Báo cáo khóa học: Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen ppt

Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen Sara Karlgren 1 , Caroline Filipsson 2 , Jonathan G. L. Mullins 3 , Roslyn M. Bill 1,4 , Markus J. Tama ´ s 1 and Stefan Hohmann 1 1 Department of Cell and Molecular Biology/Microbiology, Go ¨ teborg University, Sweden; 2 Department of Biochemistry and Biophysics, Go ¨ teborg University, Sweden; 3 Swansea Clinical School, University of Wales Swansea, UK; 4 School of Life and Health Sciences, Aston University, Birmingham, UK Aquaporins and aquaglyceroporins mediate the transport of waterandsolutesacrossbiologicalmembranes.Saccharo- myces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation. The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation. In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct resi- dues, all facing the inside of the cell. Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain. Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control. Further- more, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control. Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regula- tory domain and the B-loop could interact in channel con- trol. Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes. Keywords: aquaglyceroporin; channel; genetic screen; glycerol; osmoregulation. The discovery of the aquaporins marked a breakthrough in our understanding of water and solute transmembrane transport [1]. Aquaporins and aquaglyceroporins [the major intrinsic protein (MIP) family] have been found in archea, eubacteria, fungi, plants, animals and human [2,3]. Aqua- porins facilitate the diffusion of water across biological membranes while the closely related aquaglyceroporins mediate transport of water and solutes such as glycerol and urea. These proteins are present in membranes where rapid and controlled water or solute fluxes occur, for example, in the mammalian kidney [2,4,5] and plant roots [6,7]. The yeast Saccharomyces cerevisiae has four such MIP channels: the aquaporins Aqy1 and Aqy2 and the aquaglyceroporins Fps1 and Yfl054 [8]. Aqy1 is a strictly spore-specific aquaporin while Aqy2 may play a role in osmoregulation during cell growth (F. Sidoux-Walter & S. Hohmann, unpublished observation). Possible roles in freeze tolerance have been claimed for Aqy1 and Aqy2 [9]. The physiological role of Yfl054 has not yet been established [8,10]. Yeast cells accumulate glycerol as a compatible solute in osmoregulation [11]. The plasma membrane channel Fps1 mediates glycerol export and is required for survival of a hypo-osmotic shock when glycerol has to be rapidly exported from cells in order to prevent bursting [12,13]. On the other hand, hyperactive Fps1 causes an inability to grow at high external osmolarity because cells lose the glycerol they produce [12,13]. Moreover, it has been shown that Fps1 is required to control turgor and prevent cell lysis during cell fusion of mating yeast cells [14]. Together, these observations illustrate that Fps1 plays a central role in yeast osmoregulation. The transport function of Fps1 is controlled by osmotic changes in order to prevent glycerol loss at high osmolarity and to allow rapid export at low external osmolarity. The capacity for glycerol transmembrane flux through the yeast plasma membrane is reduced within seconds upon a hyperosmotic shock while it increases equally fast upon a shift to hypo-osmotic conditions. As Fps1 is responsible for most of the glycerol transmembrane flux [10] these obser- vations together with the phenotype caused by hyperactive Fps1 suggest that the channel is directly controlled by osmotic changes [12,13,15]. Aquaporins and aquaglyceroporins have six transmem- brane spanning domains (TMD) and five connecting loops [2,16–18]. The hydrophobic B- and E-loop, facing inside and outside, respectively, are part of the central water/solute pore. These a-helical loops dip into the membrane where their highly conserved Asn-Pro-Ala (NPA) motifs form the central pore constriction [2,16–18]. Structural analysis and Correspondence to S. Hohmann, Department of Cell and Molecular Biology/Microbiology, Go ¨ teborg University, Box 462, S-40530 Go ¨ teborg, Sweden. Fax: + 46 31 7732599, Tel.: + 46 31 7732595, E-mail: hohmann@gmm.gu.se Abbreviations: MIP, major intrinsic protein; NPA, Asn-Pro-Ala; TMD, transmembrane domains; YNB, yeast nitrogen base; YPD, yeast peptone glucose (Received 19 November 2003, revised 21 December 2003, accepted 5 January 2004) Eur. J. Biochem. 271, 771–779 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03980.x molecular dynamic modelling as well as biophysical analy- ses have revealed the mechanisms that ensure very rapid transport and at the same time high selectivity of water or glycerol transport [17–22]. Fps1 is an atypical aquaglyceroporin as the highly conserved NPA motifs in the B- and the E-loop are NPS (Asn-Pro-Ser) and NLA (Asn-Leu-Ala), respectively, sequences that are also found in the Plasmodium glycerol facilitator, although in the opposite loops [23]. While Fps1 can tolerate NPA in both positions, the Escherichia coli homologue GlpF is inactive when its NPA motifs are converted to NPS and NLA, suggesting a somewhat different and more flexible arrangement of the Fps1 channel [24]. In addition, Fps1 has unique long N- and C-terminal domains only found in orthologues from other yeasts [15]. While large parts of these extensions can be removed without apparent consequence, short domains close to the first and the last TMD seem to be required for channel control: deletions or mutations in these regions render the channel hyperactive (K. Hedfalk, R. M. Bill, J. G. Mullins, S. Karlgren, C. Filipsson, C. Bergstrom, M. J. Tama ´ s, J. Rydstro ¨ m & S. Hohmann, unpublished observation) [13,15]. The N-terminal regulatory domain may fold in a similar way as the channel forming B- and E-loops, hence dipping into the membrane. We suggested that this domain, dubbed the N-loop, might directly interact with the channel forming B-loop to control transport function [15]. As a novel approach to study the control of Fps1, we present a random genetic screen for hyperactive Fps1. Twenty independent mutants are reported here, represent- ing 17 different mutations, all facing the cell interior. The majority of the mutations are clustered in or near the already identified N-terminal regulatory domain. Mutations in the C-terminal domain resulted in premature termination. Most interestingly, five different mutations hit two con- served residues in the B-loop. The data lend support to the notion that the N-terminal regulatory domain may interact with the central pore and obstruct transport. Hence, this study provides new insight into Fps1 control and opens up for further mutational analyses this interesting aqua- glyceroporin. Materials and methods Strains and plasmids The yeast strains used in this study are YSH 642 (gpd1D::TRP1 gpd2::DURA3) [26] and YMT2 (fps1D:: HIS3) [13] in the W303-1 A background (MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0) [27]. YEpmyc-FPS1 is a 2l LEU2 plasmid expressing a c-myc epitope-tagged Fps1 and YEpmycfps1-D1 encodes a truncated version of Fps1 lacking the amino acids 12–231 [13]. Growth conditions Yeast cells were grown in medium containing 2% peptone, 1% yeast extract, 2% glucose (YPD), or for selection of transformants in yeast nitrogen base (YNB) medium [28]. Tests for hyperactive alleles of FPS1 were performed by pregrowing the gpd1D gpd2D mutant transformed with YEpmyc-FPS1 and derivatives thereof for two days on YNB agar plates, resuspending them in YNB medium to an D 600 of 0.4 and then performing 10-fold serial dilutions. Cell suspensions (5 lL) were spotted onto agar plates supple- mented with 1 M xylitol, or with 0.8 M NaCl as a negative control, and on medium without osmoticum as a positive control. Growth was monitored after 2–7 days at 30 °C. For growth tests after osmotic shifts, transformants were pregrown on YNB plates, then resuspended and spotted on the same medium as the control. To invoke hyperosmotic shock, cells were pregrown in medium without osmoticum and shifted to medium with 0.8 M NaCl. For a hypo- osmotic shock, cells were pregrown in the presence of 0.8 M NaCl and shifted to medium without salt. Growth was monitored as above. Mutagenesis and screening Random mutations in FPS1 were introduced by transform- ing YEpmyc-FPS1 into the E. coli strain XL1-Red from Stratagene (La Jolla, CA, USA) following the manufac- turer’s recommendations. Transformants were grown for approximately 24 h yielding about 200 colonies per plate. Colonies from each plate were pooled and grown in LB- medium supplemented with 100 lgÆmL )1 of ampicillin for 24 h. Plasmids were isolated (Qiagen miniprep kits) and transformedintoagpd1D gpd2D strain using the LiAc- method [29]. Yeast cells were spread on selective media (YNB) to a density of approximately 200 transformants per plate. The colonies were replica-plated onto YNB-leu (positive control), YNB-leu plus 1 M sorbitol, YNB-his (negative controls) and YNB-leu plus 1 M xylitol (selective) to ensure that a low cell density was left on the velvet before replicating onto selective plates. Cell densities that are too high make distinction between growth and no growth difficult. Cells were grown for 4–5 days and positive clones were re-tested in growth assays on 1 M xylitol. Plasmid was recovered from positive transformants, checked by restric- tion analysis, propagated in E. coli Top10 cells and then re-transformed into the gpd1D gpd2D strain for testing on 1 M xylitol plates. In addition, plasmids were also trans- formed into the fps1D mutant and tested for growth after hypo/hyper-osmotic shock. All FPS1 genes from trans- formants that scored positive in the tests were completely sequenced. Western blot analysis Cells were cultured in YNB supplemented with 2% glucose to late log phase (typically D 600 is 0.8). The total membrane fraction was isolated and visualized as described previously (S.Karlgren,N.Pettersson,R.M.Bill&S.Hohmann, unpublished observation). Glycerol transport measurements To determine glycerol influx following its concentration gradient, cells were grown in liquid YNB medium to a D 600 of approximately 0.7. Cells were harvested, washed and suspended in ice-cold Mes buffer (10 m M Mes, pH 6.0) to a density of 40–60 mg cellsÆml )1 . All subsequent steps were performed at 4 °C. Glycerol influx in the presence or 772 S. Karlgren et al. (Eur. J. Biochem. 271) Ó FEBS 2004 absence of hyperosmotic stress was measured by adding glycerol to a final concentration of 100 m M ÔcoldÕ glycerol plus 40 l M [ 14 C]glycerol (5.9 GBqÆMmol )1 ;Amersham)in a total volume of 250 lL [13,15]. Aliquots of 50 lLwere collected by filtration at 0, 15, 30, 45 and 60 s, immediately washed three times with ice-cold buffer and the radioactivity that was retained on the filters was determined. Filters with cells were dried at 80 °C overnight for dry weight deter- mination. Transport experiments were performed in tripli- cate and data are expressed in lmol per gram of dry cells. Modelling Models are based on previous analyses [15] using the structural information on E. coli GlpF as a template [17]. They were generated in MOLMOL [31] avoiding any side chain conflicts, and bringing the N-loop in as centrally as possible to the pore cavity, rotating the ends of the N-loop toward transmembrane domain 1 (the closest TMD in sequence terms). Results Screen for hyperactive Fps1 In order to identify residues within Fps1 that are important for channel control we performed a random genetic screen for mutants that render Fps1 hyperactive. The c-myc-tagged FPS1 gene on a YEp plasmid was randomly mutagenized using the DNA repair-defective E. coli strain XL1-Red. This procedure generated a series of libraries of 200 clones each, which were individually transformed into a gpd1D gpd2D double mutant. This strain is unable to produce glycerol and hence cannot grow at elevated osmolarity caused by salt [26], or by various polyols including xylitol and even glycerol (S. Karlgren, N. Pettersson, R. M. Bill & S. Hohmann, unpublished observation). However, growth of the gpd1D gpd2D mutant in the presence of polyols can be rescued when transformed with a plasmid encoding hyper- active Fps1 (FPS1-D1) that alleviates the osmotic dis- equilibrium by permitting solute influx (S. Karlgren, N. Pettersson, R. M. Bill & S. Hohmann, unpublished observation) [12,13] (Fig. 1). Although hyperactive Fps1 can rescue growth of the gpd1D gpd2D mutant through influx of various polyols (S. Karlgren, N. Pettersson, R. M. Bill & S. Hohmann, unpublished observation) we chose xylitol for the screen as it gave the clearest phenotype. We note that we actually screen for hyperactive xylitol uptake while the objective is to obtain mutants that fail to retain internally produced glycerol under hyperosmotic stress, an aspect that will be discussed when interpreting the muta- tions obtained. Transformants with mutagenized FPS1 in the gpd1D gpd2D strain were grown to colonies on YNB and then replica-plated onto plates supplemented with 1 M xylitol. Approximately 5000 colonies were screened and 31 grew on xylitol plates. These were re-tested for growth on 1 M xylitol. Plasmids were isolated from these positive yeast colonies, amplified in E. coli, checked by restriction analysis and retransformed into the gpd1D gpd2D mutant. Those trans- formants were again tested for growth on 1 M xylitol (Fig. 1) leaving a total of 20 different clones for further analysis. Mutations obtained Sequence analysis of the entire FPS1 gene from these 20 plasmids revealed 19 mutants with single amino acid replacements (Table 1) that clustered in a characteristic manner (Fig. 2). The mutation P236L was represented five times, leaving a total of 15 unique single mutations. One mutant had a point mutation in position S246P in the approach to the first TMD as well as a stop codon in Fig. 1. Growth on plates. Cells were dropped in a 1 : 10 dilution series on synthetic YNB medium with the indicated osmotica. A hyperactive Fps1 function is indicated by growth on xylitol (or sorbitol) in the gpd1D gpd2D mutant and by poor growth on NaCl in the fps1D mutant. The hypo-osmotic test (shift from 0.8 M NaCl to medium without salt) is a test for function: poor growth indicates no or reduced function. Ó FEBS 2004 Control of aquaglyceroporin Fps1 (Eur. J. Biochem. 271) 773 approximately the middle of the C-terminal extension (Q592stop). Six mutations fall within the previously char- acterized regulatory domain, which, according to our previous mutational analysis and sequence conservation among yeast Fps1 orthologues, encompasses the stretch from Met219 until about Ser248. A nine amino acid linker follows this domain to the first TMD, which is predicted to start at Leu257. Figure 2B provides an overview of the relevant mutations from previous [15] and present analyses within this region. One mutation was found in the approach to the first TMD in a lysine (K250E) that is conserved among yeast Fps1 orthologues [15]. Importantly, two residues within the channel forming B-loop, which are both highly conserved throughout the MIP family, were affected by multiple exchanges. All three mutations occurring in the C-terminal extension caused premature translation termin- ation, either by generating a stop codon or a frame shift leading to a stop some codons further downstream. As the newly isolated mutations do not hit all residues that we previously found to be critical for Fps1 control, while at the same time we identified new relevant residues, the present genetic screen is not saturated. A significant larger number of mutations, probably more than 100, will be needed for a fully comprehensive mutational map of channel control. The genetic screen employed selected for mutated versions of Fps1 that retain function, hence the proteins Table 1. Summary of mutations obtained. Mutation Nucleotide change Location K223E AAGfiGAG In front of the N-terminal regulatory domain Q227R CAGfiCGG Within the N-terminal regulatory domain T231A ACAfiGCA Within the N-terminal regulatory domain P232S CCTfiTCT Within the N-terminal regulatory domain P236L (found five times) CCCfiCTC Within the N-terminal regulatory domain S246P + Q592 stop TCTfiCCT + CAAfiTAA Between the N-terminal regulatory domain and TMD1 K250E AAAfiGAA Between the N-terminal regulatory domain and TMD1 G348D GGTfiGAT Loop B G348R GGTfiCGT Loop B G348S GGTfiAGT Loop B H350L CATfiCTT Loop B H350Y CATfiTAT Loop B L451W TTGfiTGG Loop D I531FIRVMNLQSTG T insertion at 1595 C-terminus S537QLVFTSL 1613CAGTC1617 deleted C-terminus W541stop TGGfiTAG C-terminus B (219)MVKPKTLYQNPQTPTVLPSTYHPINKWSS(248) L225A Q227A N228A P229A Q230A T231A P231A T232A P236A K223E Q227R T231A P231S P236L S246P K250E K223E Q227R T231A P323S P236L S246P + Q592stop G348D G348R G348S H350L H350Y L451W Q592stop + S246P B-loop E-loop N-terminus C-terminus W541stop + H 3 N I531FIRVMNLQSTG S537QLVFTSL - OOC A Fig. 2. Summary of mutations obtained. (A) Topology map of Fps1 indicating mutations found in the genetic screen reported here. For clarity the N- and the C-termini are not shown according to scale. (B) Summary of mutations affecting the function of the N-terminal regu- latory domain. Data shown above the sequence is from a previous study [15]. Data shown below the sequence is from this study. Underlined mutations cause particularly strong osmosensitivity. 774 S. Karlgren et al. (Eur. J. Biochem. 271) Ó FEBS 2004 should be expressed and localized to the plasma membrane. However, differences in expression levels could be relevant for the interpretation of the results. For this reason we performed Western blot analysis, making use of the C-terminal c-myc-tag (Fig. 3). This was only possible for mutants that retained a complete C-terminus. Some of the Fps1 mutants were apparently less abundant in the plasma membrane, in particular those in His350. However, this does not seem to affect their function because they mediate strong growth on xylitol and fully complement the hypo- osmosensitivity of an fps1D mutant (Fig. 1, see below). Also in previous mutational analyses we have observed that the apparent protein abundance of Fps1 does not affect performance in functional assays over a wide range [15]. One explanation may be that mutations cause different detectability in immuno blots. Another possibility is that Fps1 is present in excess such that even much lower levels can perform full function. None of the mutants was more abundant than wild type Fps1, excluding simple expression changes as a cause for the observed gain of function. Phenotypic characteristics of mutants obtained All mutations obtained rescued growth on 1 M xylitol to the gpd1D gpd2D mutant, albeit clearly to different extents (Fig. 1). The three most upstream mutations as well as the C-terminal truncations conferred the weakest growth on xylitol. The double mutant, S246P plus stop at 591, conferred particularly robust growth on xylitol and in contrast to all other mutations even allowed growth in the presence of 1 M of the C6 polyol D -sorbitol. As all other mutations in the C-terminal extension occur much further upstream and caused much weaker growth on xylitol and because we did not observe any effects conferred by truncations that far downstream in the C-terminus in a different study (K. Hedfalk, R. M. Bill, J. G. Mullins, S. Karlgren, C. Filipsson, C. Bergstrom, M. J. Tama ´ s, J. Rydstro ¨ m & S. Hohmann, unpublished observation), we believe that the effect in the double mutant is mainly due to the S246P mutation, although we can not fully exclude a synergistic effect of both mutations. As we had isolated the mutants based on their ability to mediate xylitol influx we wished to test if the novel Fps1 derivates were still fully able to exert their normal physiological function in mediating efflux of glycerol upon a hypo-osmotic shock. This is most conveniently tested by monitoring the survival after a hypo-osmotic shock, as a smaller proportion of mutants lacking Fps1 survive, and survivors start growth more poorly. We have previously shown these tests to be very reproducible and to correctly reflect the ability of Fps1 to mediate glycerol release [13,15]. All plasmids were transformed into an fps1D strain, which was wild type for GPD1 and GPD2 and therefore capable of producing glycerol. The ability of the novel alleles to complement the lack of FPS1 was tested by shifting transformants from high osmolarity medium (0.8 M NaCl) to medium without osmoticum (Fig. 1). Most of the transformants grew like wild type but in some instances the mutation reduced survival due to hypo-osmotic shock, indicating impaired glycerol export function under these conditions. This effect was most pronounced for C-terminal truncations, some of which conferred sensitivity almost like deletion of FPS1 (Fig. 1). In addition, mutations at G348 in the B-loop reduced survival upon hypo-osmotic shock. Hyperactive Fps1-D1 confers both hyperosmosensitivity in cells competent of producing glycerol and ability to grow on 1 M xylitol to a strain unable to produce glycerol. The mutants isolated here were selected for the latter phenotype and hence we wished to test if they were also affected for retention of internally produced glycerol. We have shown previously that this ability is well reflected by growth characteristics in the presence of high external osmolarity [12,13,15]. The different mutants showed varying degrees of osmo- sensitivity (Fig. 1). The strongest effects were observed for mutations within the N-terminal regulatory domain and in particular for mutations of the two prolines P232 and P236, the double mutant (S246P plus stop at Q591), K250E in the linker to the first TMD and mutations in G348 in the B-loop. Interestingly, different exchanges of the highly con- served G348 caused different effects, with G348D causing strongest osmosensitivity. The two different exchanges of the conserved His350 caused similar effects. Two residues in the N-terminal regulatory domain had previously been studied by alanine-scanning mutagenesis: Gln227 and Pro236 [13]. In order to compare the effects of the different exchanges directly they were tested side-by-side (Fig. 4). Q227A and Q227R caused similar strong osmo- sensitivity indicating that the two exchanges, although chemically very different, affected channel control in a similar way. While exchange of Pro236 with alanine only Fig. 3. Western blot analysis of the whole membrane fraction probed with an anti c-myc Ig against the C-terminal c-myc-tag of Fps1. Amounts of protein loaded from left to right: 50, 20, 30, 50, 50, 30, 50, 50, 30, 50, 50, 100, 50 and 50 lg. The lower double band is probably a degradation product. Fig. 4. Growth on plates. Cells were pregrown on YNB and dropped in a 1 : 10 dilution series on the same medium with or without NaCl. Ó FEBS 2004 Control of aquaglyceroporin Fps1 (Eur. J. Biochem. 271) 775 caused moderate osmosensitivity, exchange with the some- what larger but chemically similar leucine seemed to affect channel control much more dramatically. If the ability of the Fps1 alleles for mediating influx of xylitol and efflux of glycerol were equally affected by the novel mutations we would expect that good growth on 1 M xylitol would correlate with poor growth on 0.8 M NaCl. This was the case for most of the mutations with some exceptions. G348D, which caused poorest growth on 0.8 M NaCl only mediated moderate growth on 1 M xylitol. L451W, on the other hand, which mediated robust growth on 1 M xylitol, caused almost no sensitivity to 0.8 M NaCl. Also mutations that truncated the C-terminus caused either no or poor sensitivity to 0.8 M NaCl while permitting growth on xylitol. These observations indicate that the two functions tested here, xylitol influx and glycerol efflux, share common determinants while at the same time they can be distinguished through specific mutations. High glycerol transport capacity through the Fps1 mutant proteins We have previously shown that the ability of Fps1 derivatives to mediate glycerol transport and to down- regulate glycerol transport upon a hyperosmotic shock can be monitored by measuring the influx of radiolabelled glycerol following its concentration gradient in unstressed cells and in cells exposed to 0.8 M NaCl. We selected some mutations from the new collection that represented different characteristics including P236L, G348D, H350L and L451W. As observed previously, glycerol transport through hyperactive Fps1 is higher than that through wild type under all conditions and is down-regulated by hyperosmotic shock. This is also the case for the mutants studied here (Fig. 5). All of them, however, maintained a much higher glycerol transport capacity then wild type even after hyperosmotic shock. The apparent rate of glycerol transport is lower for H350L than for the other mutants, which is consistent with the fact that it only conferred moderate osmosensitivity, a measure for glycerol loss (Fig. 1). The three other mutants conferred similar glycerol influx while they caused different degrees of osmosensitivity. Glycerol uptake rates correlated better with the ability to grow on xylitol, suggesting that in some of the mutants isolated influx may be enhanced to a higher degree than efflux. Discussion The transmembrane transport of glycerol in yeast is rapidly controlled by osmotic changes to ensure glycerol accumu- lation under hyperosmotic stress and fast glycerol release upon a hypo-osmotic shock. Fps1, an aquaglyceroporin, mediates most of the glycerol transport through the plasma membrane. Its importance is illustrated by the fact that hyperactive Fps1 causes glycerol loss and sensitivity to hyperosmotic conditions while inactivation of Fps1 causes inability to release glycerol upon a hypo-osmotic shock and poor survival. We have observed previously that even in cells expressing hyperactive Fps1 a hyperosmotic shock mediates substantial down-regulation of glycerol transmem- brane transport. One simple explanation for this observa- tion is that cells shrink after hyperosmotic shock, which means that both their cell surface and volume rapidly diminish, which may reduce the capacity for uptake. We can, however, not exclude that even hyperactive Fps1 still retains some regulatory capacity. It also appears that hyper- active Fps1 mediates higher glycerol flux under normal as well as hypertonic conditions. This might indicate that only a subset of all Fps1 molecules is active at any given time under normal conditions while such mutations fully activate all channels. Hence, Fps1 is likely to constantly switch between open and closed conformations, and osmotic conditions alter the probability for either conformation. Mutations that render Fps1 hyperactive may therefore increase the ÔopenÕ probability. In this work we have used a novel genetic approach to identify intramolecular determinants of Fps1 control. The genetic screen we employed is based on the observation that hyperactive Fps1 allows mutants unable to produce glycerol (gpd1D gpd2D) to grow in the presence of 1 M xylitol. Hence we screen for enhanced uptakeof glycerol, while the physiological role is glycerol export. Most, although not all, mutants we obtained in this way also conferred osmosensitivity (and hence enhanced glycerol loss under these conditions). Moreover, we obtained several mutations in residues that we previously identified as important by targeted mutagenesis. These observations confirm the validity of the approach. Although we screened for enhanced uptake, all mutations faced the inside of the cell. Recently we screened for suppressors of truncated, hyperactive Fps1 and obtained mutations that reduced transport. The four mutants char- acterized faced the outside of the cell [15]. Structural analysis of AQP1 and GlpF suggested that these channels are largely symmetric (except for the tails facing the inside) [17,32]. While our mutational analysis may not yet be representa- tive, the distribution of mutations may suggest that the Fig. 5. Uptake profile of 100 m M radiolabelled glycerol by the strains indicated, before and after an osmotic shift to 0.8 M NaCl. 776 S. Karlgren et al. (Eur. J. Biochem. 271) Ó FEBS 2004 outside face is mainly important for transport and the inside face for control, at least in the case of the somewhat unusual Fps1. Some mutations, such as L451W and C-terminal trun- cations but also alterations in His350 allow solid growth in the presence of xylitol while causing only moderate osmo- sensitivity (i.e. moderate glycerol loss). We have observed previously that certain mutations affected glycerol transport in one direction more than in the other [24]. The phenotype of the mutants described here suggests that it might be possible to partly dissect uptake and efflux functions using specific genetic screens. The results of our genetic screen confirm and extend previous analyses. Mutations identified in and around the previously characterized N-terminal regulatory domain confirm its importance but at the same time also indicate that residues involved in channel control are located between this domain and the first TMD. We also confirm previous observations on the importance of the C-terminus (K. Hedfalk, R. M. Bill, J. G. Mullins, S. Karlgren, C. Filipsson, C. Bergstrom, M. J. Tama ´ s, J. Rydstro ¨ m & S. Hohmann, unpublished observation), although we note that the truncations obtained here only cause moderate if any osmosensitivity (indicative of glycerol loss during high external osmolarity). More mutations need to be charac- terized to judge if those obtained here, which all confer premature translational stop rather then specific amino acid replacements, are truly representative. A particularly significant finding of this study is that even mutations in highly conserved residues of the channel forming B-loop can mediate hyperactive transport. So far, important residues were identified on the basis of mutations that block transport. Hence, the approach used here, which is novel as it screens for gain of function, leads to completely novel insight into the structure-function relationship of MIP channels. Based on the structure of GlpF [17] and previous modelling [15] we have attempted to rationalize the mutations obtained in this study. We have shown previously that the N-terminal regulatory domain, dubbed the N-loop, has sequence and predicted structural similarity with the channel forming B- and E-loop. We suggested that B- and N-loops may interact. In the models shown (Fig. 6), the 226–236 region of the N-loop and 347–356 of the B-loop are in close proximity. Residues affected by mutation are then located for the most part in the functionally critical pore region. These include Lys223 (violet), Gln227 (white), Thr231 (brown), Pro232 and Pro236 (grey), and on the B-loop they include Gly348 (orange) and His350 (blue). Lys451 (black) is the only mutation to be clearly located away from the pore. Ser246, Lys250, Ile531 and Ser537 lie in regions of the protein that are not currently structurally modelled. K223E (violet) and K250E (not modelled) result in a charge reversal, which is likely to introduce electrostatic interactions with other nearby lysine residues. This, in turn, is likely to reduce the flexibility of the section linking the N-terminal regulatory domain with TMD1, thereby holding the pore more permanently open. Q227R (white) lies directly adjacent to the NPQ (Asn- Pro-Gln) region of the regulatory motif (which is similar to NPA of the B-loop [15]). Even slight changes in the nature of amino acids in this sensitive region are liable to affect the regulatory domain. Likewise, T231A (brown), immediately on the other side of the NPQ motif, disrupts the regulation of the pore, but more markedly so than Q227R. This is most likely due not only to its closeness to the NPQ motif but also to the major role of threonine residues in hydrogen bonding. This mutation is consistent with our previous findings regarding T231, as well the general importance of the role of threonine residues in the pore region of Fps1. T256 on the B-loop is conserved across the whole MIP family. Fig. 6. Structural modelling. The B-loop is shown in yellow and the N-loop in green. The residues involved in random mutations are found to be located for the most part in the functionally critical pore region. On the N-loop, these include Lys223 (violet), Gln227 (white), Thr231 (brown), Pro232 and Pro236 (grey), and on the B-loop they include Gly348 (orange) and His350 (blue). Leu451 (black) is the only mutation to be clearly located away from the pore. Ser246, Lys249, Ile531 and Ser537 lie in regions of the protein that are not currently structurally modelled. Ó FEBS 2004 Control of aquaglyceroporin Fps1 (Eur. J. Biochem. 271) 777 The random mutations involving proline residues of the regulatory domain, P232S and P236L (both shown in grey at either end of the N-loop a-helix), are in line with our previous inferences regarding the importance of the pro- nounced secondary structure in this region. Mutation of either proline could result in reduction or increase in the length of the helical secondary structure of the exiting N-loop, affecting flexibility and function. Similarly, the introduction of a proline residue, as at S246P (not modelled) could result in marked changes in local secondary structure. Mutations involving Gly348 (orange) on loop B, con- served across the MIP family, to larger charged or polar residues, G348D, G348R, G348S could have several effects. Substitution by a larger residue could disrupt the close arrangement with nearby residues, notably Q227 (white) on the N-loop and His350 (blue) on the B-loop. The mutation of this glycine residue could also disrupt the capacity of the B-loop for membrane insertion, as it is clearly located in the interfacial environment between the membrane face and the cytosolic compartment (the orange residue, best viewed in the side-on view of the model). Indeed, the mutations to charged or polar residues have the most capacity for disrupt- ing membrane insertion. A striking finding is the greater effect observed with G348D than with G348R. This suggests that the region surrounding Gly348 requires some flexibility and distancing from His350, and perhaps to lie more intimately with Gln227 on the N-loop to regulate normally. In the model, where the beginning and end of the N-loop region are aligned so that they face TMD1 as much as possible, and with the NPQ turn of the B-loop maximally immersed in the pore cavity, there is also a notably close arrangement between Gln227 of the N-loop (shown in white) and His350 on the B-loop (coloured blue). Both these residues are highly polar, and have complementary charges for interaction with each other. The mutations of His350 (blue) caused marked effects despite changing to similarly large residues. Hence, the positive charge of His350 appears to be critical for normal function. In this regard, its proximity in the model to the negative dipole of Gln227 is of significant interest. His350 is also conserved across the MIP family. Indeed, the random mutations reported here regarding Gly348 and His350 are likely to be of more general relevance for the understanding of the structure-function relationship of the MIP family of channel proteins. Acknowledgements This work was supported by the European Commission contract QLR3-CT2000-00778 and the Human Frontier Science Foundation. S.H. holds a research position of the Swedish Research Council. References 1. Preston, G.M., Carroll, T.P., Guggino, W.B. & Agre, P. (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256, 385–387. 2. Borgnia, M., Nielsen, S., Engel, A. & Agre, P. (1999) Cellular and molecular biology of the aquaporin water channels. Annu. Rev. Biochem. 68, 425–458. 3. Hohmann,S.,Nielsen,S.&Agre,P.(2001)Aquaporins. Academic Press, San Diego, CA. 4. Deen, P.M., Marr, N., Kamsteeg, E.J. & van Balkom, B.W. (2000) Nephrogenic diabetes insipidus. Curr. Opin. Nephrol. Hypertens. 9, 591–595. 5. Deen, P.M.T. & Brown, D. (2001) Trafficking of native and mutant mammalian MIP proteins. In Aquaporins (Hohmann, S., Nielsen, S. & Agre, P., eds), pp. 235–277. Academic Press, San Diego, CA. 6. Chrispeels, M.J., Morillon, R., Maurel, C., Gerbeau, P., Kjellbom, P. & Johansson, I. (2001) Aquaporins in plants: struc- ture, function, regulation, and role in plant water relations. In Aquaporins (Hohmann, S., Nielsen, S. & Agre, P., eds), pp. 277– 334. Academic Press, San Diegi, CA. 7. Tournaire-Roux, C., Sutka, M., Javot, H., Gout, E., Gerbeau, P., Luu, D.T., Bligny, R. & Maurel, C. (2003) Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. Nature 425, 393–397. 8. Hohmann, S., Bill, R.M., Kayingo, I. & Prior, B.A. (2000) Microbial MIP channels. Trends Microbiol. 8, 33–38. 9. Tanghe, A., Van Dijck, P., Dumortier, F., Teunissen, A., Hohmann, S. & Thevelein, J.M. (2002) Aquaporin expression correlates with freeze tolerance in baker’s yeast, and overexpression improves freeze tolerance in industrial strains. Appl. Environ. Microbiol. 68, 5981–5989. 10. Oliveira, R., Lages, F., Silva-Graca, M. & Lucas, C. (2003) Fps1p channel is the mediator of the major part of glycerol passive dif- fusion in Saccharomyces cerevisiae: artefacts and re-definitions. Biochim. Biophys. Acta. 1613, 57–71. 11. Hohmann, S. (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol. Mol. Biol. Rev. 66, 300–372. 12. Luyten, K., Albertyn, J., Skibbe, F., Prior, B.A., Ramos, J., Thevelein, J.M. & Hohmann, S. (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. 13. Tama ´ s, M.J., Luyten, K., Sutherland, F.C.W., Hernandez, A., Albertyn,J.,Valadi,H.,Li,H.,Prior,B.A.,Kilian,S.G.,Ramos,J., Gustafsson, L., Thevelein, J.M. & Hohmann, S. (1999) Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation. Mol. Microbiol. 31, 1087–1104. 14. Philips, J. & Herskowitz, I. (1997) Osmotic balance regulates cell fusion during mating in Saccharomyces cerevisiae. J. Cell Biol. 138, 961–974. 15. Tama ´ s, M.J., Karlgren, S., Bill, R.M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J.M., Rydstrom, J., Mullins, J.G. & Hohmann, S. (2003) A short regulatory domain restricts glycerol transport through yeast Fps1p. J. Biol. Chem. 278, 6337–6345. 16. Stahlberg, H., Heymann, B., Mitsuoka, K., Fuyijoshi, Y. & Engel, A. (2001) The aquaporin superfamily: structure and function. In Aquaporins (Hohmann, S., Nielsen, S. & Agre, P., eds), pp. 39–119. Academic Press, San Diego, CA. 17. Fu, D., Libson, A., Miercke, L.J., Weitzman, C., Nollert, P., Krucinski, J. & Stroud, R.M. (2000) Structure of a glycerol- conducting channel and the basis for its selectivity. Science 290, 481–486. 18. Sui, H., Han, B.G., Lee, J.K., Walian, P. & Jap, B.K. (2001) Structural basis of water-specific transport through the AQP1 water channel. Nature 414, 872–878. 19. de Groot, B.L. & Grubmuller, H. (2001) Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF. Science 294, 2353–2357. 20. Zeuthen, T. (2001) How water molecules pass through aqua- porins. Trends Biochem. Sci. 26, 77–79. 21. de Groot, B.L., Frigato, T., Helms, V. & Grubmuller, H. (2003) The mechanism of proton exclusion in the aquaporin-1 water channel. J. Mol. Biol. 333, 279–293. 778 S. Karlgren et al. (Eur. J. Biochem. 271) Ó FEBS 2004 22. Fujiyoshi, Y., Mitsuoka, K., de Groot, B.L., Philippsen, A., Grubmuller, H., Agre, P. & Engel, A. (2002) Structure and function of water channels. Curr. Opin. Struct. Biol. 12, 509–515. 23. Hansen, M., Kun, J.F., Schultz, J.E. & Beitz, E. (2002) A single, bi-functional aquaglyceroporin in blood-stage Plasmodium falci- parum malaria parasites. J. Biol. Chem. 277, 4874–4882. 24. Bill, R.M., Hedfalk, K., Karlgren, S., Mullins, J.G., Rydstrom, J. & Hohmann, S. (2001) Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p. J. Biol. Chem. 276, 36543– 36549. 25. Reference withdrawn. 26. Ansell, R., Granath, K., Hohmann, S., Thevelein, J. & Adler, L. (1997) The two isoenzymes for yeast NAD-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2,have distinct roles in osmoadaption and redox regulation. EMBO J. 16, 2179–2187. 27. Thomas, B.J. & Rothstein, R.J. (1989) Elevated recombination rates in transcriptionally active DNA. Cell. 56, 619–630. 28. Sherman, F., Fink, G.R. & Hicks, J.B. (1983) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 29.Gietz,D.,StJean,A.,Woods,R.A.&Schiestl,R.H.(1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425. 30. Reference withdrawn. 31. Koradi, R., Billeter, M. & Wuthrich, K. (1996) MOLMOL : a pro- gram for display and analysis of macromolecular structures. J. Mol. Graph. 14 (51–5), 29–32. 32. Murata,K.,Mitsuoka,K.,Hirai,T.,Walz,T.,Agre,P.,Heymann, J.B., Engel, A. & Fujiyoshi, Y. (2000) Structural determinants of water permeation through aquaporin-1. Nature 407, 599–605. Ó FEBS 2004 Control of aquaglyceroporin Fps1 (Eur. J. Biochem. 271) 779 . Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen Sara Karlgren 1 , Caroline Filipsson 2 ,. channels: the aquaporins Aqy1 and Aqy2 and the aquaglyceroporins Fps1 and Yfl054 [8]. Aqy1 is a strictly spore-specific aquaporin while Aqy2 may play a role