino W.B. Agre P. J. Biol. Chem. 1994; 269: 14648-14654Abstract Full Text PDF PubMed Google Scholar) reported that following expression in Xenopus oocytes, mutants in loop E sedimented with s values slightly higher than native AQP1 suggesting an importance of this loop in oligomerization and water channel function. The second subgroup of proteins in the MIP family is constituted by the glycerol facilitators (20Muramatsu S. Mizumo T. Nucleic Acids Res. 1989; 17: 4378-4379Crossref PubMed Scopus (15) Google Scholar). Although the structural feature of one aquaporin is known (12Walz T. Hirai T. Murata K. Heyman J.B. Mitsouka K. Fujiyosi Y. Smith B.L. Agre P. Engel A. Nature. 1997; 387: 624-627Crossref PubMed Scopus (377) Google Scholar, 13Heymann J. Agre P. Engel A. J. Struct. Biol. 1998; 121: 191-206Crossref PubMed Scopus (76) Google Scholar), no data are yet available about the three-dimensional structure and oligomerization state of GlpFs. However, based on the similarities in their amino acid sequences and hydrophobicity profiles, the monomers of glycerol facilitators and of water channels likely exhibit similar three-dimensional structural organizations. A topological model for MIPs and a comparison of sequences in loop E between an aquaporin, AQPcic of Cicadella viridis, and the GlpF of Escherichia coli are shown in Fig. 1. We recently detected five amino acid positions that distinguish clearly two groups in MIPs (21Froger A. Tallur B. Thomas D. Delamarche C. Protein Sci. 1998; 7: 1458-1468Crossref PubMed Scopus (185) Google Scholar). These amino acids have been designated as “discriminant.” Interestingly, these two groups correspond to the two functionally different groups within MIPs, namely aquaporins and glycerol facilitators. One of these positions is located in loop C and the four others are found in or close to loop E. To study the oligomerization state of MIPs, we analyzed the behavior on sucrose density gradients of wild type or mutated AQPcic and of GlpF. We show that the heterologous AQPcic is tetrameric whereas GlpF is in a monomeric form either in OG or in Triton X-100 micelles. Moreover, we demonstrate that the substitution of a conserved amino acid of the aquaporin by a conserved amino acid of GlpF leads to the passage of AQPcic from a tetrameric to a monomeric form. pSP-AQPcic corresponds to the full-length AQPcic coding sequence subcloned into plasmid pXβG-ev1 (22Le Cahérec F. Deschamps S. Delamarche C. Pellerin I. Bonnec G. Guillam M-T. Thomas D. Gouranton J. Hubert J.F. Eur. J. Biochem. 1996; 241: 707-715Crossref PubMed Scopus (73) Google Scholar). The pY60-AQPcic contains the AQPcic cDNA coding sequence placed under control of a GAL10-CYC promoter and a phosphoglycerate kinase terminator (19Lagrée V. Pellerin I. Hubert J.F. Tacnet F. Le Cahérec F. Roudier N. Thomas D. Gouranton J. Deschamps S. J. Biol. Chem. 1998; 273: 12422-12426Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The AQPcic mutants were obtained by performing a two-step polymerase chain reaction using pSP-AQPcic as a template and sets of appropriate primers that overlap in the region of the mutation (23Horton R.M. Pease L.R. McPherson M.J. Directed Mutagenesis, a Practical Approach. IRL Press, Oxford1991: 217-248Google Scholar). The mutated AQPcic cDNAs were cloned either into plasmid pXβG-ev1 (constructions are named pSP-AQPS205D, pSP-AQPA209K, and pSP-AQPS205D/A209K) or into the yeast expression vector pYeDP60 (constructions are named pY60-AQPS205D, pY60-AQPA209K, and pY60-AQPS205D/A209K). The constructs pSP-AQPC134S and pY60-AQPC134S were described in Ref. 19Lagrée V. Pellerin I. Hubert J.F. Tacnet F. Le Cahérec F. Roudier N. Thomas D. Gouranton J. Deschamps S. J. Biol. Chem. 1998; 273: 12422-12426Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar. The coding region of E. coli glpF was amplified from the pglpF vector, a generous gift of Dr. Mizumo (20Muramatsu S. Mizumo T. Nucleic Acids Res. 1989; 17: 4378-4379Crossref PubMed Scopus (15) Google Scholar), by polymerase chain reaction and cloned either into the bacterial plasmid pUC18 (pUC-glpF) or into pXβG-ev1 (pSP-glpF). Yeast cells (W3031B strain) were transformed with pY60-AQPcic, pY60-AQPS205D, pY60-AQPA209K, pY60AQPS205D/A209K, and pY60-AQPC134S. Expression of wild type or mutated forms of AQPcic was then performed as described previously (19Lagrée V. Pellerin I. Hubert J.F. Tacnet F. Le Cahérec F. Roudier N. Thomas D. Gouranton J. Deschamps S. J. Biol. Chem. 1998; 273: 12422-12426Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). For the production of GlpF in bacteria, the E. coli strain GD236 (JM103 (24Sweet G. Gandor C. Voegele R. Winttekindt N. Beuerle J. Truniger V. Lin E. Boos W. J. Bacteriol. 1990; 175: 1087-1094Google Scholar)) was transformed with pUC-glpF and was grown at 37 °C in LB with ampicillin (100 μg/ml). At midlog phase (A 600 ∼0.6), expression of GlpF was induced by adding 1 mmisopropyl-1-thio-β-d-galactopyranoside in the culture medium for 30 min. Production of proteins in Xenopus oocytes and subsequent water and glycerol transport assays were achieved as described (22Le Cahérec F. Deschamps S. Delamarche C. Pellerin I. Bonnec G. Guillam M-T. Thomas D. Gouranton J. Hubert J.F. Eur. J. Biochem. 1996; 241: 707-715Crossref PubMed Scopus (73) Google Scholar). After protein expression, total membrane fractions from yeast cells were prepared as described previously (25Centeno F. Deschamps S. Lompré A-M. Anger M. Moutin M-J. Dupont Y. Palmgren M.G. Villalba J.M. Møller J.V. Falson P. le Maire M. FEBS Lett. 1994; 354: 117-122Crossref PubMed Scopus (37) Google Scholar). Bacteria and Xenopusoocyte membranes were prepared as described in Refs. 26Osborn M.J. Gander J.E. Parisi E. Carson J. J. Biol. Chem. 1972; 247: 3962-3972Abstract Full Text PDF PubMed Google Scholar and 9Preston G. Jung J.S. Guggino W.B. Agre P. J. Biol. Chem. 1993; 268: 17-20Abstract Full Text PDF PubMed Google Scholar, respectively. Membranes were incubated in TB buffer (20 mmTris-HCl, pH 7.4, 1 mm dithiothreitol) containing 2% OG or 1% Triton X-100 for 12 h at 4 °C or 1% SDS for 12 h at room temperature. Insoluble materials were eliminated by a 100,000 × g centrifugation for 45 min at 15 °C. Linear 2–20% (w/v) sucrose density gradients were prepared from 2 and 20% sucrose stock in TB buffer containing 2% 1% Triton X-100, or SDS. proteins were on of and to was performed at 100,000 × g for h at °C. fractions were then from the of and analyzed by Nature. PubMed Scopus Google Scholar) and P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). AQPcic was performed using a the native C. protein F. Le Cahérec F. Guillam M-T. Cavalier A. Garret A. Tassan J-P. Delamarche C. Schultz P. Mallouh V. Rolland J-P. Hubert J-F. Gouranton J. Thomas D. J. Biol. Chem. 1995; 270: 17414-17422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). GlpF was detected using a in a of GlpF to analysis was performed as described (22Le Cahérec F. Deschamps S. Delamarche C. Pellerin I. Bonnec G. Guillam M-T. Thomas D. Gouranton J. Hubert J.F. Eur. J. Biochem. 1996; 241: 707-715Crossref PubMed Scopus (73) Google Scholar). were incubated with either or were and analyzed using were obtained using The of wild type and mutant proteins in membrane cells was analyzed by shown in yeast cells transformed by appropriate AQPcic 2), AQPcic-S205D and no is detected in membranes of control yeast In membranes of Xenopus oocytes, AQPcic AQPcic-S205D and are clearly detected and in control oocytes Fig. 2 shows that the a protein in bacteria transformed by pUC-glpF vector and in glpF oocytes is detected in control 1 and The of this specific demonstrates that the GlpF protein is expressed in membranes of oocytes and In to the GlpF we a protein in bacteria membrane The of this protein from the expression of an sequence to GlpF cDNA and the of cDNA in the pUC-glpF To the and of assays were performed on oocytes with wild type or mutants of AQPcic and GlpF 2 water is in wild type AQPcic oocytes when with control oocytes. of water is in oocytes, or GlpF oocytes. The of has been previously demonstrated in (19Lagrée V. Pellerin I. Hubert J.F. Tacnet F. Le Cahérec F. Roudier N. Thomas D. Gouranton J. Deschamps S. J. Biol. Chem. 1998; 273: 12422-12426Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). the other glycerol is about in oocytes, whereas no of glycerol is in oocytes and In in assays of glycerol have demonstrated that the GD236 E. coli strain transformed with pUC-glpF plasmid a glycerol channel not We previously demonstrated that the native AQPcic is a homotetramer in OG and in Triton X-100 and that SDS from membranes of the protein F. Le Cahérec F. Guillam M-T. Cavalier A. Garret A. Tassan J-P. Delamarche C. Schultz P. Mallouh V. Rolland J-P. Hubert J-F. Gouranton J. Thomas D. J. Biol. Chem. 1995; 270: 17414-17422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). we the to and AQPcic and mutants of AQPcic and GlpF. To the form of the proteins in the nondenaturing or detergents, membrane from oocytes, or bacteria were analyzed by sucrose and of proteins in fractions was by shown in Fig. the AQPcic protein at sedimentation fractions This corresponds to a sedimentation and with the form of AQPcic F. Le Cahérec F. Guillam M-T. Cavalier A. Garret A. Tassan J-P. Delamarche C. Schultz P. Mallouh V. Rolland J-P. Hubert J-F. Gouranton J. Thomas D. J. Biol. Chem. 1995; 270: 17414-17422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). AQPcic is performed in sedimentation occurs at fractions of that correspond to the monomeric form of the These show that heterologous AQPcic expressed either in yeast or in Xenopus oocytes in the tetrameric state as the native We then analyzed on sucrose gradients the GlpF protein in E. In expression systems, GlpF in OG or in SDS at an sedimentation of in fractions This is of sedimentation is obtained when the is performed coli GlpF extracted by Triton X-100 The of amino acid substitution in loop E on oligomerization of MIPs was then We substituted the serine 205 or the of AQPcic with the amino acids located in the positions in an aspartic acid and a sedimentation analysis of these mutants expressed in yeast and in Xenopus oocytes is shown in Fig. In expression systems, the AQPcic-S205D mutation the protein in a monomeric state in whereas the mutation A209K in AQPcic does not modify the in the nondenaturing We have also demonstrated that AQPcic-S205D and mutants are monomeric in SDS and that the mutant is monomeric in OG and in SDS not In in yeast membrane solubilized in the mutant which is a aquaporin (19Lagrée V. Pellerin I. Hubert J.F. Tacnet F. Le Cahérec F. Roudier N. Thomas D. Gouranton J. Deschamps S. J. Biol. Chem. 1998; 273: 12422-12426Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), is found In the we the oligomerization state of proteins of the two subgroups of the MIP For this we analyzed the aquaporin AQPcic expressed in yeast or in Xenopus oocytes and the glycerol facilitator of E. coli expressed in bacteria or in oocytes. We also the oligomerization state of AQPcic when of aquaporins are substituted with of the glycerol facilitator family and that the amino acid in position 205 is involved in tetramerization or stability of the AQPcic The native tetrameric state has been previously demonstrated for AQP0 T. H. de J. J. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, N. J. Biol. 1997; PubMed Scopus (37) Google Scholar), AQP1 (3Smith B.L. Agre P. J. Biol. Chem. 1991; 266: 6407-6415Abstract Full Text PDF PubMed Google Scholar), or AQPcic F. Le Cahérec F. Guillam M-T. Cavalier A. Garret A. Tassan J-P. Delamarche C. Schultz P. Mallouh V. Rolland J-P. Hubert J-F. Gouranton J. Thomas D. J. Biol. Chem. 1995; 270: 17414-17422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) in by on the that native MIP tetramers are in with nondenaturing such as OG or Triton AQP1 T. Smith B.L. M.L. Engel A. Agre P. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) and AQPcic (19Lagrée V. Pellerin I. Hubert J.F. Tacnet F. Le Cahérec F. Roudier N. Thomas D. Gouranton J. Deschamps S. J. Biol. Chem. 1998; 273: 12422-12426Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) solubilized in OG have been in we performed protein and sedimentation analysis in 2% OG or 1% Triton X-100, values and in 1% a of the to native AQPcic F. Le Cahérec F. Guillam M-T. Cavalier A. Garret A. Tassan J-P. Delamarche C. Schultz P. Mallouh V. Rolland J-P. Hubert J-F. Gouranton J. Thomas D. J. Biol. Chem. 1995; 270: 17414-17422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We previously that AQPcic is a water channel when expressed in oocytes (22Le Cahérec F. Deschamps S. Delamarche C. Pellerin I. Bonnec G. Guillam M-T. Thomas D. Gouranton J. Hubert J.F. Eur. J. Biochem. 1996; 241: 707-715Crossref PubMed Scopus (73) Google Scholar) or in yeast (19Lagrée V. Pellerin I. Hubert J.F. Tacnet F. Le Cahérec F. Roudier N. Thomas D. Gouranton J. Deschamps S. J. Biol. Chem. 1998; 273: 12422-12426Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The study demonstrates that in heterologous expression systems, the wild type aquaporin AQPcic is In the aquaporin mutant is also To GlpF we the as for We the of the protein following expression oocytes and in the specific glycerol transport through GlpF has been The monomeric state of GlpF that we have in oocytes is not likely to correspond to a form of the assays in bacteria GlpF about a monomeric GlpF To the between the sedimentation and the oligomerization state of a membrane protein in the of be Although the of OG to GlpF in the highly conserved hydrophobicity of MIPs, we that OG has the behavior on GlpF as on other of the MIP Moreover, of hydrophobic transmembrane to the likely the monomeric forms of MIPs to than the forms in micelles. The of OG to AQP0 was found from to of protein T. H. de J. J. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, N. J. Biol. 1997; PubMed Scopus (37) Google Scholar). For we previously an OG of of protein F. Le Cahérec F. Guillam M-T. Cavalier A. Garret A. Tassan J-P. Delamarche C. Schultz P. Mallouh V. Rolland J-P. Hubert J-F. Gouranton J. Thomas D. J. Biol. Chem. 1995; 270: 17414-17422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). GlpF was in a or form in OG with an of OG of of the of the be at This is with the sedimentation in sucrose gradients for and only with a monomer of the protein with We also that in yeast the wild type or the mutants of AQPcic are expressed in and no in the have with the of GlpF was performed with two nondenaturing from membranes of two different we that GlpF is in monomeric form. the mutation systematically of these data that the monomeric state of GlpF is not a The aquaporin AQP1 been previously shown to in Triton X-100 as constituted by one and G. Jung J.S. Guggino W.B. Agre P. J. Biol. Chem. 1993; 268: 17-20Abstract Full Text PDF PubMed Google Scholar). and van Hoek van Hoek A.N. Biochem. Physiol. B. Biochem. Biol. 1997; PubMed Scopus Google Scholar) recently obtained OG of AQP1 and AQP1 from The that different and the behavior of some aquaporins in nondenaturing In the we which is a aquaporin, T. Guillam and J-F. and GlpF for which no For that using the not high forms of GlpF coli or in Interestingly, this study shows that the serine residue in position 205 of the loop E in the aquaporin AQPcic be essential for whereas and are found essential for the water transfer function. The AQPcic-S205D mutant does not in to an This of be to the in the oligomerization state in the membrane or to a The mutation was following sequence analysis of MIPs (21Froger A. Tallur B. Thomas D. Delamarche C. Protein Sci. 1998; 7: 1458-1468Crossref PubMed Scopus (185) Google Scholar). The 205 and positions to a and a residue for the glycerol facilitators and two small amino acids for the aquaporins The of AQPcic-S205D to form a be to the of the residue in position 205 than the small The mutation the of loop and this the structural However, in the the mutation A209K that also the of loop E not modify the tetrameric form of the heterologous the of the structural map of AQP1 by electron (12Walz T. Hirai T. Murata K. Heyman J.B. Mitsouka K. Fujiyosi Y. Smith B.L. Agre P. Engel A. Nature. 1997; 387: 624-627Crossref PubMed Scopus (377) Google Scholar), in the tetrameric native form of the six transmembrane domains a zone that is to some of loops B, C, and E. In this the serine 205 and the in loop E likely not at the between We not have to the mutation oligomerization and the A209K substitution does The and transport of MIPs and the for of aquaporins in have to be In the we demonstrate that the substitution of a amino acid in an aquaporin by the residue located at the position in GlpF the aquaporin to the oligomerization state of GlpF. Because the serine and the aspartic acid in this position are highly conserved in aquaporins and in GlpFs, we postulate that glycerol facilitators are monomers in whereas aquaporins are organized in tetramers. Our suggest that loop E a major structural and role in the MIP family proteins. We for