aquaporins in yeasts and filamentous fungi

Biol. Cell (2005) 97, 487–500 (Printed in Great Britain) Review

Aquaporins in yeasts andfilamentous fungiNina Pettersson*, Caroline Filipsson†, Evren Becit*, Lars Brive* and Stefan Hohmann*1

*Department of Cell and Molecular Biology, Goteborg University, Box 462, S-40530 Goteborg, Sweden, and †Department of Chemistry,

Goteborg University, Box 462, S-40530 Goteborg, Sweden

Recently, genome sequences from different fungi have become available. This information reveals that yeastsand filamentous fungi possess up to five aquaporins. Functional analyses have mainly been performed in budd-ing yeast, Saccharomyces cerevisiae, which has two orthodox aquaporins and two aquaglyceroporins. WhereasAqy1 is a spore-specific water channel, Aqy2 is only expressed in proliferating cells and controlled by osmoticsignals. Fungal aquaglyceroporins often have long, poorly conserved terminal extensions and differ in the other-wise highly conserved NPA motifs, being NPX and NXA respectively. Three subgroups can be distinguished. Fps1-like proteins seem to be restricted to yeasts. Fps1, the osmogated glycerol export channel in S. cerevisiae, playsa central role in osmoregulation and determination of intracellular glycerol levels. Sequences important for gatinghave been identified within its termini. Another type of aquaglyceroporin, resembling S. cerevisiae Yfl054, has along N-terminal extension and its physiological role is currently unknown. The third group of aquaglyceroporins,only found in filamentous fungi, have extensions of variable size. Taken together, yeasts and filamentous fungi area fruitful resource to study the function, evolution, role and regulation of aquaporins, and the possibility to compareorthologous sequences from a large number of different organisms facilitates functional and structural studies.

IntroductionDuring the last few years, a number of fungalgenomes have been sequenced. This has provided op-portunities to study important principles of genomeevolution and to employ sequence conservation oforthologous proteins as a suitable tool for functionalanalysis. Comparative genomics can provide inform-ation about structurally and functionally importantresidues and domains, serving as a basis for design-ing mutational studies. In the present study, we haveinvestigated the presence and conservation of aqua-porins in fungal genomes. Orthodox aquaporins me-diate rapid and selective flux of water across biologicalmembranes and hence play important roles in theosmoregulation of cells and organisms. Aquaglycero-porins on the other hand, facilitate transmembranetransport of small uncharged molecules like polyols,urea, arsenite and many more, thereby playing roles in

1To whom correspondence should be addressed ([email protected]).Key words: Fps1, genome sequence, glycerol transport, osmoregulation,water transport.Abbreviations used: MAPK, mitogen-activated protein kinase; TMD,transmembrane domain.

nutrient uptake, osmoregulation and probably otherprocesses (Borgnia et al., 1999; Hohmann et al.,2001). The aquaglyceroporins are divided into Fps1-like (defined by a conserved regulatory region in theN-terminus), Yfl054-like (having a very long N-ter-minal extension including a conserved stretch) and athird group of proteins, not falling into any of thesecategories. Detailed studies on the precise physio-logical roles and functional properties have been re-ported only for a small number of the fungal water andglycerol channels. The present study tries to moti-vate future studies on fungal aquaporins as they bearpotential to reveal novel information on the functionand physiological roles of members of this ancientprotein family.

Yeasts and filamentous fungiFungi form one of the five kingdoms of life and are alarge and diverse group of eukaryotic organisms. Onlya very small fraction of all fungal species have beendescribed to date. Fungi, ranging from single-celledorganisms (yeasts) to the edible mushrooms, haveenormous ecological importance and they encompasspathogens for humans, animals and plants. Fungi

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thersTable 1 Fungal aquaporinsFunctional studies have been performed on the proteins from S. cerevisiae (cited in the text), S. pombe (Kayingo et al., 2004), Z. rouxii (Wang et al., 2002; Neves et al., 2004) andC. albicans (Carbrey et al., 2001b). Fps1 orthologues from K. lactis and K. marxianus complement the S. cerevisiae fps1� mutant (Neves et al., 2004). The others are predicted fromgenome sequences.

Orthodox Fps1-like Yfl054-like Other

Species aquaporins* aquaglyceroporins* aquaglyceroporins* aquaglyceroporins* Comment Names

S. cerevisiae Aqy1 (305) Fps1 (669) Yfl054 (646) S.cer Aqy1,2

Aqy2 (289) S.cer Fps1

S.cer Yfl054

S. bayanus Aqy1 (278) Fps1 (661) Yfl054 (654) S.bay Aqy1

S.bay Fps1

S.bay Yfl054

S. paradoxus Aqy1 (305) Fps1 (673) Yfl054 (648) S.par Aqy1

S.par Fps1

S.par Yfl054

S. mikatae Aqy1 (305) Fps1 (218) Yfl054 (648) Fps1 incomplete S.mik Aqy1

S.mik Fps1

S.mik Yfl054

S. kluyveri Aqy1 (266) Fps1 (427) Fps1 incomplete S.klu Aqy1

S.klu Fps1

S. kudriavzevii Aqy1 (306) Fps1 (259) Fps1 incomplete S.kud Aqy1

S.kud Fps1

S. castelii Aqy1 (245) S.cas Aqy1

S. pombe SPAC977.17 (598) S.pom SPAC977.17

K. lactis KLLA0B10010g (193) KLLA0E00550g (563) K.lac Aqp1 incomplete K.lac KLLA0B10010g

K.lac KLLA0E00550g

K. marxianus Q6QHK5 (571) No genome sequence K.mar Fps1

Z. rouxii Q6RW11 (692) No genome sequence Z.rou Fps1

K. waltii K.wal 20572 (600) K.wal 15269 (666) K.wal 20572

K.wal 15269

C. glabrata CAGL0A01221g (293) CAGL0C03267g (652) C.Gla CAGL0A01221g

CAGL0D00154g (290) CAGL0E03894g (602) C.Gla CAGL0D00154g

C.Gla CAGL0C03267g

C.Gla CAGL0E03894g

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C. albicans CaAqy1 (273) C.alb Aqy1

D. hansenii DEHA0F28787g (310) D.han DEHA0F28787g

A. gossypii AGL266C (450) Acl068w (476) A.gos AGL266C

A.gos ACL068W

Y. lipolytica YALI0F01210g (284) YALI0F00462g (392) Y.lip YALI0F01210g

YALI0E05665g (385) Y.lip YALI0F00462g

Y.lip YALI0E05665g

N. crassa NCU08052.1 (239) N.cra NCU08052.1

A. nidulans AN7168.2 (959) AN2822.2 (463) AN3915.2 (386) AN7168.2 in A.nid AN7168.2

AN7618.2 (612) AN0830.2 (286) annotation fused to A.nid AN2822.2

amino acid transporter A.nid AN7618.2

A.nid AN3915.2

A.nid AN0830.2

F. gramineum FG10816.1 (547) FG03780.1 (548) FG03248.1 (343) F.gra FG10816.1

FG00811.1 (318) F.gra FG00811.1

FG03680.1 (286) F.gra FG03680.1

F.gra FG03780.1

F.gra FG03248.1

M. grisea MG03904.4 (536) MG05880.4 (390) M.gri MG03904.4

MG04162.4 (270) M.gri MG04162.4

MG10783.4 (278) M.gri MG10783.4

M.gri MG05880.4

U. maydis UM00223.1 (420) UM01508.1 (363) U.may UM00223.1

UM02842.1 (503) UM02169.1 (396) U.may UM02842.1

UM01930.1 (570) U.may UM01508.1

U.may UM02169.1

U.may UM01930.1

*Name of the aquaporin/aquaglyceroporin is given along with the number of amino acids in parentheses.

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are useful, not only for eating, but also in vari-ous biotechnological processes, including productionof antibiotics, pharmaceutical proteins and, last butnot the least, beer, wine, cheese and bread. Becauseof these reasons, fungi have attracted the interest ofscientists. Several fungi are excellent experimentalorganisms. Recently, fungal genome sequencing hasbecome popular because of the rather small genomes(10–40 Mbp). Some 20 genomes have so far been (par-tially) sequenced and many more are in the pipeline,generating a valuable resource of sequence inform-ation for comparative genomics. Efforts will, how-ever, have to be invested into proper annotation ofthe genome information.

The yeast species whose genomes have so farbeen sequenced belong to the hemiascomycete family(Table 1). They include four Saccharomyces species: Sac-charomyces cerevisiae, S. paradoxus, S. mikatae and S. bay-anus (Kellis et al., 2004), which are closely related toeach other. Lower coverage genome information isalso available for S. kluyveri, S. kudriavzevii and S. cas-telli (Cliften et al., 2003). The genome of S. cerevisiae,the most important cellular model organism, wasalready completely sequenced 10 years ago (Goffeauet al., 1996) and is by far the best annotated. Can-dida albicans (Jones et al., 2004) is the most commonhuman pathogenic yeast, whereas C. glabrata is thesecond most important causative agent (Dujon et al.,2004). Although these two species share a commonfamily name, C. glabrata is clearly more closely re-lated to S. cerevisiae. Kluyveromyces lactis (Dujon et al.,2004) is phylogenetically placed in the middle of thehemiascomycetes and used for genetic studies andin industrial applications (the name stands for ‘milkyeast’). The K. waltii genome has recently been se-quenced for studies on genome duplications (Kelliset al., 2004). Ashbya gossypii is a filamentous yeast usedas a model to study polar growth. It has industrialimportance for the production of vitamin B2. TheA. gossypii genome is the smallest of all free-livingeukaryotes yet sequenced (9.2 Mb, encoding 4718proteins; Dietrich et al., 2004). Debaromyces hanseniiis a halotolerant yeast found on fish and salted dairyproducts (Dujon et al., 2004). Yarrowia lipolytica isan alkane-using yeast (Dujon et al., 2004). As is alsoapparent from aquaporin sequences, this yeast sharesproperties with filamentous fungi. It is used in ge-netic studies and for heterologous protein production.Finally, the fission yeast Schizosaccharomyces pombe

(Wood et al., 2002) is quite distinct from all otheryeasts (as distinct from S. cerevisiae as either of the twofrom human!) and is like budding yeast S. cerevisiae,a widely used model system in molecular cellbiology.

There are five filamentous fungi included in thepresent study, the first four all being ascomycetes(like yeasts). Neurospora crassa was first describedin 1843, as the causative agent of a mould infection inFrench bakeries. It is a multicellular fungus compris-ing 28 morphologically distinct cell types (Borkovichet al., 2004). It has a long history as an experimentalorganism: Beadle and Tatum in the 1940s discoveredthat genes affect enzymes using N. crassa genetics(Horowitz, 1991). Aspergillus nidulans is a widely usedexperimental organism (the genome sequence has notbeen published). It is a multicellular fungus in whichnuclei migrate from a central spore out into newlyformed filaments after division. Fusarium gramineum(the genome sequence has not been published), alsoknown as Gibberella zeae, is a mycotoxin-producingfilamentous fungus causing scab of wheat and barley,with big impact on U.S. agriculture in the past de-cade. Magnaporte grisea is a haploid filamentous asco-mycete with a relatively small genome (genomesequence has not been published; 40 Mb). It is anexcellent model organism for studying fungal phyto-pathogenicity and host–parasite interactions. It is thecausal agent of rice blast disease and such a big threatto the world food supply that it is considered as a bio-logical weapon. Ustilago maydis (genome sequence hasnot been published) is a basidiomycete (like commonmushrooms) inducing tumours on host plants, e.g.maize. The haploid stage can be propagated in cultureas yeast-like cells. It is an important model systemfor studies on phytopathogenicity and host–parasiteinteractions as well as for the discovery of antifungaldrugs and has been studied by plant pathologists formore than 100 years.

Aquaporin sequences in fungal genomesTable 1 summarizes the aquaporin protein sequenceswe found to be encoded in the fully sequencedgenomes of yeasts and filamentous fungi. The aqua-glyceroporins were divided into three subgroups: pro-teins similar to S. cerevisiae Fps1, proteins similar toS. cerevisiae Yfl054 and a third group of proteins thatdo not seem to resemble any of the first two.

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Aquaporins in yeasts and filamentous fungi Review

Interestingly, the number of aquaporins seemsto vary significantly even between closely relatedspecies. There are several yeasts and one of the fila-mentous fungi (N. crassa) that only possess a singleprotein of the family. Those organisms that appearto lack orthodox aquaporins (note that the genomesequences of K. marxianus and Zygosaccharomyces rouxiiare incomplete) usually possess an aquaglyceroporin.Perhaps this is a bifunctional water/solute channel,as was found in the malaria parasite Plasmodiumfalciparum (Hansen et al., 2002). Even proteins thatappear to be orthodox aquaporins based on sequencesimilarity might have a wider substrate spectrum, asshown for a Toxoplasma protein (Pavlovic-Djuranovicet al., 2003). Overall, the number of aquaporinfamily members can range from one to five, witha tendency for higher numbers in filamentous fungi.Such proteins may therefore play a role in differen-tiation processes or be expressed in different celltypes. In organisms with five aquaporins, the numberfor each subtype differs: A. nidulans has four aqua-glyceroporins and F. gramineum has three orthodoxaquaporins. It certainly would be most interestingto study the localization, expression and function ofthese proteins.

Orthodox aquaporins in SaccharomycescerevisiaeFungal orthodox aquaporins have so far been studiedonly in S. cerevisiae (Bonhivers et al., 1998; Laizeet al., 1999, 2000a, b; Carbrey et al., 2001a;Meyrial et al., 2001) and in C. albicans (Carbrey et al.,2001b). Remarkably, most of the S. cerevisiae labora-tory strains have spontaneous inactivating mutationsin both their orthodox aquaporin genes. In Aqy1,there are three point mutations in residues that areconserved among human and plant aquaporins, andone frameshift mutation leading to an 18 residuesshorter C-terminus (Bonhivers et al., 1998). Inter-estingly, truncation of the C-terminus of Aqy1 fromthe reference strain �1278b (functional aquaporins)results in increased water permeability in Xenopusoocytes (Laize et al., 1999). The AQY2 gene in mostlaboratory strains is interrupted by an 11 bp dele-tion, leading to a premature stop codon and hence anon-functional protein (Carbrey et al., 2001a, b). Inaddition, when comparing S.cer Aqy2 with Aqy2 ofS. chevaleri (probably a strain of S. cerevisiae), which is

a functional water channel when expressed in Xenopusoocytes, it differs only at position 141 [proline versusserine residue; Carbrey et al., 2001a). Substitution(P141S) improved localization of S.cer Aqy2 tothe plasma membrane. A serine or threonine residueoccurs in 11 out of 22 fungal water channels inthis position, whereas all the remaining 11 possesshydrophobic amino acids (alanine, glycine andvaline).

Investigation by a PCR approach of orthodox aqua-porin alleles in 52 laboratory, wild and industrialyeast strains showed that a functional Aqy1 allelewas present in all wild and industrial strains but onlyin a small number of laboratory strains (Laize et al.,2000b). On the other hand, the Aqy2 allele was non-functional in most strains, indicating that there seemsto be a selective pressure to maintain Aqy1 but notAqy2 (Laize et al., 2000b). This appears to fit withthe observation that most yeast species related toS. cerevisiae have only one orthodox aquaporin. On thebasis of available information, it is known that theaquaporin gene is in a syntenic position (and henceprobably functionally equivalent) to AQY1. Aqy1and Aqy2 are highly similar (88% identical) indi-cating a recent gene duplication. On the other hand,as discussed below, AQY1 and AQY2 expression isregulated differently, indicating functional special-ization. More genome sequences, especially of highsequence quality, will be needed to study aquaporinevolution in yeasts thoroughly.

Although the fungal water channels possess all theknown sequence characteristics of orthodox aqua-porins, they show a minor divergence in the NPAmotifs, which are part of the pore constriction formedby loops B and E. The water channel in Y. lipolyticahas an NPC in the B loop and F. gramineum andN. crassa have NPV in the E loop (see discussionon NPA motifs below). It appears that several fungalwater channels have N- and C-terminal extensions ofunknown function and poor sequence conservation.In the case of the N. crassa aquaporin, the C-terminalextension is almost certainly due to a sequence or an-notation mistake, as this extension by itself has simi-larity to a complete amino acid transporter with 12TMDs (transmembrane domains), probably derivedfrom a different gene.

The fungal orthodox aquaporins are an amazinglydiverse group of proteins. Overall, only 20 residuesare completely conserved. Still, BLAST searches with



these fungal aquaporins detect the other fungal waterchannels with highest similarity, even if the sequenceidentity between yeast and fungal aquaporins de-creases to approx. 35%. This suggests that fungalorthodox aquaporins have evolved from a commonancestor since the eukarya split into three kingdoms.Orthodox aquaporins from yeasts appear to be moresimilar to plant plasma membrane intrinsic proteinscompared with animal water channels, whereas thesituation is just the opposite for those of filamentousfungi.

S. cerevisiae Aqy1The AQY1 gene becomes abundantly expressed whendiploid S. cerevisiae are shifted to sporulation con-ditions (nutrient starvation; Chu et al., 1998). Thegene is only poorly expressed in haploid and diploidvegetative cells. Sporulation of diploid yeast cells istightly coupled with meiosis and results in the form-ation of four haploid spores, surrounded by an ascuswall. Spores differ from vegetative cells in their differ-ently composed cell wall, their decreased water con-tent and diminished metabolic activity. This makesthem much more resistant to harsh environmentalconditions. Overall, the yeast sporulation process re-sembles in many respects gametogenesis in animals(Engebrecht, 2003).

When a heterozygous AQY1/AQY1-GFP strainundergoes sporulation, the green fluorescent proteinsignal is detected only in two of the four spores(Sidoux-Walter et al., 2004). Therefore Aqy1 is pro-duced during the later stages of sporulation, i.e. oncespores have formed and separated from each other.Mutants lacking Aqy1 show a 30% decreased sporeviability and results suggest that this is due to eventsoccurring during spore formation rather than duringspore maintenance or germination (Sidoux-Walteret al., 2005). One possible explanation for these ob-servations is that Aqy1 plays a role in decreasing thespore water content, which is approx. half of that ofvegetative cells. This is partly due to spores accumu-lating large amounts of trehalose. Although thispotentially generates a driving force for water intothe developing spore, this force is counteracted by therigid spore wall and hence turgor pressure. Thereforethe combination of production of material inside thecell combined with a rigid wall and an active waterchannel could be a means to decrease cell/sporewater content (Sidoux-Walter et al., 2004).

S. cerevisiae Aqy2The expression of AQY2 also appears to be tightlyregulated. The gene is expressed in exponentiallygrowing cells, but not at all in resting cells (F. Sidoux-Walter and S. Hohmann, unpublished data). It hasbeen reported that AQY2 expression is stimulatedby the protein kinase A isoform Tpk2, which couldpotentially explain growth phase regulation(Robertson et al., 2000). In addition, expression ofAQY2 is diminished when cells are shifted to highosmolarity and it reappears when cells are againshifted to lower osmolarity. Down-regulation byosmoshock at least partly depends on the osmo-sensing high osmolarity glycerol MAPK (mitogen-activated protein kinase) pathways (F. Sidoux-Walterand S. Hohmann, unpublished data; Hohmann,2002). The Aqy2 protein is located mainly inthe plasma membrane, although localization in theendoplasmic reticulum occurs in laboratory strainsthat carry the Pro141 allele (see above; Carbrey et al.,2001a).

A phenotype for the deletion of AQY2, which fitswith the observed expression pattern has not been re-ported. Agre and co-workers observed that deletionof either AQY1 or AQY2 renders yeast cells moretolerant to repeated osmotic shifts (Bonhivers et al.,1998; Carbrey et al., 2001a). Such conditions are,according to our data, associated with poor expressionof both water channels. Thevelein and co-workers re-ported diminished survival of rapid freezing regimesfor deletion of either orthodox aquaporins, whereasoverexpression enhanced survival. It was not reportedwhether these two aquaporins are normally expressedunder the conditions employed in these experiments(Tanghe et al., 2002, 2004).

Aquaglyceroporins in yeasts andfilamentous fungiAquaglyceroporins efficiently facilitate specificpassive permeation of small uncharged solutes acrossbiological membranes such as glycerol, other polyols,urea, arsenite, antimonite and probably more, com-monly in preference to water (Heller et al., 1980;Maurel et al., 1994; Gerbeau et al., 1999; Fu et al.,2000; Wysocki et al., 2001; Liu et al., 2002). GlpF,the glycerol facilitator in Escherichia coli, is best stud-ied and a crystal structure of 2.2 A resolution has beenreported (Fu et al., 2000). Yeasts and filamentous



Figure 1 Cladogram of fungal aquaporinsAquaglyceroporins can be divided into three subgroups;

Fps1-like proteins (defined by a conserved regulatory region

in the N-terminus), Yfl054-like proteins (defined by a long

N-terminal extension including a conserved stretch) and a

third group containing proteins not matching any of these

criteria. As shown in the cladogram proteins divided accord-

ing to these criteria indeed cluster together. The cladogram

was made in CLUSTAL W using default parameters and then

reconstructed in ‘Tree view’. Abbreviations are explained in

Table 1.

fungi possess a range of genes that encode aqua-glyceroporins. Again, those of S. cerevisiae are beststudied; Fps1 was, in fact, one of the first five aqua-porins discovered approx. 15 years ago (Van Aelstet al., 1991). As discussed below, the role of Fps1in yeast osmoregulation is very well characterized.Genome sequencing revealed the existence of a secondyeast aquaglyceroporin with the systematic nameYfl054 (Hohmann et al., 2000).

The fungal aquaglyceroporins seem to belong tothree groups (Figure 1, Table 1): the Fps1-like pro-teins, the Yfl054-like proteins and those that do notresemble either of these groups. Whereas Fps1-like

proteins are found only in yeasts and the third grouponly in filamentous fungi, Yfl054-like proteins arefound in both.

Fps1-like proteinsFps1 plays a central role in the osmoregulation ofS. cerevisiae (Hohmann, 2002). The protein controlsthe intracellular level of glycerol, the compatibleosmolyte of proliferating yeast cells. Under hyper-osmotic conditions, yeast cells stimulate the activityof the MAPK Hog1 through an elaborate sensing-signalling system (Hohmann, 2002). Activation ofHog1 in turn stimulates, among many others, genesencoding enzymes in glycerol biosynthesis (Albertynet al., 1994; Rep et al., 2000). Together with theactivation of glycolysis (Dihazi et al., 2004), this res-ults in enhanced glycerol production. In order forglycerol to be accumulated in the cell, the activityof Fps1 diminishes under hyperosmotic conditionswithin seconds (Tamas et al., 1999). Once the cell hasaccumulated sufficient glycerol or is shifted to hypo-osmotic conditions, Fps1 opens again to release gly-cerol and hence turgor pressure (Tamas et al., 1999).This picture is consistent with phenotypes associatedwith FPS1 mutations. Deletion of FPS1 renders yeastcells sensitive to hypo-osmotic shock because ex-cessive turgor pressure cannot be released and hyper-active, unregulated Fps1 causes sensitivity tohyperosmotic stress because the lost turgor pressurecannot be restored.

There is no evidence that Fps1 requires additionalproteins for its regulation, although this needs to bedemonstrated with reconstituted protein in vitro. Sofar, however, expression and purification of Fps1 hasnot been successful. In any case, it appears that Fps1is an aquaglyceroporin whose activity is regulated byosmotic changes. This regulation requires an approx.20 amino acid long regulatory domain right in frontof the first TMD as well as a short stretch locatedright downstream of the sixth TMD (Figures 2 and3) (Tamas et al., 1999; Hedfalk et al., 2004; Karlgrenet al., 2004).

The N-terminal regulatory domain was initiallyidentified by truncation analysis. Remarkably, largeportions of the 250 residues long N-terminus couldbe removed without affecting regulation (Tamas et al.,1999, 2003). However, deletion of a short elementbetween amino acids 225 and 236, just 20 residues infront of the first TMD, rendered Fps1 constitutively



Figure 2 Conserved domains in Fps1-like proteinsLocation of the domains with reference to the topology of Fps1 (Figure 3) are as follows: (A) residues 205–280 (S. cerevisiae

sequence) correspond to the first TMD 1 and the sequences proximal to it; (B) residues 501–548 correspond to the sixth TMD

and sequences distal to it. Alignments were carried out by CLUSTAL W, using default parameters. Abbreviations are explained

in Table 1.

open. This effect can be scored easily as yeast cellsexpressing hyperactive Fps1 fail to grow on highosmolarity medium. Mutational analysis narrowedthe important region to a 12 amino acid long domain(LYQNPQTPTVLP), which is extremely well con-served among yeast species and is a part of the approx.25 amino acid stretch constituting the most con-served part within the N-termini among Fps1-likesequences (Figures 2 and 3). The distance betweenthis domain and the first TMD, which is also con-served, is important for proper channel regulation(Tamas et al., 2003).

The C-terminal regulatory domain was identifiedby employing a similar truncation analysis, whereparts of the approx. 150 residue long C-terminuswere eliminated (Hedfalk et al., 2004). This identi-fied 12 amino acids, residues 535–546, as importantfor controlling the Fps1 function. Expression of Fps1lacking this domain also resulted in delayed intracel-lular glycerol accumulation and sensitivity to hyper-osmotic conditions. The first half of this 12 aminoacid long domain (HESPVN) is very well conservedamong yeast species (Figure 2). In fact, the regu-latory elements in the N- and C-terminal extensions



Figure 3 Topology map of Fps1 highlighting important residuesConserved residues in the MIP family (�) and the C-terminal myc-tag (�) used for detection on Western blot are highlighted.

Enlarged and bold residues correspond to the regulatory domains important for proper channel closure. Squared residues (�)

were identified in a genetic screen for hyperactive mutants. Model revised by courtesy of Kristina Hedfalk, Chalmers University

of Technology, Goteborg, Sweden.

are more highly conserved compared with loops Band E.

A genetic screen for hyperactive Fps1 has contri-buted further information about residues involved inthe regulation (Karlgren et al., 2004). The screen wasbased on the use of a gpd1∆ gpd2∆ mutant that cannotproduce glycerol and therefore does not grow in thepresence of, for example, 1 M xylitol. However, incells expressing a constitutively open Fps1, xylitol canbe taken up into the cell resulting in equal concen-trations on both sides of the plasma membrane, re-lieving osmotic stress and allowing growth. This wasused to screen for random mutations rendering Fps1constitutively open. So far, mutations in 14 distinctresidues have been identified (Figure 3; Karlgrenet al., 2004). Five of those are located within the

N-terminal regulatory domain, whereas three causetruncation of the C-terminus, thereby confirmingprevious studies on the importance of the termini.Two conserved residues in the B loop also appearto be critical for channel control. Interestingly, allmutations identified hit residues that are completelyconserved among the ten aligned yeast Fps1 pro-teins. Random mutational analysis and generation ofdouble point mutations will provide a framework forfurther genetic and structural analysis to understandbetter the mechanisms that control Fps1 regulation.

The present view on how Fps1 might be regulatedis also based on a structural prediction of the con-served N-terminal regulatory domain. The conservedNPQ motif in the centre of this domain reminds ofthe NPA motif of loops B and E discussed above.



Indeed, it seems probable that the regulatory do-main can form a loop structurally similar to the Bloop (Karlgren et al., 2004). This has stimulated theidea that the regulatory domain could fold back intothe membrane and block the B loop. The observ-ation that residues in the B loop itself also contrib-ute to regulation could be accommodated in such amodel. The C-terminal regulatory domain may havea more general role on properly positioning theTMDs within the membrane and facilitate an inter-action of the N-terminal regulatory domain and theB loop. Eventually, structural information and stud-ies in a reconstituted system will be needed to fullyexplain the mechanism that controls Fps1.

By searching whole or partial genome sequences,we identified nine proteins that resemble Fps1. Inaddition, the Fps1 gene from Z. rouxii, an osmo-tolerant yeast, has been sequenced. It appears thatFps1-like proteins are restricted to yeasts since theyhave not been found so far in filamentous fungi.This is remarkable, given the important role playedby Fps1 in yeast osmoregulation and the apparentlysophisticated mechanism of its regulation. As a cri-teria for Fps1-like proteins, we used the highly con-served, unique regulatory sequence located in frontof the first TMD (no other hits in the databases),the long, variable hydrophilic extensions of differ-ent lengths and an unusually long (approx. 35 aminoacids) but poorly conserved A loop. The predictedproteins differ in length by as much as 216 aminoacids from the shortest, A. gossypii Fps1 (476 residues),to the longest, Z. rouxii Fps1 (692 residues). C. glab-rata is unique in having two Fps1 homologues. Inter-estingly, both C. glabrata proteins seem to be some-what more similar to S.cer Fps1 than to each other,which is quite unusual for paralogous genes. One pos-sible explanation could be that both C. glabrata genesoriginate from independent horizontal gene transferevents from the S. cerevisiae gene.

The six TMDs show high similarity in agree-ment with conserved residues in the aquaporin family(Park and Saier, 1996), as well as the AEF motif inTMD1 and its counterpart D in TMD6 (Zardoya andVillalba, 2001). The TMDs are flanked by conservedand often charged or polar amino acids. Althoughthe overall sequence conservation of the termini ispoor, there are some not previously recognized con-served sequence elements. In the N-terminus, thereis an acidic region around position 75 (S. cerevisiae)

followed by a conserved motif around position 110(FPIQEVIPS). In the C-terminus, there is a conservedelement (FKSV) around 75 amino acids distal fromthe sixth TMD and clustering of acidic and basicamino acids seems to be conserved as well. The se-quence of these elements does not reveal any func-tional role. Since deletion of these sections does notaffect channel regulation (Tamas et al., 2003; Hedfalket al., 2004), the conserved elements might indicateadditional, possible regulatory roles of Fps1.

Alterations in NPA motifsAmong aquaporins, the NPA motifs in loops Band E respectively are highly conserved. These loopsfold back into the membrane and form part of thecentral pore constriction (Fu et al., 2000; Nollertet al., 2001; de Groot et al., 2003). Together, theNH donor groups from the asparagine side chainsform hydrogen bonds with the hydroxy groups ofglycerol (Fu et al., 2000).

Remarkably, in fungal aquaglyceroporins, there areseveral alterations in the NPA motifs (Figure 4A).In the B loop, only NP is perfectly conserved (oneexception, which shows an HPA), and in the E loop,only the asparagine and alanine residues are conserved(one exception with an NPS). In the B loop, the thirdposition of the motif can be taken by alanine, ser-ine, threonine or isoleucine, the central position ofthe motif in the E loop can be taken by proline,leucine, methionine, phenylalanine, glycine or alan-ine residue. Since the asparagine residues crucial forglycerol transport are perfectly conserved in fungalaquaglyceroporins, a similar glycerol pathway as forGlpF can be anticipated. It has been shown that Fps1(S. cerevisiae) tolerates ‘restored’ NPA in both loops(Bill et al., 2001), which is in line with the fact thatonly the asparagine residues are in contact with gly-cerol. At the same time, GlpF does not tolerate theNPS and NLA sequences found in Fps1, indicatingthat altered NPA motifs are compensated by otherchanges (Bill et al., 2001). Several other residues im-portant for glycerol transport, i.e. Arg206 in the con-striction region of GlpF (Sui et al., 2001) and residuesknown to interact with glycerol (Fu et al., 2000) arewell conserved in fungal aquaglyceroporins.

The strictly conserved proline and alanine (Pro/Ala)residues are Pro69 (Pro353) and Ala205 (Ala482) in the



Figure 4 (A) Sequences of loops B and E of fungalaquaglyceroporins, showing alterations in theNPA motifsAlignments were carried out by CLUSTAL W, using default

parameters. Abbreviations are explained in Table 1. (B) Struc-

tural details of the region near the Asn-Pro-Ala motifs in the

crystal structure of E. coli GlpF (PDB code 1fx8; Fu et al.,

2000). The view is approximately along the quasi-2-fold sym-

metry axis, along the centre of the membrane plane, look-

ing towards the core of the protein. The periplasmic side is

up. The thin black line separates the well-conserved Pro/

Ala pair (right of the line; Pro69/Ala205) from the less-conserved

pair (left; Pro204/Ala70). Selected residues are shown as stick

models, with those of the NPA motifs having green carbons.

Two hydrogen bonds from Glu14 to the B loop main-chain

amides of residues His66 and Leu67 (grey carbons) and one

hydrogen bond to Gln93 are indicated with grey lines. Also

shown is the selectivity filter residue Arg206 (grey carbons).

Glycerol molecules in the channel are shown as space-filling

models.

B and E loop NPA motifs respectively (numberedaccording to GlpF with Fps1 numbers in paren-theses). The less conserved Pro/Ala pair is composedof Ala70 (Ser354) and Pro204 (Leu481) residues. In con-trast with the asparagine residues of the NPA motifs,the proline and alanine residues do not contribute tothe channel surface, but are located roughly 7 A awayfrom the pore.

A possible explanation for the difference in se-quence conservation lies in the asymmetry of the localenvironment near the NPA motifs. The two Pro/Alapairs are located close in space near the quasi-2-foldaxis, illustrated in Figure 4(B). Within van der Waalscontact distance from the side chains of the well-conserved Pro/Ala pair are Val94 (Leu378) and thestrictly conserved Gln93 (Gln377) in TMD3, as wellas the remainder of the NPA residues. Gln93 (Gln377)is buried in the protein core, and hydrogen bondsto Glu14 (Glu259), which is also well buried and in-volved in an extensive hydrogen bond network. Hy-drogen bonds are also present between the side-chaincarboxy group of Glu14 (Glu259) and the backboneamides of His66 (His350) and Leu67 (Leu351), whosecarbonyl groups form hydrogen bonds with glycerolmolecules that traverse the channel. A mutation ofPro69 (Pro353) or Ala205 (Ala482) may therefore res-ult in structural changes that propagate through thehydrogen bond network and alter the function of thechannel. In addition, changes in backbone geometryat Ala205 (Ala482) would also affect the succeedingresidue Arg206 (Arg483), which contributes directlyto the selectivity filter and is strictly conserved.

The residues within van der Waals contact distancefrom the side chains of the less well-conserved Pro/Ala pair [Ala70 (Ser354)/Pro204 (Leu481)] are Pro240

(Pro516), Ile241 (Phe517) and Ala244 (Ala519) in TMD6.Side chains of none of these residues line the channel



or contain polar groups and are therefore expected tointeract less rigidly with the channel residues andto be more tolerant to structural variation withoutloss of channel function. Ala70 (Ser354) is succeededin sequence by Val71 (Ile355), which contributes tothe channel surface but in an unspecific manner, re-flected in its replacement by other hydrophobicresidues across species. The substitution of Ala70

(Ser354) or Pro204 (Leu481) can therefore lead to a func-tional channel and may be a way to fine-tune channelproperties.

Yfl054-like aquaglyceroporinsYfl054 was identified following sequencing of thegenome of S. cerevisiae. Proteins similar to Yfl054 havebeen found by database searches in other yeasts anda Yfl054-like protein seems to be the only aquaporinin the fission yeast S. pombe.

Yfl054-like proteins are characterized by an ap-prox. 350 amino acids long N-terminal extension andan approx. 50 amino acid C-terminal extension. Thecore transmembrane part is strikingly well conservedamong the different yeast proteins, even when includ-ing the homologue from the only distantly relatedfission yeast. The long N-terminal extension is lesswell conserved but shows one stretch of signifi-cant sequence similarity. This sequence (PVWSLNQ-PLPV) is perfectly conserved among yeasts, whereasit is partly conserved for the filamentous fungi (PV-WSLXXPLPV for A. nidulans and PXXSLXXPLPXfor F. gramineum). However, the non-conserved re-sidues generally have the same chemical properties asthe yeast sequence.

There have been attempts to associate a physio-logical role to S. cerevisiae Yfl054 and S. pombeSPAC977.17, so far without success (M.J. Tamasand S. Hohmann, unpublished data; Kayingo et al.,2004), although one report indicated that it couldhave a function redundant to Fps1 in ethanol-affectedglycerol transport (Oliveira et al., 2003). Attemptsto identify the cellular membrane in which Yfl054 islocalized have also been unsuccessful. It seems, how-ever, that Yfl054 has a function that is distinct fromthat of Fps1. Lucas and co-workers reported that de-letion of either FPS1 or YFL054 causes enhancedpassive diffusion of ethanol (Oliveira et al., 2003),which could be due to altered membrane compo-sition. Such an effect has been reported for the dele-tion of FPS1 (Toh et al., 2001).

Given the conservation of domain structure and se-quence, it is clear that Yfl054 confers a specific role as-sociated with transmembrane solute fluxes and it maybe involved in regulatory processes through its longN-terminus. More work is needed to decipher suchroles.

Other aquaglyceroporinsThe third group of aquaglyceroporins is found onlyin filamentous fungi. This group appears to consist ofmembers that share limited sequence similarity anddiffer in size. Whereas BLAST searches using suchaquaglyceroporin sequences identify members of thisgroup among the top scores, other aquaglyceroporins,such as those from Trypanosoma or Yfl054 scoreequally high. Several members of this group alsohave long extensions, especially at the N-terminus,but those appear to be unrelated to that of Yfl054.Although the Trypanosoma proteins have recentlybeen characterized (Uzcategui et al., 2004), to ourknowledge no research has been performed on thesefungal proteins and hence nothing is known aboutexpression patterns, localization, cell-type specificityor physiological function.

ConclusionsFungal aquaporins seem to be quite a diverse groupof proteins with unique functional, regulatory andphysiological properties. When genome sequences ofsufficient coverage and quality become available, itwill again be possible to draw more detailed pic-tures of the evolution and occurrence of aquaporinsin fungi. What is clearly needed are further studieson the function, expression, localization and physio-logical roles of fungal aquaporins, which seems per-fectly feasible given the fact that several of the fungilisted in the present study are experimental organ-isms in which genetic manipulation can be performedfast and with high precision. On the other hand,given the difficulty in associating physiological func-tions to several of the yeast aquaporins, it apparentlyrequires some scientific creativity to elucidate theprecise cellular role of these proteins. In the labor-atory, aquaporins are not usually needed for survivalor fitness.

This should, however, rather encourage furtherstudies on fungal aquaporins. Given the diversity ofthese proteins and the knowledge collected so far,for instance on yeast Fps1, it is clear that studies on



yeast and filamentous fungi bear potential to discovernew principles of aquaporin regulation and physio-logical function, as well as osmoregulation and cellphysiology in general.

Sequence filesThe sequences used in the present study as well asseveral multiple alignments can be viewed and down-loaded at http://www.gmm.gu.se/groups/hohmann/fungalMIP.

AcknowledgmentsThis work was supported by grants from the EuropeanCommission (QLK3-CT2000-00778, QLK3-CT2000-52116 and QLK3-CT2001-00987), theHuman Frontier Science Organisation (RG-0021-2000-M) and the Swedish Research Council (researchposition to S.H.).

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Received 24 September 2004; accepted 30 October 2004

Published on the Internet 23 June 2005, DOI 10.1042/BC20040144


aquaporins in yeasts and filamentous fungi

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