- Email: [email protected]
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24 Norris, F.A. et al. (1998) SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase. Proc. Natl. Acad. Sci. U. S. A. 95, 14057–14059 25 Hong, K.H. and Miller, V.L. (1998) Identification of a novel Salmonella invasion locus homologous to Shigella IpgDE.
J. Bacteriol. 180, 1793–1802 26 Eckmann, L. et al. (1997) D-myo-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signalling pathways. Proc. Natl. Acad. Sci. U. S. A. 94, 14456–14460
Microbial MIP channels Stefan Hohmann, Roslyn M. Bill, Gerald Kayingo and Bernard A. Prior
I
t has been known for many MIP channels occur in all classes of Structure–function years that the water permeorganism ranging from bacteria to man. relationships and transport ability of specialized bioThere are two major categories of MIP specificity logical membranes is much channels, aquaporins and glycerol All MIP channels share highly higher than that of artificial facilitators, which facilitate the diffusion conserved residues and are lipid bilayers. At the beginning across biological membranes of water or predicted to have six transof the 1990s, the first bona glycerol and other uncharged compounds, membrane domains (Fig. 1). fide water channel was characrespectively. As a result of their Residues present in loops B terized from red blood cells1,2. involvement in osmoregulation and and E comprise the family’s This protein, now known as metabolism, MIP channels are believed to signature sequences: Ser-Glyaquaporin 1 (AQP1), was affect a wide range of biological processes. X-His-X-Asn-Pro-Ala-Val-Thr found to be related to MIP, the (where X 5 any amino acid) S. Hohmann* and R.M. Bill are in the Dept of Cell major intrinsic protein of and Asn-Pro-Ala-Arg, respecand Molecular Biology/Microbiology, Göteborg mammalian lens fibre; to TIPs, tively; each contains a canoniUniversity, Box 462, S-40530 Göteborg, Sweden; the tonoplast intrinsic proteins cal NPA (Asn-Pro-Ala) box. G. Kayingo and B.A. Prior are in the Dept of from plants; and to GlpF, These loops are believed to ‘dip Microbiology, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa. the glycerol facilitator from into’ the membrane bilayer, *tel: 146 31 773 2595, Escherichia coli. The protein forming a continuous solute fax: 146 31 773 2599, family was eponymously named channel (the so-called houre-mail: [email protected] MIP, although the major inglass), lined by a chain of hytrinsic protein of mammalian drogen-bonding amino acids7. lens fibre has since been renamed AQP0. To date, To date, structural data (at 6Å) are only available more than 200 members of the MIP family have been for AQP1 (Ref. 7). Even though the most divergent identified. In fact, higher organisms possess an amaz- members of the MIP family are less than 20% identiing number of isoforms, which are expressed in dif- cal8, it is expected that the gross structural features of ferent subcellular compartments and tissues under AQP1 will also be present in other water and glycerol different environmental conditions or during different channels. For example, AQP1 is functionally homo developmental stages. For example, ten genes encod- tetrameric, a quaternary structure which is therefore ing MIP channels are currently known in humans2, anticipated for all other MIPs. Comparison of the 30 have been described in the plant Arabidopsis crystal structure of MIP with that of E. coli AqpZ and thaliana3, and the nematode Caenorhabditis elegans GlpF confirms the close overall structural relationhas nine4. The best-documented example of an MIP ship between MIP channels9,10, although it has been channel that transports water is AQP2, which is re- proposed recently that glycerol channels could be quired for urine concentration in the mammalian kid- functionally monomeric11. ney5. In A. thaliana, antisense RNA studies have demonTransport studies using MIP channels expressed in strated the effects of aquaporin function on root Xenopus oocytes (Box 1) and detailed sequence comdevelopment3,6. These few examples hint at the parisons have led to the classification of MIP chansignificant impact MIP channel function can have on nels into two major categories: (1) aquaporins sensu diverse biological processes. Recently, MIP channels stricto, which are highly specific for water and (2) that transport water and/or uncharged solutes have glycerol facilitators, which transport glycerol and also been found in many pro- and eukaryotic micro- possibly other solutes in addition to, or even in organisms. The elucidation of their specific roles pro- preference to, water2,12. However, the molecular devides a considerable challenge to investigators in this terminants that influence this specificity are not well emerging field. understood. 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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Repeat 2
Repeat 1
E A
C
Extracellular 1
3
2
5 6
4
Intracellular
D
NH2
B
NPA boxes Conserved between repeats
COOH
Conserved throughout the family Residues influencing specificity trends in Microbiology
Fig. 1. Predicted topology for members of the MIP family. MIP channel genes are believed to be the products of an ancient duplication event as a number of residues are conserved between the two repeats. Moreover, there are a number of residues which, despite not being conserved between the repeats, are conserved within the MIP family. The group of residues highlighted with an asterisk are predicted to be involved in the determination of whether a MIP channel is an aquaporin or a glycerol facilitator. Froger and co-workers12 have shown that the two subgroups of the MIP family can be very clearly distinguished according to these five residues and that a switch from an aquaporin to an glycerol facilitator can be achieved by substitution of these amino acid residues11. The transmembrane domains are numbered 1–6 and the loops lettered A–E.
As for other channel-protein families, the genes encoding MIP channels are apparently the products of an ancestral duplication event. The amino-terminal half of the encoded protein is thought to be associated with general functions such as membrane localization and pore formation, whereas the carboxy-terminal half could determine substrate specificity8,13. Indeed, in the sixth transmembrane domain of the insect water channel AQPcic, substitution of Tyr and Trp by Pro and Leu, respectively, leads to a switch in channel selectivity from water to glycerol11,12. Microbial MIP channels Table 1 lists the protein and/or gene sequence accession numbers for microbial MIP channels that were deposited in the publicly accessible databases up to October 1999. It can be seen that glycerol facilitators occur in bacteria from all subgroups, whereas aquaporins are found in some Gram-negative bacteria and two archaeal species. To date, Enterococcus faecalis is the only Gram-positive bacterium in which an aquaporin has been found. Although the reason for this is unclear, it could be that some glycerol facilitators are also able to transport water, albeit with lower permeability than bona fide aquaporins, and hence could fulfil both functions in Gram-positive bacteria. There are some microorganisms that apparently lack MIP
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channels altogether, such as the archaea Methanococcus jannaschii and Pyrococcus horikoshii and the bacteria Aquifex aeolicus, Helicobacter pylori, Mycobacterium tuberculosis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Rickettsia prowazekii and Campylobacter jejuni. These organisms include animal pathogens and deep-sea inhabitants. It is possible that, in such environments, microorganisms do not experience significant changes in osmolarity and thus facilitated water or solute transport through MIP channels is not required. Alternatively, other currently undefined mechanisms for water or solute transport might exist in these organisms. Remarkably, no MIP-encoding genes have yet been detected in the fission yeast Schizosaccharomyces pombe from the .5500 loci sequenced to date; the physiological significance of this is unclear. Phylogenetic analysis of the sequences listed in Table 1 confirms their division into aquaporins and glycerol facilitators8,13. Within these major clusters, the glycerol facilitators from Gram-positive and Gram-negative bacteria form distinct groups, indicating that these proteins were present when the two bacterial classes separated. The propanediol facilitator of Salmonella typhimurium clusters with the glycerol faciliators of Gram-negative bacteria, and the fungal glycerol facilitators form another distinct cluster. The aquaporins, however, do not show such clear clustering according to different organism classes, suggesting that gene transfer might have contributed to their evolution. For example, the aquaporin from the photosynthetic bacterium Chlorobium tepidum is more closely related to eukaryotic aquaporins than to prokaryotic aquaporins. The two MIP channels from Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum are closely related and cluster with aquaporins, indicating that the initial classification of the A. fulgidus protein as a glycerol facilitator might be incorrect. Uptake by microbial glycerol facilitators Glycerol and other uncharged small molecules are able to cross microbial membranes by simple diffusion. Additionally, glycerol is taken up via facilitated diffusion mediated by MIP channels such as GlpF (Ref. 14). glpF in both E. coli and Pseudomonas aeruginosa forms part of an operon composed of the promoter-proximal glpF, glpK and glpX. glpK and glpX encode a glycerol kinase and a putative transcription factor, respectively15,16. The glycerol kinase appears to be closely associated with the facilitator, resulting in glycerol phosphorylation during uptake17. This operon organization is well conserved in both Gram-positive and Gram-negative bacteria, providing additional evidence for a role of GlpF proteins in glycerol utilization. In addition, mutants defective in glpF show perturbed utilization of glycerol at low concentrations. S. typhimurium PduF is associated with the pdu operon, which suggests its involvement in propanediol uptake and metabolism18. Saccharomyces cerevisiae Fps1p mediates measurable uptake of glycerol into yeast cells by facilitated
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diffusion19–22 (Box 1). However, no growth defects have been found in S. cerevisiae fps1 mutants cultivated on glycerol as a sole carbon source22. As outlined below, the main transport function of Fps1p appears to be glycerol export. Interestingly, mutation of glpF in E. coli and deletion of FPS1 in S. cerevisiae reduces passive diffusion of glycerol and influences the cellular lipid composition20,23. Hence, these proteins could play a role in controlling the availability of glycerol-3-phosphate, a precursor of phospholipid biosynthesis24. GlpF and Fps1p mediate transport of polyols, glyceraldehyde, glycine and urea, suggesting low specificity (Ref. 14; H. Li and S.G. Kilian, unpublished), whereas only insignificant water transport has been observed through either protein25,26. The low transport specificity of these channels, together with the apparently higher rate for efflux than uptake14,22, suggests that gating mechanisms might control the loss of low Mr metabolites from the cell. Glycerol export So far, S. cerevisiae Fps1p is unique among the glycerol facilitators for a number of reasons. First, neither of the NPA motifs in loops B and E is fully preserved, changes that could be compensated for by other residues. Second, Fps1p has long amino- and carboxyterminal hydrophilic extensions, resulting in a protein of 669 amino acids, compared with 281 amino acids for E. coli GlpF. Third, Fps1p controls the export of glycerol produced by yeast cells19,22 (Box 1): glycerol accumulated during growth in high-osmolarity medium (Box 2) is released upon hypo-osmotic shock in an Fps1p-dependent fashion19,22, and mutants lacking Fps1p recover poorly from hypo-osmotic shock22. Fourth, Fps1p is involved in cell fusion during yeast mating. This process requires local degradation of the cell wall, which could lead to bursting if turgor is not released27. Finally, Fps1p affects signal transduction in yeast osmoregulation (Box 2) in a manner consistent with its role in controlling the intracellular glycerol content28. These observations establish Fps1p as primarily a glycerol exporter involved in osmoregulation. Export pathways for osmolytes exist in bacterial cells and also cells of higher organisms, but apart from Fps1p and E. coli MscL/MscS29,30 (the latter are not MIP channels), no such export pathway has been characterized at the molecular level. The role of Fps1p in an osmolyte export pathway suggests that other MIP channels might have similar functions. Fps1p-dependent glycerol export from yeast cells is regulated19,22. The mechanisms triggering rapid channel closing following hyperosmotic shock and opening following hypo-osmotic shock are unknown. The involvement of several candidate protein kinases has been excluded and there is no evidence for the involvement of membrane stretching. However, a regulatory domain required for gating has been identified within the amino-terminal hydrophilic extension. Deletion of this 20-amino-acid domain results in loss of glycerol from the cell and poor growth in highosmolarity medium19,22.
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Box 1. Evaluating the transport function of MIP channels Transport by MIP channels is frequently studied using Xenopus oocytes. This heterologous system is used to demonstrate that the observed transport is truly mediated by the expressed protein. Homologous systems are used to show that the predicted transport also occurs in that organism; for this purpose, a mutant lacking a specific MIP channel is required to compare with the corresponding wild-type organism. The Xenopus oocyte heterologous expression system • Oocytes are isolated from Xenopus (which survive this procedure and are used repeatedly). • The gene encoding the MIP channel of interest is transcribed in vitro. • The mRNA is injected into oocytes; the amount of protein produced by the oocytes is proportional to the amount of mRNA injecteda. After three days the oocytes are used for transport studies. • Water transport is studied by monitoring the swelling and shrinking of oocytes (determined spectroscopically) in response to changes in the osmolarity of the incubation buffera. The rate of swelling and shrinking provides a measure of water transport and can be used to calculate single-channel conductance provided the number of water channels can be estimated, for example by counting particles in a freeze-fracture electron micrograph of the oocyte membraneb. • Transport of glycerol and other solutes can be studied by monitoring the uptake of a relevant radiolabelled compoundc. Transport studies in a homologous system • Water transport is measured by monitoring osmotic swelling in whole cellsd, spheroplastsb or membrane vesiclese. Swelling is followed by light scattering or the use of a concentrationdependent fluorescent dyef, procedures that also allow quantification, or by cryoelectron microscopyg. • The uptake of solutes such as glycerol into cells is monitored quantitatively using the relevant radiolabelled compoundh. • The release of solutes such as glycerol is measured by determining the intracellular and/or extracellular concentration of that compound, usually by biochemical methodsi,j. References a Preston, G.M. et al. (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP 28 protein. Science 256, 385–387 b Laizé, V. et al. (1999) Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain. Biochem. Biophys. Res. Commun. 257, 139–144 c Maurel, C. et al. (1994) Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. J. Biol. Chem. 269, 11869–11872 d Roudier, N. et al. (1998) Evidence for the presence of aquaporin-3 in human red blood cells. J. Biol. Chem. 273, 8407–8412 e Zhang, R. et al. (1993) Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28. J. Cell Biol. 120, 359–369 f Chang, A. et al. (1994) Permeability properties of the mammalian bladder apical membrane. Am. J. Physiol. 267, C1483–C1492 g Delamarche, C. et al. (1999) Visualization of AqpZ-mediated water permeability in Escherichia coli by cryoelectron microscopy. J. Bacteriol. 181, 4193–4197 h Sutherland, F.C.W. et al. (1997) Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae. J. Bacteriol. 179, 7790–7795 i Luyten, K. et al. (1995) Fps1, a yeast member of the MIP-family of channel proteins, is a facilitator for glycerol uptake and efflux and it is inactive under osmotic stress. EMBO J. 14, 1360–1371 j Tamás, M.J. et al. (1999) Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol. Microbiol. 31, 1087–1104
Glycerol is also produced by S. cerevisiae cells under anaerobic conditions to dispose of excess NADH in redox regulation31–33. Mutants lacking
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Table 1. Microbial MIP-family channel proteinsa Organism
Gene
Acc. No.
Predicted function
Amino acids
C5f02a1.r1b CNS01A8X Stanford 5476 wacA aqpA NCSM1G3T3b FPS1d YFL054 AQY1-1f AQY1-2 f AQY2 f RPCI93g
AA783486 AL112633 Contig4-2389 U68246 AB032841 AI392589 P23900 P43549 AAC69713 P53386 AAD25168 AQ641778
ND GlpF AQP AQP AQP AQP GlpFe GlpF AQPe AQP AQP GlpF
118c 239c 273 277 279 195c 669 646 327 305 289 160c
glpF GTC 1488 glpF TIGR 1299 TIGR 1351 TIGR 1351d TIGR 1351 ydp1 TIGR 1280 glpF TIGR 1313 TIGR 1313 OUACGT 1315 OUACGT 1315 gylA glpF
P18156 AE001437 X86492 8796 gef 6204 gef 6176 gef 6403 P22094 4410 U12567 sp 42 sp 16 Contig115 Contig104 P19255 AAD36499
GlpFe GlpF GlpF GlpF GlpF GlpF AQP GlpF1AQPe GlpF GlpF GlpF ND GlpF GlpF GlpF GlpF
274 242 164c 271 239 236 233 289 272 233 289 269c 233 282 80c 234
Yersinia pestis
glpF Sanger 518 TIGR 1097d glpF aqpZ glpF glpF glpF glpF glpF glpF CBCUMN 747 ORF10P glpF glpF pduF WUGSC 99287 TIGR 24 glpF aqpZ Stanford 382 smpX aqpZ TIGR 920 TIGR 666 TIGR 666 Sanger 632
AAC66629 Contig 2552 gct 5 P11244 U38664 P44826 U32782 Z33098 P52280 P47279 P75071 Contig341 AB025970 Q51389 AB015973 P37451 Contig79 4323 P31140 AAC12651 423050H01 D43774 BAA17863 1538 asm814 1741 Contig630
GlpF AQP AQP GlpFe AQPe GlpFe GlpF GlpF GlpF GlpF GlpF GlpF AQP GlpFe GlpF PduFe GlpF AQP GlpFe AQP ND GlpF AQP ND GlpF GlpF GlpF
254 236 268 281 231 264 213c 89c 205c 258 264 261 233 279 285 264 288 231 281 231 154c 269 247 144c 261 285 282
Archaea Archaeoglobus fulgidus Methanobacterium thermoautotrophicum
glpF aqp
AAB89820 AAB84602
GlpF AQP
246 246
Eukarya Aspergillus nidulans Botrytis cinerea Candida albicans Dictyostelium discoideum
Neurospora crassa Saccharomyces cerevisiae
Trypanasoma brucei Gram-positive Bacteria Bacillus subtilis Clostridium acetobutylicum Clostridium perfringens Deinococcus radiodurans Enterococcus faecalis
Lactococcus lactis Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Streptomyces coelicolor Thermotoga maritima Gram-negative Bacteria Borrelia burgdorferi Bordetella bronchiseptica Chlorobium tepidum Escherichia coli
Haemophilus influenzae Mycoplasma capricolum Mycoplasma gallisepticum Mycoplasma genitalium Mycoplasma pneumoniae Pasteurella multocida Plesiomonas shigelloides Pseudomonas aeruginosa Pseudomonas tolaasii Salmonella typhimurium Shewanella putrefaciens Shigella flexneri Sinorhizobium meliloti Synechococcus sp. PCC7942 Synechocystis sp. PCC6803 Thiobacillus ferrooxidans Vibrio cholerae
a
Abbreviations: Acc. No., accession number; AQP, aquaporin; GlpF, glycerol facilitator/transporter; ND, not determined; PduF, propanediol facilitator. bDeduced from mRNA sequence. cIncomplete sequence obtained from GenBank. dOne or both NPA motifs have a different sequence. e Function confirmed experimentally. fOnly in certain strains. gDeduced from genome survey sequence. Sequences were retrieved from the SwissProt, EMBL, GenBank and PROSITE databases. Preliminary sequence data were obtained from The Institute for Genomic Research (http://www.tigr.org) and the Sanger Centre (http://www.sanger.ac.uk).
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Fps1p accumulate glycerol to high levels under anaerobic conditions and grow poorly, presumably owing to unfavourably high turgor22. Under these growth conditions, Fps1p apparently serves as an export pathway for a metabolic waste product. In fact, MIP channels such as Fps1p could play a role in the control of metabolism by facilitating the exit of metabolic end products along a concentration gradient, but it is unknown if this is a general feature. Microbial aquaporins E. coli AqpZ (Ref. 34) and S. cerevisiae Aqy1p (Refs 35,36) can mediate water transport in Xenopus oocytes (Box 1). Additionally, it has been demonstrated by cryoelectron microscopy of osmotically shocked E. coli cells that AqpZ mediates water transport into and out of the cell37. Similarly, functional aquaporins have been detected in S. cerevisiae by observing spheroplast swelling36. The growth conditions under which microbial cells need water channels, despite their high surface-to-volume ratio and the relatively rapid passive diffusion rate of water through the lipid bilayer, are yet to be elucidated. E. coli aqpZ mutants appear to grow more poorly on low-osmolarity medium34. As turgor pressure is probably high under these conditions, AqpZ might be important for the export of water. This would be consistent with the observed stimulated expression of aqpZ during growth in low-osmolarity medium34. Whether AqpZ plays a role in water homeostasis throughout growth or only in specific stages of the cell cycle is not known. Bakers’ yeast has two genes that potentially encode aquaporins, AQY1 and AQY2, which are .80% identical35,36. Curiously, most laboratory strains possess irrevertable mutations in both these genes35. Only strains recovered from natural or industrial environments appear to possess a functional Aqy1p (Refs 35,36). It therefore appears that laboratory conditions might be disadvantageous to cells possessing functional aquaporins, suggesting that they play a role only under very specific conditions35. Elevated expression of AQY1 during sporulation hints at a role for aquaporins in spore formation and/or germination, but as yet there is no evidence for this. Conclusions and perspectives In combination with a specific lipid composition, which limits the passive diffusion of water and/or glycerol, MIP channels enable the cell to control the passage of these compounds through cellular membranes. MIP channels might also enable the cell to coordinate transport with other processes, such as the phosphorylation of glycerol or signalling osmotic changes. In addition, the control of MIP-channel function, expression or localization provides the cell with a means to regulate transmembrane water and solute flux according to its physiological needs. Such regulation has been demonstrated in the case of certain aquaporins from mammals5, plants3 and for yeast Fps1p (Refs 19,22), and is important for the control of cellular and whole-organism osmoregulation, and
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Box 2. Osmoregulation: compatible solutes The control of relative cellular water content is extremely important to ensure proper functioning of biochemical processes and constant turgor pressure, which in turn is relevant for the morphology and shape of cells and organisms. Consequently, osmoregulation is complex and includes many processes, some of which are conserved in evolution, whereas others are a reflection of the specific environment of an organism. A common strategy is the accumulation of compatible solutes to adjust the intracellular water activity without disturbing biochemical processesa. Compatible solutes can be ions, sugars, sugar alcohols or amino acids or derivatives thereof. Such solutes can be actively accumulated from the environment or can be produced by the cell. For example, Escherichia coli uses potassium ions, trehalose and glycine-betaine as compatible solutesb, whereas Saccharomyces cerevisiae uses glycerolc,d. Once the osmolarity of the surrounding environment drops, the cell has to decrease its solute content, for example by specifically releasing compatible solutes into the mediume. Regulation of solute accumulation occurs at the level of gene expression, leading to changes in metabolic capacity and the modulation of transport activity. There is a substantial research interest in the mechanisms of sensing changes in the osmolarity of the cellular environment and the signal transduction pathways that lead to the observed responsesf,g. References a Yancey, P.H. et al. (1982) Living with water stress: evolution of osmolyte systems. Science 217, 1214–1222 b Kempf, B. and Bremer, E. (1998) Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolarity environments. Arch. Microbiol. 170, 319–330 c Hohmann, S. (1997) Shaping up: the response of yeast to osmotic stress. In Yeast Stress Responses (Hohmann, S. and Mager, W.H., eds), pp. 101–145, R.G. Landes Co. d Nevoigt, E. and Stahl, U. (1997) Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 21, 231–241 e Booth, I.R. and Louis, P. (1999) Managing hypo-osmotic stress: aquaporins and mechanosensitive channels in Escherichia coli. Curr. Opin. Microbiol. 2, 166–169 f Wood, J.M. (1999) Osmosensing by bacteria: signals and membrane-based sensors. Microbiol. Mol. Biol. Rev. 63, 230–262 g Gustin, M.C. et al. (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62, 1264–1300
Questions for future research • What are the structural features of aquaporins and glycerol facilitators that determine transport specificity? • When is aquaporin-mediated water transport relevant for microorganisms? Why do some microorganisms have water channels and not others? • Is the export of solutes and/or metabolic end products a feature common to many glycerol facilitators? • How common is regulation of MIP channel function and what are the specific mechanisms of this regulation? • To what extent can microorganisms be employed as useful models to study MIP channel function? • Can MIP channels be exploited, for example for the engineering of the performance of microorganisms under stressful industrial conditions and/or engineering metabolism towards specific metabolic end products?
for metabolism. Therefore, an understanding of the molecular details of MIP channel regulation is an important goal for future analysis. MIP channels appear to affect a wide range of processes via their roles in osmoregulation and
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metabolism, and although much effort has focused on water transport, many MIP channels could control the export of compatible solutes and/or metabolic waste products. As more biological roles of MIP channels are being discovered, their potential in medicine, pharmacology, agrobiotechnology and metabolic engineering is also becoming clear. The availability of genetically tractable microbial systems for the study of MIP-channel function and regulation will contribute to the exploitation of the special characteristics of this exciting protein family. Acknowledgements We thank P. Agre, G. Calamita, A. Engel, D. Thomas, E. Bremer, R. Krämer and S.G. Kilian for communicating unpublished results and manuscripts in press, and V. Laizé for help with Fig. 1. Work in the laboratory of S.H. is supported by the Commission of the European Union via contracts FMRX-CT97-0128 and BIO4-CT98-0024. B.A.P. acknowledges support from the National Research Foundation, South Africa. References 1 Preston, G.M. et al. (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP 28 protein. Science 256, 385–387 2 Borgnia, M. et al. (1999) Cellular and molecular biology of aquaporin water channels. Annu. Rev. Biochem. 68, 425–458 3 Kjellbom, P. et al. (1999) Aquaporins and water homeostasis in plants. Trends Plant Sci. 4, 308–314 4 Kuwahara, M. et al. (1998) A water channel of the nematode C. elegans and its implications for channel selectivity of MIP proteins. Am. J. Physiol. 275, C1459–C1464 5 Deen, P.M.T. and van Knoers, N. (1998) Physiology and pathophysiology of the aquaporin-2 water channel. Curr. Opin. Nephrol. Hyperten. 7, 37–42 6 Kaldenhoff, R. et al. (1998) Significance of plasmalemma aquaporins for water-transport in Arabidopsis thaliana. Plant J. 14, 121–128 7 Heymann, J.B. et al. (1998) Progress on the structure and function of aquaporin 1. J. Struct. Biol. 121, 191–206 8 Park, J.H. and Saier, M.H. (1996) Phylogenetic characterization of the MIP family of transmembrane channel proteins. J. Membr. Biol. 153, 171–180 9 Hasler, L. et al. (1998) Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution. J. Mol. Biol. 279, 855–864 10 Ringler, P. et al. (1999) Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography. J. Mol. Biol. 291, 1181–1190 11 Lagree, V. et al. (1999) Switch from an aquaporin to a glycerol channel by two amino acids substitution. J. Biol. Chem. 274, 6817–6819 12 Froger, A. et al. (1998) Prediction of functional residues in water channels and related proteins. Protein Sci. 7, 1458–1468 13 Reizer, J. et al. (1993) The MIP family of integral membrane channel proteins: sequence comparison, evolutionary relationship, reconstructed pathway of evolution and proposed functional differentiation of the two repeated halves of the proteins. Crit. Rev. Biochem. Mol. Biol. 28, 235–257 14 Heller, K.B. et al. (1980) Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli. J. Bacteriol. 144, 274–278 15 Truniger, V. et al. (1992) Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri. J. Bacteriol. 174, 6981–6991 16 Schweizer, H.P. et al. (1997) Structure and gene-polypeptide
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17 18 19
20 21 22 23 24 25 26 27 28 29
30 31
32 33 34 35 36 37
relationships of the region encoding glycerol diffusion facilitator (glpF) and glycerol kinase (glpK) of Pseudomonas aeruginosa. Microbiology 143, 1287–1297 Voegele, R.T. et al. (1993) Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator. J. Bacteriol. 175, 1087–1094 Chen, P. et al. (1994) The control region of the pdu/cob regulon in Salmonella typhimurium. J. Bacteriol. 176, 5474–5482 Luyten, K. et al. (1995) Fps1, a yeast member of the MIP-family of channel proteins, is a facilitator for glycerol uptake and efflux and it is inactive under osmotic stress. EMBO J. 14, 1360–1371 Sutherland, F.C.W. et al. (1997) Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae. J. Bacteriol. 179, 7790–7795 Lages, F. and Lucas, C. (1997) Contribution to the physiological characterization of glycerol active uptake in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1322, 8–18 Tamás, M.J. et al. (1999) Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol. Microbiol. 31, 1087–1104 Truniger, V. and Boos, W. (1993) Glycerol uptake in Escherichia coli is sensitive to membrane lipid composition. Res. Microbiol. 144, 565–574 Daum, G. et al. (1998) Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14, 1471–1510 Maurel, C. et al. (1994) Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. J. Biol. Chem. 269, 11869–11872 Coury, L.A. et al. (1999) Water transport across yeast vacuolar and plasma membrane-targeted secretory vesicles occurs by passive diffusion. J. Bacteriol. 181, 4437–4440 Philips, J. and Herskowitz, I. (1997) Osmotic balance regulates cell fusion during mating in Saccharomyces cerevisiae. J. Cell Biol. 138, 961–974 Tao, W. et al. (1999) Intracellular glycerol levels modulate the activity of Sln1p, a Saccharomyces cerevisiae two-component regulator. J. Biol. Chem. 274, 360–367 Levina, N. et al. (1999) Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 18, 1730–1737 Booth, I.R. and Louis, P. (1999) Managing hypo-osmotic stress: aquaporins and mechanosensitive channels in Escherichia coli. Curr. Opin. Microbiol. 2, 166–169 Ansell, R. et al. (1997) The two isoenzymes for yeast NADdependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2, have distinct roles in osmoadaption and redox regulation. EMBO J. 16, 2179–2187 Hohmann, S. (1997) Shaping up: the response of yeast to osmotic stress. In Yeast Stress Responses (Hohmann, S. and Mager, W.H., eds), pp. 101–145, R.G. Landes Co. Nevoigt, E. and Stahl, U. (1997) Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 21, 231–241 Calamita, G. et al. (1998) Regulation of the Escherichia coli water channel gene aqpZ. Proc. Natl. Acad. Sci. U. S. A. 95, 3627–3631 Bonhivers, M. et al. (1998) Aquaporins in Saccharomyces. Genetic and functional distinctions between laboratory and wild-type strains. J. Biol. Chem. 273, 27565–27572 Laizé, V. et al. (1999) Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain. Biochem. Biophys. Res. Commun. 257, 139–144 Delamarche, C. et al. (1999) Visualization of AqpZ-mediated water permeability in Escherichia coli by cryoelectron microscopy. J. Bacteriol. 181, 4193–4197
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24 Norris, F.A. et al. (1998) SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase. Proc. Natl. Acad. Sci. U. S. A. 95, 14057–14059 25 Hong, K.H. and Miller, V.L. (1998) Identification of a novel Salmonella invasion locus homologous to Shigella IpgDE.
J. Bacteriol. 180, 1793–1802 26 Eckmann, L. et al. (1997) D-myo-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signalling pathways. Proc. Natl. Acad. Sci. U. S. A. 94, 14456–14460
Microbial MIP channels Stefan Hohmann, Roslyn M. Bill, Gerald Kayingo and Bernard A. Prior
I
t has been known for many MIP channels occur in all classes of Structure–function years that the water permeorganism ranging from bacteria to man. relationships and transport ability of specialized bioThere are two major categories of MIP specificity logical membranes is much channels, aquaporins and glycerol All MIP channels share highly higher than that of artificial facilitators, which facilitate the diffusion conserved residues and are lipid bilayers. At the beginning across biological membranes of water or predicted to have six transof the 1990s, the first bona glycerol and other uncharged compounds, membrane domains (Fig. 1). fide water channel was characrespectively. As a result of their Residues present in loops B terized from red blood cells1,2. involvement in osmoregulation and and E comprise the family’s This protein, now known as metabolism, MIP channels are believed to signature sequences: Ser-Glyaquaporin 1 (AQP1), was affect a wide range of biological processes. X-His-X-Asn-Pro-Ala-Val-Thr found to be related to MIP, the (where X 5 any amino acid) S. Hohmann* and R.M. Bill are in the Dept of Cell major intrinsic protein of and Asn-Pro-Ala-Arg, respecand Molecular Biology/Microbiology, Göteborg mammalian lens fibre; to TIPs, tively; each contains a canoniUniversity, Box 462, S-40530 Göteborg, Sweden; the tonoplast intrinsic proteins cal NPA (Asn-Pro-Ala) box. G. Kayingo and B.A. Prior are in the Dept of from plants; and to GlpF, These loops are believed to ‘dip Microbiology, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa. the glycerol facilitator from into’ the membrane bilayer, *tel: 146 31 773 2595, Escherichia coli. The protein forming a continuous solute fax: 146 31 773 2599, family was eponymously named channel (the so-called houre-mail: [email protected] MIP, although the major inglass), lined by a chain of hytrinsic protein of mammalian drogen-bonding amino acids7. lens fibre has since been renamed AQP0. To date, To date, structural data (at 6Å) are only available more than 200 members of the MIP family have been for AQP1 (Ref. 7). Even though the most divergent identified. In fact, higher organisms possess an amaz- members of the MIP family are less than 20% identiing number of isoforms, which are expressed in dif- cal8, it is expected that the gross structural features of ferent subcellular compartments and tissues under AQP1 will also be present in other water and glycerol different environmental conditions or during different channels. For example, AQP1 is functionally homo developmental stages. For example, ten genes encod- tetrameric, a quaternary structure which is therefore ing MIP channels are currently known in humans2, anticipated for all other MIPs. Comparison of the 30 have been described in the plant Arabidopsis crystal structure of MIP with that of E. coli AqpZ and thaliana3, and the nematode Caenorhabditis elegans GlpF confirms the close overall structural relationhas nine4. The best-documented example of an MIP ship between MIP channels9,10, although it has been channel that transports water is AQP2, which is re- proposed recently that glycerol channels could be quired for urine concentration in the mammalian kid- functionally monomeric11. ney5. In A. thaliana, antisense RNA studies have demonTransport studies using MIP channels expressed in strated the effects of aquaporin function on root Xenopus oocytes (Box 1) and detailed sequence comdevelopment3,6. These few examples hint at the parisons have led to the classification of MIP chansignificant impact MIP channel function can have on nels into two major categories: (1) aquaporins sensu diverse biological processes. Recently, MIP channels stricto, which are highly specific for water and (2) that transport water and/or uncharged solutes have glycerol facilitators, which transport glycerol and also been found in many pro- and eukaryotic micro- possibly other solutes in addition to, or even in organisms. The elucidation of their specific roles pro- preference to, water2,12. However, the molecular devides a considerable challenge to investigators in this terminants that influence this specificity are not well emerging field. understood. 0966-842X/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. TRENDS
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Repeat 2
Repeat 1
E A
C
Extracellular 1
3
2
5 6
4
Intracellular
D
NH2
B
NPA boxes Conserved between repeats
COOH
Conserved throughout the family Residues influencing specificity trends in Microbiology
Fig. 1. Predicted topology for members of the MIP family. MIP channel genes are believed to be the products of an ancient duplication event as a number of residues are conserved between the two repeats. Moreover, there are a number of residues which, despite not being conserved between the repeats, are conserved within the MIP family. The group of residues highlighted with an asterisk are predicted to be involved in the determination of whether a MIP channel is an aquaporin or a glycerol facilitator. Froger and co-workers12 have shown that the two subgroups of the MIP family can be very clearly distinguished according to these five residues and that a switch from an aquaporin to an glycerol facilitator can be achieved by substitution of these amino acid residues11. The transmembrane domains are numbered 1–6 and the loops lettered A–E.
As for other channel-protein families, the genes encoding MIP channels are apparently the products of an ancestral duplication event. The amino-terminal half of the encoded protein is thought to be associated with general functions such as membrane localization and pore formation, whereas the carboxy-terminal half could determine substrate specificity8,13. Indeed, in the sixth transmembrane domain of the insect water channel AQPcic, substitution of Tyr and Trp by Pro and Leu, respectively, leads to a switch in channel selectivity from water to glycerol11,12. Microbial MIP channels Table 1 lists the protein and/or gene sequence accession numbers for microbial MIP channels that were deposited in the publicly accessible databases up to October 1999. It can be seen that glycerol facilitators occur in bacteria from all subgroups, whereas aquaporins are found in some Gram-negative bacteria and two archaeal species. To date, Enterococcus faecalis is the only Gram-positive bacterium in which an aquaporin has been found. Although the reason for this is unclear, it could be that some glycerol facilitators are also able to transport water, albeit with lower permeability than bona fide aquaporins, and hence could fulfil both functions in Gram-positive bacteria. There are some microorganisms that apparently lack MIP
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channels altogether, such as the archaea Methanococcus jannaschii and Pyrococcus horikoshii and the bacteria Aquifex aeolicus, Helicobacter pylori, Mycobacterium tuberculosis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Rickettsia prowazekii and Campylobacter jejuni. These organisms include animal pathogens and deep-sea inhabitants. It is possible that, in such environments, microorganisms do not experience significant changes in osmolarity and thus facilitated water or solute transport through MIP channels is not required. Alternatively, other currently undefined mechanisms for water or solute transport might exist in these organisms. Remarkably, no MIP-encoding genes have yet been detected in the fission yeast Schizosaccharomyces pombe from the .5500 loci sequenced to date; the physiological significance of this is unclear. Phylogenetic analysis of the sequences listed in Table 1 confirms their division into aquaporins and glycerol facilitators8,13. Within these major clusters, the glycerol facilitators from Gram-positive and Gram-negative bacteria form distinct groups, indicating that these proteins were present when the two bacterial classes separated. The propanediol facilitator of Salmonella typhimurium clusters with the glycerol faciliators of Gram-negative bacteria, and the fungal glycerol facilitators form another distinct cluster. The aquaporins, however, do not show such clear clustering according to different organism classes, suggesting that gene transfer might have contributed to their evolution. For example, the aquaporin from the photosynthetic bacterium Chlorobium tepidum is more closely related to eukaryotic aquaporins than to prokaryotic aquaporins. The two MIP channels from Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum are closely related and cluster with aquaporins, indicating that the initial classification of the A. fulgidus protein as a glycerol facilitator might be incorrect. Uptake by microbial glycerol facilitators Glycerol and other uncharged small molecules are able to cross microbial membranes by simple diffusion. Additionally, glycerol is taken up via facilitated diffusion mediated by MIP channels such as GlpF (Ref. 14). glpF in both E. coli and Pseudomonas aeruginosa forms part of an operon composed of the promoter-proximal glpF, glpK and glpX. glpK and glpX encode a glycerol kinase and a putative transcription factor, respectively15,16. The glycerol kinase appears to be closely associated with the facilitator, resulting in glycerol phosphorylation during uptake17. This operon organization is well conserved in both Gram-positive and Gram-negative bacteria, providing additional evidence for a role of GlpF proteins in glycerol utilization. In addition, mutants defective in glpF show perturbed utilization of glycerol at low concentrations. S. typhimurium PduF is associated with the pdu operon, which suggests its involvement in propanediol uptake and metabolism18. Saccharomyces cerevisiae Fps1p mediates measurable uptake of glycerol into yeast cells by facilitated
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diffusion19–22 (Box 1). However, no growth defects have been found in S. cerevisiae fps1 mutants cultivated on glycerol as a sole carbon source22. As outlined below, the main transport function of Fps1p appears to be glycerol export. Interestingly, mutation of glpF in E. coli and deletion of FPS1 in S. cerevisiae reduces passive diffusion of glycerol and influences the cellular lipid composition20,23. Hence, these proteins could play a role in controlling the availability of glycerol-3-phosphate, a precursor of phospholipid biosynthesis24. GlpF and Fps1p mediate transport of polyols, glyceraldehyde, glycine and urea, suggesting low specificity (Ref. 14; H. Li and S.G. Kilian, unpublished), whereas only insignificant water transport has been observed through either protein25,26. The low transport specificity of these channels, together with the apparently higher rate for efflux than uptake14,22, suggests that gating mechanisms might control the loss of low Mr metabolites from the cell. Glycerol export So far, S. cerevisiae Fps1p is unique among the glycerol facilitators for a number of reasons. First, neither of the NPA motifs in loops B and E is fully preserved, changes that could be compensated for by other residues. Second, Fps1p has long amino- and carboxyterminal hydrophilic extensions, resulting in a protein of 669 amino acids, compared with 281 amino acids for E. coli GlpF. Third, Fps1p controls the export of glycerol produced by yeast cells19,22 (Box 1): glycerol accumulated during growth in high-osmolarity medium (Box 2) is released upon hypo-osmotic shock in an Fps1p-dependent fashion19,22, and mutants lacking Fps1p recover poorly from hypo-osmotic shock22. Fourth, Fps1p is involved in cell fusion during yeast mating. This process requires local degradation of the cell wall, which could lead to bursting if turgor is not released27. Finally, Fps1p affects signal transduction in yeast osmoregulation (Box 2) in a manner consistent with its role in controlling the intracellular glycerol content28. These observations establish Fps1p as primarily a glycerol exporter involved in osmoregulation. Export pathways for osmolytes exist in bacterial cells and also cells of higher organisms, but apart from Fps1p and E. coli MscL/MscS29,30 (the latter are not MIP channels), no such export pathway has been characterized at the molecular level. The role of Fps1p in an osmolyte export pathway suggests that other MIP channels might have similar functions. Fps1p-dependent glycerol export from yeast cells is regulated19,22. The mechanisms triggering rapid channel closing following hyperosmotic shock and opening following hypo-osmotic shock are unknown. The involvement of several candidate protein kinases has been excluded and there is no evidence for the involvement of membrane stretching. However, a regulatory domain required for gating has been identified within the amino-terminal hydrophilic extension. Deletion of this 20-amino-acid domain results in loss of glycerol from the cell and poor growth in highosmolarity medium19,22.
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Box 1. Evaluating the transport function of MIP channels Transport by MIP channels is frequently studied using Xenopus oocytes. This heterologous system is used to demonstrate that the observed transport is truly mediated by the expressed protein. Homologous systems are used to show that the predicted transport also occurs in that organism; for this purpose, a mutant lacking a specific MIP channel is required to compare with the corresponding wild-type organism. The Xenopus oocyte heterologous expression system • Oocytes are isolated from Xenopus (which survive this procedure and are used repeatedly). • The gene encoding the MIP channel of interest is transcribed in vitro. • The mRNA is injected into oocytes; the amount of protein produced by the oocytes is proportional to the amount of mRNA injecteda. After three days the oocytes are used for transport studies. • Water transport is studied by monitoring the swelling and shrinking of oocytes (determined spectroscopically) in response to changes in the osmolarity of the incubation buffera. The rate of swelling and shrinking provides a measure of water transport and can be used to calculate single-channel conductance provided the number of water channels can be estimated, for example by counting particles in a freeze-fracture electron micrograph of the oocyte membraneb. • Transport of glycerol and other solutes can be studied by monitoring the uptake of a relevant radiolabelled compoundc. Transport studies in a homologous system • Water transport is measured by monitoring osmotic swelling in whole cellsd, spheroplastsb or membrane vesiclese. Swelling is followed by light scattering or the use of a concentrationdependent fluorescent dyef, procedures that also allow quantification, or by cryoelectron microscopyg. • The uptake of solutes such as glycerol into cells is monitored quantitatively using the relevant radiolabelled compoundh. • The release of solutes such as glycerol is measured by determining the intracellular and/or extracellular concentration of that compound, usually by biochemical methodsi,j. References a Preston, G.M. et al. (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP 28 protein. Science 256, 385–387 b Laizé, V. et al. (1999) Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain. Biochem. Biophys. Res. Commun. 257, 139–144 c Maurel, C. et al. (1994) Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. J. Biol. Chem. 269, 11869–11872 d Roudier, N. et al. (1998) Evidence for the presence of aquaporin-3 in human red blood cells. J. Biol. Chem. 273, 8407–8412 e Zhang, R. et al. (1993) Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28. J. Cell Biol. 120, 359–369 f Chang, A. et al. (1994) Permeability properties of the mammalian bladder apical membrane. Am. J. Physiol. 267, C1483–C1492 g Delamarche, C. et al. (1999) Visualization of AqpZ-mediated water permeability in Escherichia coli by cryoelectron microscopy. J. Bacteriol. 181, 4193–4197 h Sutherland, F.C.W. et al. (1997) Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae. J. Bacteriol. 179, 7790–7795 i Luyten, K. et al. (1995) Fps1, a yeast member of the MIP-family of channel proteins, is a facilitator for glycerol uptake and efflux and it is inactive under osmotic stress. EMBO J. 14, 1360–1371 j Tamás, M.J. et al. (1999) Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol. Microbiol. 31, 1087–1104
Glycerol is also produced by S. cerevisiae cells under anaerobic conditions to dispose of excess NADH in redox regulation31–33. Mutants lacking
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Table 1. Microbial MIP-family channel proteinsa Organism
Gene
Acc. No.
Predicted function
Amino acids
C5f02a1.r1b CNS01A8X Stanford 5476 wacA aqpA NCSM1G3T3b FPS1d YFL054 AQY1-1f AQY1-2 f AQY2 f RPCI93g
AA783486 AL112633 Contig4-2389 U68246 AB032841 AI392589 P23900 P43549 AAC69713 P53386 AAD25168 AQ641778
ND GlpF AQP AQP AQP AQP GlpFe GlpF AQPe AQP AQP GlpF
118c 239c 273 277 279 195c 669 646 327 305 289 160c
glpF GTC 1488 glpF TIGR 1299 TIGR 1351 TIGR 1351d TIGR 1351 ydp1 TIGR 1280 glpF TIGR 1313 TIGR 1313 OUACGT 1315 OUACGT 1315 gylA glpF
P18156 AE001437 X86492 8796 gef 6204 gef 6176 gef 6403 P22094 4410 U12567 sp 42 sp 16 Contig115 Contig104 P19255 AAD36499
GlpFe GlpF GlpF GlpF GlpF GlpF AQP GlpF1AQPe GlpF GlpF GlpF ND GlpF GlpF GlpF GlpF
274 242 164c 271 239 236 233 289 272 233 289 269c 233 282 80c 234
Yersinia pestis
glpF Sanger 518 TIGR 1097d glpF aqpZ glpF glpF glpF glpF glpF glpF CBCUMN 747 ORF10P glpF glpF pduF WUGSC 99287 TIGR 24 glpF aqpZ Stanford 382 smpX aqpZ TIGR 920 TIGR 666 TIGR 666 Sanger 632
AAC66629 Contig 2552 gct 5 P11244 U38664 P44826 U32782 Z33098 P52280 P47279 P75071 Contig341 AB025970 Q51389 AB015973 P37451 Contig79 4323 P31140 AAC12651 423050H01 D43774 BAA17863 1538 asm814 1741 Contig630
GlpF AQP AQP GlpFe AQPe GlpFe GlpF GlpF GlpF GlpF GlpF GlpF AQP GlpFe GlpF PduFe GlpF AQP GlpFe AQP ND GlpF AQP ND GlpF GlpF GlpF
254 236 268 281 231 264 213c 89c 205c 258 264 261 233 279 285 264 288 231 281 231 154c 269 247 144c 261 285 282
Archaea Archaeoglobus fulgidus Methanobacterium thermoautotrophicum
glpF aqp
AAB89820 AAB84602
GlpF AQP
246 246
Eukarya Aspergillus nidulans Botrytis cinerea Candida albicans Dictyostelium discoideum
Neurospora crassa Saccharomyces cerevisiae
Trypanasoma brucei Gram-positive Bacteria Bacillus subtilis Clostridium acetobutylicum Clostridium perfringens Deinococcus radiodurans Enterococcus faecalis
Lactococcus lactis Staphylococcus aureus Streptococcus pneumoniae Streptococcus pyogenes Streptomyces coelicolor Thermotoga maritima Gram-negative Bacteria Borrelia burgdorferi Bordetella bronchiseptica Chlorobium tepidum Escherichia coli
Haemophilus influenzae Mycoplasma capricolum Mycoplasma gallisepticum Mycoplasma genitalium Mycoplasma pneumoniae Pasteurella multocida Plesiomonas shigelloides Pseudomonas aeruginosa Pseudomonas tolaasii Salmonella typhimurium Shewanella putrefaciens Shigella flexneri Sinorhizobium meliloti Synechococcus sp. PCC7942 Synechocystis sp. PCC6803 Thiobacillus ferrooxidans Vibrio cholerae
a
Abbreviations: Acc. No., accession number; AQP, aquaporin; GlpF, glycerol facilitator/transporter; ND, not determined; PduF, propanediol facilitator. bDeduced from mRNA sequence. cIncomplete sequence obtained from GenBank. dOne or both NPA motifs have a different sequence. e Function confirmed experimentally. fOnly in certain strains. gDeduced from genome survey sequence. Sequences were retrieved from the SwissProt, EMBL, GenBank and PROSITE databases. Preliminary sequence data were obtained from The Institute for Genomic Research (http://www.tigr.org) and the Sanger Centre (http://www.sanger.ac.uk).
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Fps1p accumulate glycerol to high levels under anaerobic conditions and grow poorly, presumably owing to unfavourably high turgor22. Under these growth conditions, Fps1p apparently serves as an export pathway for a metabolic waste product. In fact, MIP channels such as Fps1p could play a role in the control of metabolism by facilitating the exit of metabolic end products along a concentration gradient, but it is unknown if this is a general feature. Microbial aquaporins E. coli AqpZ (Ref. 34) and S. cerevisiae Aqy1p (Refs 35,36) can mediate water transport in Xenopus oocytes (Box 1). Additionally, it has been demonstrated by cryoelectron microscopy of osmotically shocked E. coli cells that AqpZ mediates water transport into and out of the cell37. Similarly, functional aquaporins have been detected in S. cerevisiae by observing spheroplast swelling36. The growth conditions under which microbial cells need water channels, despite their high surface-to-volume ratio and the relatively rapid passive diffusion rate of water through the lipid bilayer, are yet to be elucidated. E. coli aqpZ mutants appear to grow more poorly on low-osmolarity medium34. As turgor pressure is probably high under these conditions, AqpZ might be important for the export of water. This would be consistent with the observed stimulated expression of aqpZ during growth in low-osmolarity medium34. Whether AqpZ plays a role in water homeostasis throughout growth or only in specific stages of the cell cycle is not known. Bakers’ yeast has two genes that potentially encode aquaporins, AQY1 and AQY2, which are .80% identical35,36. Curiously, most laboratory strains possess irrevertable mutations in both these genes35. Only strains recovered from natural or industrial environments appear to possess a functional Aqy1p (Refs 35,36). It therefore appears that laboratory conditions might be disadvantageous to cells possessing functional aquaporins, suggesting that they play a role only under very specific conditions35. Elevated expression of AQY1 during sporulation hints at a role for aquaporins in spore formation and/or germination, but as yet there is no evidence for this. Conclusions and perspectives In combination with a specific lipid composition, which limits the passive diffusion of water and/or glycerol, MIP channels enable the cell to control the passage of these compounds through cellular membranes. MIP channels might also enable the cell to coordinate transport with other processes, such as the phosphorylation of glycerol or signalling osmotic changes. In addition, the control of MIP-channel function, expression or localization provides the cell with a means to regulate transmembrane water and solute flux according to its physiological needs. Such regulation has been demonstrated in the case of certain aquaporins from mammals5, plants3 and for yeast Fps1p (Refs 19,22), and is important for the control of cellular and whole-organism osmoregulation, and
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Box 2. Osmoregulation: compatible solutes The control of relative cellular water content is extremely important to ensure proper functioning of biochemical processes and constant turgor pressure, which in turn is relevant for the morphology and shape of cells and organisms. Consequently, osmoregulation is complex and includes many processes, some of which are conserved in evolution, whereas others are a reflection of the specific environment of an organism. A common strategy is the accumulation of compatible solutes to adjust the intracellular water activity without disturbing biochemical processesa. Compatible solutes can be ions, sugars, sugar alcohols or amino acids or derivatives thereof. Such solutes can be actively accumulated from the environment or can be produced by the cell. For example, Escherichia coli uses potassium ions, trehalose and glycine-betaine as compatible solutesb, whereas Saccharomyces cerevisiae uses glycerolc,d. Once the osmolarity of the surrounding environment drops, the cell has to decrease its solute content, for example by specifically releasing compatible solutes into the mediume. Regulation of solute accumulation occurs at the level of gene expression, leading to changes in metabolic capacity and the modulation of transport activity. There is a substantial research interest in the mechanisms of sensing changes in the osmolarity of the cellular environment and the signal transduction pathways that lead to the observed responsesf,g. References a Yancey, P.H. et al. (1982) Living with water stress: evolution of osmolyte systems. Science 217, 1214–1222 b Kempf, B. and Bremer, E. (1998) Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolarity environments. Arch. Microbiol. 170, 319–330 c Hohmann, S. (1997) Shaping up: the response of yeast to osmotic stress. In Yeast Stress Responses (Hohmann, S. and Mager, W.H., eds), pp. 101–145, R.G. Landes Co. d Nevoigt, E. and Stahl, U. (1997) Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 21, 231–241 e Booth, I.R. and Louis, P. (1999) Managing hypo-osmotic stress: aquaporins and mechanosensitive channels in Escherichia coli. Curr. Opin. Microbiol. 2, 166–169 f Wood, J.M. (1999) Osmosensing by bacteria: signals and membrane-based sensors. Microbiol. Mol. Biol. Rev. 63, 230–262 g Gustin, M.C. et al. (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62, 1264–1300
Questions for future research • What are the structural features of aquaporins and glycerol facilitators that determine transport specificity? • When is aquaporin-mediated water transport relevant for microorganisms? Why do some microorganisms have water channels and not others? • Is the export of solutes and/or metabolic end products a feature common to many glycerol facilitators? • How common is regulation of MIP channel function and what are the specific mechanisms of this regulation? • To what extent can microorganisms be employed as useful models to study MIP channel function? • Can MIP channels be exploited, for example for the engineering of the performance of microorganisms under stressful industrial conditions and/or engineering metabolism towards specific metabolic end products?
for metabolism. Therefore, an understanding of the molecular details of MIP channel regulation is an important goal for future analysis. MIP channels appear to affect a wide range of processes via their roles in osmoregulation and
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metabolism, and although much effort has focused on water transport, many MIP channels could control the export of compatible solutes and/or metabolic waste products. As more biological roles of MIP channels are being discovered, their potential in medicine, pharmacology, agrobiotechnology and metabolic engineering is also becoming clear. The availability of genetically tractable microbial systems for the study of MIP-channel function and regulation will contribute to the exploitation of the special characteristics of this exciting protein family. Acknowledgements We thank P. Agre, G. Calamita, A. Engel, D. Thomas, E. Bremer, R. Krämer and S.G. Kilian for communicating unpublished results and manuscripts in press, and V. Laizé for help with Fig. 1. Work in the laboratory of S.H. is supported by the Commission of the European Union via contracts FMRX-CT97-0128 and BIO4-CT98-0024. B.A.P. acknowledges support from the National Research Foundation, South Africa. References 1 Preston, G.M. et al. (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP 28 protein. Science 256, 385–387 2 Borgnia, M. et al. (1999) Cellular and molecular biology of aquaporin water channels. Annu. Rev. Biochem. 68, 425–458 3 Kjellbom, P. et al. (1999) Aquaporins and water homeostasis in plants. Trends Plant Sci. 4, 308–314 4 Kuwahara, M. et al. (1998) A water channel of the nematode C. elegans and its implications for channel selectivity of MIP proteins. Am. J. Physiol. 275, C1459–C1464 5 Deen, P.M.T. and van Knoers, N. (1998) Physiology and pathophysiology of the aquaporin-2 water channel. Curr. Opin. Nephrol. Hyperten. 7, 37–42 6 Kaldenhoff, R. et al. (1998) Significance of plasmalemma aquaporins for water-transport in Arabidopsis thaliana. Plant J. 14, 121–128 7 Heymann, J.B. et al. (1998) Progress on the structure and function of aquaporin 1. J. Struct. Biol. 121, 191–206 8 Park, J.H. and Saier, M.H. (1996) Phylogenetic characterization of the MIP family of transmembrane channel proteins. J. Membr. Biol. 153, 171–180 9 Hasler, L. et al. (1998) Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution. J. Mol. Biol. 279, 855–864 10 Ringler, P. et al. (1999) Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography. J. Mol. Biol. 291, 1181–1190 11 Lagree, V. et al. (1999) Switch from an aquaporin to a glycerol channel by two amino acids substitution. J. Biol. Chem. 274, 6817–6819 12 Froger, A. et al. (1998) Prediction of functional residues in water channels and related proteins. Protein Sci. 7, 1458–1468 13 Reizer, J. et al. (1993) The MIP family of integral membrane channel proteins: sequence comparison, evolutionary relationship, reconstructed pathway of evolution and proposed functional differentiation of the two repeated halves of the proteins. Crit. Rev. Biochem. Mol. Biol. 28, 235–257 14 Heller, K.B. et al. (1980) Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli. J. Bacteriol. 144, 274–278 15 Truniger, V. et al. (1992) Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri. J. Bacteriol. 174, 6981–6991 16 Schweizer, H.P. et al. (1997) Structure and gene-polypeptide
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relationships of the region encoding glycerol diffusion facilitator (glpF) and glycerol kinase (glpK) of Pseudomonas aeruginosa. Microbiology 143, 1287–1297 Voegele, R.T. et al. (1993) Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator. J. Bacteriol. 175, 1087–1094 Chen, P. et al. (1994) The control region of the pdu/cob regulon in Salmonella typhimurium. J. Bacteriol. 176, 5474–5482 Luyten, K. et al. (1995) Fps1, a yeast member of the MIP-family of channel proteins, is a facilitator for glycerol uptake and efflux and it is inactive under osmotic stress. EMBO J. 14, 1360–1371 Sutherland, F.C.W. et al. (1997) Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae. J. Bacteriol. 179, 7790–7795 Lages, F. and Lucas, C. (1997) Contribution to the physiological characterization of glycerol active uptake in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1322, 8–18 Tamás, M.J. et al. (1999) Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol. Microbiol. 31, 1087–1104 Truniger, V. and Boos, W. (1993) Glycerol uptake in Escherichia coli is sensitive to membrane lipid composition. Res. Microbiol. 144, 565–574 Daum, G. et al. (1998) Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14, 1471–1510 Maurel, C. et al. (1994) Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. J. Biol. Chem. 269, 11869–11872 Coury, L.A. et al. (1999) Water transport across yeast vacuolar and plasma membrane-targeted secretory vesicles occurs by passive diffusion. J. Bacteriol. 181, 4437–4440 Philips, J. and Herskowitz, I. (1997) Osmotic balance regulates cell fusion during mating in Saccharomyces cerevisiae. J. Cell Biol. 138, 961–974 Tao, W. et al. (1999) Intracellular glycerol levels modulate the activity of Sln1p, a Saccharomyces cerevisiae two-component regulator. J. Biol. Chem. 274, 360–367 Levina, N. et al. (1999) Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. EMBO J. 18, 1730–1737 Booth, I.R. and Louis, P. (1999) Managing hypo-osmotic stress: aquaporins and mechanosensitive channels in Escherichia coli. Curr. Opin. Microbiol. 2, 166–169 Ansell, R. et al. (1997) The two isoenzymes for yeast NADdependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2, have distinct roles in osmoadaption and redox regulation. EMBO J. 16, 2179–2187 Hohmann, S. (1997) Shaping up: the response of yeast to osmotic stress. In Yeast Stress Responses (Hohmann, S. and Mager, W.H., eds), pp. 101–145, R.G. Landes Co. Nevoigt, E. and Stahl, U. (1997) Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol. Rev. 21, 231–241 Calamita, G. et al. (1998) Regulation of the Escherichia coli water channel gene aqpZ. Proc. Natl. Acad. Sci. U. S. A. 95, 3627–3631 Bonhivers, M. et al. (1998) Aquaporins in Saccharomyces. Genetic and functional distinctions between laboratory and wild-type strains. J. Biol. Chem. 273, 27565–27572 Laizé, V. et al. (1999) Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain. Biochem. Biophys. Res. Commun. 257, 139–144 Delamarche, C. et al. (1999) Visualization of AqpZ-mediated water permeability in Escherichia coli by cryoelectron microscopy. J. Bacteriol. 181, 4193–4197
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