Kidney International, Vol. 60 (2001), pp. 422–426

Oligomerization of water and solute channels of the majorintrinsic protein (MIP) family

LAURENCE DUCHESNE, STEPHANE DESCHAMPS, ISABELLE PELLERIN, VALERIE LAGREE,ALEXANDRINE FROGER, DANIEL THOMAS, PATRICK BRON, CHRISTIAN DELAMARCHE,and JEAN-FRANCOIS HUBERT

Universite de Rennes 1, UMR CNRS 6026, Campus de Beaulieu, Rennes, Bretagne, France

Oligomerization of water and solute channels of the major (250 to 290 amino acids), and the high conservationintrinsic protein (MIP) family. Water and small solute fluxes throughout the MIP family may indicate a common fold:through cell membranes are ensured in many tissues by selec- a NH2 cytosolic portion followed by a hydrophobic stretchtive pores that belong to the major intrinsic protein family

of six transmembrane helices (Fig. 1A). Among highly(MIP). This family includes the water channels or aquaporinsconserved amino acids in the family, two repetitions of(AQP) and the neutral solute facilitators such as the glycerol

facilitator (GlpF). We have compared the characteristics of Asp, Pro, Ala residues (NPA box) localized in the B andrepresentatives of each subfamily. Following solubilization in E loops draw the sequence signature of the family (Fig.the nondenaturing detergents n-octyl-glucoside (OG) and Tri-

1B). The folding of these two loops in the membraneton X-100 (T-X100), AQPs remain in their native homotetra-bilayer is proposed to be responsible of the pore forma-meric state, while GlpF always behaves as a monomer. Solute

facilitators are fully solubilized by the detergent N-lauroyl sar- tion and solute movement across the membrane [2, 3].cosine (NLS), while AQPs are not. Analyses of mutants and Interestingly, AQPs are widely distributed in bacteria,chimeras demonstrate a close correlation between the water plants, and animals, while GlpFs have been characterizedtransport function and the resistance to NLS solubilization.

only within microorganisms such as bacteria or yeast. InThus, AQPs and solute facilitators exhibit different behaviorsmammals, ten MIPs have been cloned and functionallyin mild detergents; this could reflect differences in quaternary

organization within the membranes. We propose that the oligo- characterized. Some of them, such as AQP1, AQP3, andmerization state or the strength of self-association is part of the AQP4, are widely distributed in the body [4]. In contrast,mechanisms used by MIP proteins to ensure solute selectivity. AQP0, AQP2, and AQP6 are tissue specific [5–7]. To

date, the regulation of MIP expression is poorly docu-mented except for AQP2, the vasopressin-regulated wa-

Water and small solute movements across the cell ter channel expressed selectively in the principal cells ofmembranes are necessary for fundamental cell function the renal collecting duct. Activation of both expressionsuch as reabsorption, secretion, or homeostasis behavior. and apical membrane integration of AQP2 induced byThe flow of small molecules into and out of cells is medi- vasopressin is responsible for water reabsorption andated by various classes of membrane proteins: pumps, urine concentration in the kidney [5].transporters, and channels. Among such proteins, the An epithelial complex highly specialized in water trans-major intrinsic protein (MIP) family represents a specific fer is present in the digestive tract of homopteran sapmembranous channels group. To date, more than 350 sucking insects such as Cicadella: the “filter chamber,”MIP family members have been identified essentially from which allows rapid sap concentration and water move-amino acid sequences homologies [1]. However, func- ment [8]. The large excess of water ingested in the sap istional data subdivided the family into three major groups.

rapidly transferred from the initial midgut to the terminalAquaporins (AQPs) are water-selective channels, glyc-

midgut or Malpighian tubules, down a transepithelialerol facilitators (GlpF) allow glycerol and small solute

osmotic gradient (Fig. 2). Analysis of membranes in thistransport, and aquaglyceroporins transport both mole-water-shunting complex revealed the presence of a majorcules. The primary structures of these proteins are similar25 kD protein that was associated in homotetramersand organized in regular arrays of particles in the cellmembranes (Fig. 2) [9, 10]. We have cloned the corre-Key words: membrane channels, aquaporin, glycerol facilitator, water

transport, N-lauroyl sarcosine. sponding cDNA and showed that it encodes a 255 aminoacid polypeptide that we named AQPcic (for AQuaPorin 2001 by the International Society of Nephrology

422

Duchesne et al: Oligomerization of MIP proteins 423

Fig. 1. (A) Topological model of the major intrinsic protein (MIP) family with six transmembrane domains and NPA boxes localized in loops Band E. (B) The two NPA-containing loops folding in the lipid bilayer are structural components for the pore formation.

of Cicadella). This protein, which is specifically expressed membranes. When AQPcic was overexpressed in thesein filter chamber cells, exhibits a high sequence homology cells, orthogonal particles arrays identical to those ob-with the MIP family members [11, 12]. Overexpression served in native membranes were specifically detected.and functional studies of AQPcic in Xenopus oocytes or The main diameter of these particles was 8.2 nm andin Saccharomyces cerevisiae have clearly demonstrated corresponded to the tetrameric quaternary structure ofthe selective water channel function of the protein [11, 13]. the AQP, as previously measured in native insect mem-

To investigate the molecular basis of MIP protein sol- branes. Similar results have been observed previouslyute selectivity, we analyzed the structural characteristics for AQP1 [17]. Conversely, the functional expression ofof different functional members of the family. Human GlpF correlated to the presence of a specifically inducedAQP1 and AQPcic, as representative members of the subpopulation of membranous particles in which the di-AQP family, were compared with the GlpF of Esche- ameter size of 5.8 nm best fits with a monomeric formrichia coli. This article reports the results of our work of GlpF protein [18].on AQP1, AQPcic, and GlpF proteins overexpressed in Moreover, such differences in organization betweenyeast, bacteria, or Xenopus oocytes. AQPs and GlpF in lipid bilayers were suggested by ana-

lyzing the behavior of the proteins toward 2% of thenondenaturing detergent, N-lauroyl sarcosine (NLS).OLIGOMERIZATION STATE OF MIPSNative AQPs, like AQP1 from red blood cells or AQPcicSeveral observations have clearly demonstrated the ho-from insect membranes, are particularly resistant to solu-motetrameric organization of AQPs in either native mem-bilization by NLS (Fig. 3). This property allows an effi-branes [10, 14–16] or proteoliposomes after reconstitutioncient first step of AQP purification [12, 19]. This notablewith purified proteins [12, 15]. Hydrodynamic studiesresistance to NLS solubilization is also observed whenperformed in our laboratory have shown that the AQPsAQPs are overexpressed in yeast or oocyte cells [13, 20].AQP1 and AQPcic always remain in their native homo-In contrast, the GlpF, either native or overexpressed intetrameric state when solubilized in 1% Triton X-100 oryeast and oocyte, is always fully solubilized by NLS.2% n-octyl-glucoside (OG). The same quaternary struc-

Our data demonstrate that, despite a high degree ofture was determined for recombinant AQPs [10, 16]. Inhomology, AQPs and GlpFs have a differential comport-contrast, the E. coli GlpF always behaves as a monomerment in nondenaturing detergents and probably have ain these nondenaturing detergents, suggesting that AQPsdifferent organization within cell membranes. From theseand GlpF have a different oligomerization state in cellu-observations, we propose that oligomeric state or strengthlar membranes [16]. These results were corroborated byof self-association of MIP proteins could be a key elementan in vivo analysis of the particle size distribution inin transport selectivity and/or in regulation of MIP proper-membranes of Xenopus oocytes overexpressing AQPcicties. However, a recent three-dimensional crystallographicor GlpF. The size of the intramembraneous particles

was investigated by freeze fracture of oocyte plasma analysis of the E. coli GlpF indicates that histidine-

Duchesne et al: Oligomerization of MIP proteins424

Fig. 2. The filter chamber of Cicadella viridis is a highly specialized part of the digestive tract, allowing a rapid transfer of water ingested in sapfrom the initial midgut to the terminal midgut. Studies of filter chamber cell membranes after cryofracture, sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) analysis, and image processing reveal an organized array of particles (OAP) constituted by the aquaporin ofCicadella (AQPcic), a 25 kD protein associated in homotetramers [9, 10].

tagged GlpF crystallizes as a symmetric arrangement of pensity for self-association. These data may reflect a sig-nificant physiological mechanism [24].four channels [3]. The authors nicely demonstrate that

the nature of the pore, within the monomer, is closelyresponsible for the selective transport of glycerol mole- ROLE OF DISCRIMINANTcules and water exclusion. Indeed, in the three-dimen- AMINO ACID RESIDUESsional structure, a clear difference of nature and configu-

Multiple sequence alignments of MIP proteins allowedration of amino acids surrounding the solute pore isus to identify five major positions corresponding toobserved when compared with amino acids surroundingamino acid residues that are highly conserved in AQPs

the AQP1 water pore [2]. Nevertheless, the oligomericor in GlpFs, but with very different physicochemical

organization of GlpF in native bacterial membranes re- properties between the two subgroups [25]. Four or fivemains unsolved. Some data indicate that the oligomeric of these amino acids are located either in the loop E orstate of membrane proteins observed following crystalli- at the upper part of the sixth transmembrane domain.zation does not necessarily correlate with their native We constructed mutants of AQPcic in which four oforganization in the lipid bilayer [21–23]. Moreover, as these discriminating amino acids were substituted by thededuced from structural analysis, the interfaces between amino acids for GlpF (Fig. 3). Mutations in these charac-GlpF subunits are almost as hydrophobic as the exterior, teristic positions abolished or modified solute transport,suggesting that a monomer could be stable in the mem- indicating a direct or indirect involvement of these aminobrane [3]. In addition, a recent analysis using chemical acids in the functional properties of protein. In particu-cross-linking and mass spectrometry on purified GlpF lar, substitution of two of these amino acids (AQP-has confirmed our main observations with a major frac- Y222P/W223L) induced the transformation of AQP intotion of GlpF being organized in monomers [24]. The a glycerol channel [26].presence of a small fraction of GlpF oligomers observed Analysis of both oligomeric states in OG and NLSby those authors after the chemical cross-link on native solubilizations of AQPcic or GlpF mutants strengthens

our hypothesis on the relationship between oligomeriza-membranes suggests that folded GlpF could have a pro-

Duchesne et al: Oligomerization of MIP proteins 425

Fig. 3. Comparison of water channel function, oligomerization state in N-octyl-glucoside (OG) and N-lauroyl sarcosine (NLS) solubilization resistanceof MIP proteins, AQPcic mutants and chimeras. Localization of mutations and AQPcic/GlpF chimeras are described. In the AAG chimera mutant,the half of the sixth transmembrane domain and the C-terminal domain of AQPcic were replaced by the domains for GlpF and reciprocally placedin the GGA chimera. Water or glycerol permeability measurements were performed in the Xenopus oocyte system 48 hours following cRNAmicroinjection [11, 16] or by stopped-flow experiments on liposomes containing the purified recombinant proteins [13]. Oligomeric states in OGwere deduced from the velocity of sedimentation on 2 to 20% sucrose gradients [10, 16]. For solubilization studies, membranes were treated by2% NLS, and the presence of the proteins in 100,000 g pellets or supernatants was determined by Western blotting. *The AQP-245Z is found ina tetrameric form, but higher multimeric forms are also observed. nd is not determined.

tion and function of MIP proteins. Indeed, the functional especially by introducing a proline residue in the primarystructure of AQPcic, likely induces distal structural re-water channels, AQP1, AQPcic, and AQP-C134S, ex-

hibit a tetrameric oligomerization state in OG and are arrangements that lead to the switch from a water to aNLS-resistant. AQP-C134S is a mistargeted mutant in glycerol channel.oocytes but is expressed as a functional water channelin yeast cells [13]. Conversely, GlpF, AQP-S205D, AQP-

ROLES OF C-TERMINAL AND HALFY222P, and AQP-Y222P/W223L, which are unable toOF THE SIXTH TRANSMEMBRANEtransport water, are in a monomeric form in OG and areDOMAINS IN AQPCIC AND GLPFsoluble in NLS detergent. In contrast, the nonfunctional

The importance of the sixth transmembrane domainmutants AQP-A209K and -W223L remain tetrameric,in function and selectivity of MIP family proteins waslike the wild-type AQP, but have lost the particular com-investigated by analyzing the truncated forms of AQPcicportment toward NLS, suggesting a modification in theor AQPcic/GlpF chimeras (Fig. 3). Deletion of theprotein organization into the membrane. These data in-C-terminal cytoplasmic domain of AQPcic (AQP-245Z)dicate that the loss of water channel function of AQPcichad no effect on the AQP function, but deletion of themutants is closely correlated to the modification of oligo-entire sixth transmembrane domain (AQP-216Z) led tomeric organization in OG and/or resistance to NLS solu-a monomeric protein that was soluble in NLS like thebilization. Interestingly, AQP-Y222P/W223L mutantsnonfunctional mutants of AQPcic. To investigate theare permeable to glycerol, monomeric in OG, and solu-involvement of this domain in MIP properties, substitu-ble in 2% NLS as the wild-type GlpF. However, ac-tions of half of the sixth transmembrane domains ofcording to the three-dimensional structures of AQP1 andAQPcic (AAG chimera) and GlpF (GGA chimera) wereGlpF, the Y222P-W223L positions do not correspond toperformed. In AQPcic, substitution was performed inareas directly involved in the structure of the water orposition 228 in the primary structure, after the aminoglycerol pores [2, 3]. These two positions are located atacids Y222 and W223, which already have been identifiedthe junction between loop E and the sixth transmem-to be involved in AQPcic function. In GlpF, the substitu-brane domain on the extracellular side of the protein.

Substitution of two amino acids in this critical zone, tion was performed after the residue 242 (Fig. 3). Inter-

Duchesne et al: Oligomerization of MIP proteins426

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