UMR CNRS, Interactions Cellulaires et Moléculaires, Equipe Canaux et Récepteurs Membranaires, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes cedex, France1
Author for correspondence: Christian Delamarche. Tel.: +33 2 99 28 61 22. Fax: +33 2 99 28 14 77. e-mail: christian.delamarche{at}univ-rennes1.fr
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ABSTRACT |
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Keywords: glycerol transport, water transport, Lactococcus lactis
Abbreviations: MIP; major intrinsic protein
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INTRODUCTION |
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Aquaporins are highly specific for water. The most studied aquaporin, AQP1, has been analysed by electron crystallography and a three-dimensional reconstruction at 0·380·4 nm resolution has been obtained (Ren et al., 2000 ; Murata et al., 2000
). AQP1 is a homotetramer of 28 kDa subunits, each containing six transmembrane helices.
Glycerol facilitators are permeable to glycerol or small uncharged molecules. The crystal structure of the E. coli glycerol facilitator (GlpF) has been resolved at 0·22 nm by X-ray crystallography (Fu et al., 2000 ). GlpF crystallizes as a symmetric arrangement of four channels with three glycerol molecules in each.
As expected from their sequence similarities, AQPs and GlpFs exhibit a similar structural organization. However, differences in the channel-lining side chains and the residues at the narrowest parts of the channels create two different environments which should be responsible for the channel selectivity.
Aquaglyceroporins, such as AQP3, AQP7and AQP9, describe a new class of water channels which are also permeable to glycerol, but to a lesser degree than GlpF (Echevarria et al., 1994 ; Ishibashi et al., 1994
; Ma et al., 1994
; Ishibashi et al., 1997
; Kuriyama et al., 1997
; Ishibashi & Sasaki, 1998
; Tsukagushi et al., 1998
). Aquaglyceroporins are of particular interest for the investigation of the molecular basis of selectivity for both water and solutes and to address the question of a distinct molecular mechanism for such mixed channels.
Using statistical sequence analysis we have pointed out that only few key residues could distinguish aquaporins from glycerol facilitators and thus could contribute to their functional properties (Froger et al., 1998 ; Delamarche, 2000
). This finding was supported by an experimental approach where a substitution of two key residues in an aquaporin abolished water transfer and conferred selectivity to glycerol associated with monomerization of the protein (Lagrée et al., 1999
).
To bring new insights to elucidating the determination of MIP specificity, it is of primary importance to analyse the structural and functional properties of homologues, chimaeras, mutants and particularly members bearing unconventional functional properties. Presently, only a few microbial MIPs have been studied functionally (Maurel et al., 1994 ; Calamita et al., 1995
, 1998
; Delamarche et al., 1999
; Borgnia et al., 1999b
, Calamita, 2000
) and most microbial members of the MIP family have been functionally classified by sequence homology (Hohmann et al., 2000
). Thus, the physiological roles of prokaryote MIP channels are still largely undefined.
A multiple sequence alignment analysis conducted between bacterial members of the MIP family separates the sequences into three major clusters, one corresponding to aquaporins, one to glycerol facilitators and a third to a subgroup not yet correlated to a defined function, suggesting that some microbial MIPs bear unorthodox functional properties (Fig. 1).
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METHODS |
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Glycerol transport assays in E. coli.
Glycerol transport assays were performed as described by Sweet et al. (1990) with the following modifications. Bacteria were grown in M9 modified medium containing maltose (10 mM) at 30 °C to an OD600 of 0·3. Cells were harvested, pelleted, washed twice with M9 and then resuspended in M9. Assays were performed at room temperature with 6x108 cells at a final volume of 500 µl M9 containing 0·3 µM [U-14C]glycerol (final activity 5·92 Gbq mmol-1; Amersham). After 1 min of incubation, cells were vacuum-filtered through 0·45 µm cellulose nitrate membrane filters (Whatman), washed with 2 ml cold M9 and the radioactivity was counted.
Glycerol transport assays in Xenopus oocytes.
Plasmids pSP-glpF, pSP-aqpZ and pSP-Llac were linearized with XbaI and transcribed with T3 RNA polymerase by means of the mCAP cRNA capping kit (Stratagene). Stage VI Xenopus oocytes were microinjected with 40 nl water for controls or with in vitro mRNA transcripts (1 µg µl-1) and incubated in OR2 buffer (Le Cahérec et al., 1996 ) for 4872 h at 1618 °C.
At 4872 h after microinjection, the oocytes were incubated in OR2/2 supplemented with 85 mM glycerol to adjust the osmolarity to 176 mosM and with [U-14C]glycerol (final activity 0·3 Mbq ml-1). After 10 min, the oocytes were rapidly rinsed four times in 2 ml ice-cold solution (half strength solution of OR2 supplemented with 85 mM glycerol) and lysed in 10% SDS at room temperature. Radioactivity was measured by using a liquid scintillation counter.
Swelling of Xenopus oocytes.
Osmotic water permeability (Pf) was measured from the time course of oocytes swelling in response to a threefold dilution of extracellular OR2. To calculate the activation energy (Ea) the Pf was measured at three different temperatures, 10, 20 and 30 °C, as described previously (Le Cahérec et al., 1996 ).
Cryoelectron microscopy.
E. coli SK46, either with or without the plasmid pUC-Llac, was grown overnight in M9 modified minimal medium supplemented with maltose (10 mM). These cultures were diluted and grown at 37 °C until the exponential phase (OD600=0·8) in M9 containing maltose. The bacteria were then pelleted rapidly and resuspended in M9 (240 mosM) at room temperature. A 2·5 µl drop of the cell suspension was placed directly on a copper grid coated with a thin carbon film, upon which osmotic challenges were performed. Osmotic up-shocks were induced by rapidly mixing 2·5 µl of a 1·2 M sucrose-M9 solution with the cell suspension on the grid (final osmolarity 1000 mosM). After 10 s the grid was briefly blotted with filter paper and plunged into liquid ethane held at liquid nitrogen temperature. Specimens were examined at -170 °C in a Philips CM12 microscope with a Gatan model 626 cryoholder (Delamarche et al., 1999 ). Micrographs were recorded on Kodak SO 163 film under low-dose conditions at a nominal magnification of 6300.
Sequence analysis.
The MIP sequences, retrieved from the EMBL and SWISS-PROT databases, were aligned with PILEUP or CLUSTAL W (Devereux et al., 1984 ; Thompson et al., 1994
). Computing was performed using Infobiogen resources (http://www.infobiogen.fr). A score (i, j) is the sum of the elementary scores between two aligned sequences i and j, using the BLOSUM matrix (Henikoff & Henikoff, 1992
). By default, a score of 8 is attributed for the gap insertions. The similarity scores presented in Fig. 1
were calculated at Infobiogen with the program EDTALN: percentage score (i, j)=100*{score (i, j)/max [score (i, i), score (j, j)]}.
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RESULTS |
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DISCUSSION |
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E. coli uses glycerol as a carbon source for glycolysis and for lipid biogenesis. Glycerol enters the cytoplasm by passive diffusion across the lipid bilayer (Sweet et al., 1990 ) or by facilitative diffusion mediated by GlpF (Heller et al., 1980
). The E. coli glycerol facilitator, GlpF, has been shown to selectively transport glycerol and not water or ions (Maurel et al., 1994
). GlpF contributes directly to bacterial growth as illustrated by the complementation experiments presented in this paper. When expressed in E. coli, GlaLlac can play a role in bacterial growth like E. coli GlpF. We have therefore analysed the transport of glycerol mediated by GlaLlac both in bacteria and in Xenopus oocytes. We found that GlaLlac displays the same characteristics as E. coli GlpF for glycerol transport, allowing us to conclude that GlaLlac is a glycerol facilitator.
So far, AqpZ, the aquaporin of E. coli, is the only bacterial water channel which has been extensively functionally studied (Calamita et al., 1995 , 1998
; Delamarche et al., 1999
; Borgnia et al., 1999b
; Scheuring et al., 1999
; Ringler et al., 1999
; Calamita, 2000
). Although puzzling questions on the physiological necessity of fast water transport in bacteria remain, it appears that aquaporins could be directly involved in cell proliferation (Calamita et al., 1998
). This is supported by our observations in E. coli, in which two null mutations in glpF and aqpZ obviated growth. Growth was partly restored when E. coli GlpF was expressed and was completely restored with the expression of GlaLlac. This suggests that GlaLlac can mimic AqpZ function to restore growth in bacteria. Such complementation experiments using E. coli strain SK46 can be used to test the function of bacterial MIPs. We previously characterized bacterial aquaporins using cryoelectron microscopy and E. coli as an expression system (Delamarche et al., 1999
; Rodriguez et al., 2000
). In the present study we show that GlaLlac significantly mediates water fluxes. Moreover, the water channel properties of GlaLlac were demonstrated when the protein was expressed in Xenopus oocytes. The calculated Pf for GlaLlac has the same magnitude as AqpZ. Moreover the low activation energy calculated for oocytes expressing GlaLlac corresponds to that of a water channel. Therefore GlaLlac is a mixed channel, like aquaglyceroporins described in mammals, and the first one to be characterized in bacteria. Unlike mammalian aquaglyceroporins (Kuriyama et al., 1997
; Tsukagushi et al., 1998
; Echevarria et al., 1996
), GlaLlac imparts a high permeability to glycerol to the cell membrane.
Protein sequence alignments can be used to predict the function of an MIP (Froger et al., 1998 ; Delamarche, 2000
). In Fig. 1
, a high score between two sequences suggests that the two corresponding proteins have a similar function. For Gram-negative bacteria, the scores suggest that glycerol and water transport are assumed to occur independently by two distinct channels. We propose that Gram-positive bacteria contain a single MIP that possesses the two functions of glycerol facilitator and water channel. Recent studies on Bacillus subtilis, another Gram-positive bacterium, have confirmed this prediction (A. Froger & C. Delamarche, unpublished). According to the key residues predicted to distinguish the functional subgroups of MIP (Froger et al., 1998
), it can be noted that the sequences of the third group bears the signature of glycerol channels. Thus, it would be interesting to define other key residues or motifs that could determine the properties of mixed MIPs. For example, among residues that interact with glycerol in the GlpF channel (Fu et al., 2000
), the proline residue at position 246 of E. coli GlpF is found in all the glycerol facilitator group defined in Fig. 1
. Intriguingly, this proline is substituted by a glycine residue in all the sequences of the third group of putative mixed channels. In mammalian aquaglyceroporins, this proline is also substituted by a leucine, alanine, methionine or phenylalanine.
The determination of the structure of GlpF has been a major factor in the elucidation of the mechanism of selective permeability for glycerol. However, the proposed mechanism for water transport by AQP1 still requires a higher resolution structure. Moreover, the molecular mechanism for mixed channels has still to be cleared up. Analysis of factors affecting the specificity of mixed channels conducted together with high resolution structural studies should provide some key answers to this phenomenon. In that way GlaLlac from L. lactis should be an interesting tool for solving this problem.
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ACKNOWLEDGEMENTS |
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Received 11 October 2000;
revised 20 December 2000;
accepted 2 January 2001.