Comparative Analysis of Sequences, Polymorphisms and Topology of Yeasts Aquaporins and Aquaglyceroporins

FEMS Yeast Research, 16, 2016, fow025

doi: 10.1093/femsyr/fow025 Advance Access Publication Date: 20 March 2016 Minireview

MINIREVIEW

Comparative analysis of sequences, polymorphisms Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 and topology of yeasts aquaporins and aquaglyceroporins Farzana Sabir1,2,∗,†, Maria C. Loureiro-Dias1 and Catarina Prista1

1LEAF, Instituto Superior de Agronomia, University of Lisbon, Tapada da Ajuda, 1349-017 Lisbon, Portugal and 2Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, 1649-003 Lisbon, Portugal

∗Corresponding author: LEAF, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal. Tel: (+351)-213653207; Fax: (+351)-213653195; E-mail: [email protected] One sentence summary: This review summarizes a detailed analysis of water and glycerol channels in yeasts and explores the relationship between their existence and adaptation of yeasts in various ecological niches. Editor: Jens Nielsen †Farzana Sabir, http://orcid.org/0000-0002-0181-989X

ABSTRACT

Efficient homeostasis of water and glycerol is a prerequisite for osmoregulation and other aspects of yeasts life. The cellular status of these molecules is often associated with functional presence of aquaporins and aquaglyceroporins. The present study provides a detailed updated analysis of aquaporins and aquaglyceroporins in 47 yeast species. A comprehensive analysis of aquaporins and aquaglyceroporins in 38 strains of Saccharomyces cerevisiae from different ecological niches is also presented. The functionality of specific aquaporins in yeasts has been associated with their adaptation requirements in different environmental conditions. In the present study, various inactivating mutations in aquaporin sequences were found in strains of S. cerevisiae. Likewise, several new interesting polymorphisms in aquaglyceroporin sequences of some commercial wine and brewing strains, vineyard and bakery strains were also observed. Conceivably, both in the case of aquaporins and aquaglyceroporins inactivating mutations resulted in competitive advantage in selected environments. Topology and conservation of important regulatory residues within all sequences are also analyzed. We expect that the present review may contribute to establish the functional relevance of aquaporins/aquaglyceroporins for various aspects of yeasts physiology.

Keywords: Saccharomyces cerevisiae; yeasts; aquaporin; aquaglyceroporin; water transport

INTRODUCTION rapid membrane water transport through water channels was suggested in mammalian red blood cells (Paganelli and Solomon Water equilibrium is a fundamental necessity of all living organ- 1957). Only after 30 years, the discovery by Agre and co-workers isms for maintenance of their cellular shape and turgor, and also of the first water channel in red blood cells known as aqua- for providing suitable conditions for various intracellular bio- porin 1 (AQP1) (Preston et al. 1992) popularized the concept of chemical processes. Formerly, simple diffusion through lipid bi- facilitated membrane water transport through aquaporins. Dur- layer was assumed to be the only mode of water flux in cells. In ing the following 20 years, numerous remarkable breakthroughs late 1950s, a vague idea of existence of an alternative route for

1 2 FEMS Yeast Research, 2016, Vol. 16, No. 3

have been achieved to understand the structural and func- The number of aquaporins present in various yeasts is gen- tional component of membrane water transport in almost all life erally restricted to one, with the exception of S. cerevisiae and forms including protozoa, bacteria, fungi, plants and animals Candida glabrata (Soveral et al. 2011). Retention of both genes of (Beitz 2005; Kaldenhoff and Fischer 2006; Kruse, Uehlein and orthodox aquaporins in S. cerevisiae is probably a consequence Kaldenhoff 2006; Nehls and Dietz 2014). Now, aquaporins are of the whole-genome duplication, resulting in the prototype unambiguously considered as the molecular component of fast with ∼10 000 genes (Wolfe and Shields 1997). Further, this an- reversible transmembrane water flux. They provide the unique cestor massively lost around 85% of duplicated copies, result- molecular entry point of water (Maurel and Chrispeels 2001), de- ing in modern-day species with ∼6000 genes, of which only pending on its chemical potential gradient (osmotic and/or hy- around 15% are duplicated copies. Why these remaining dupli- drostatic pressure) between either side of the membrane, and cated copies are still retained in the genome? Two possible func- their function seems crucial particularly at lower temperature tional fates have been proposed: (1) neo-functionalization, when (Soveral et al. 2006). They selectively allow rapid inward and out- the duplicated paralog acquires a novel function or altered reg-

ward flux of uncharged water molecules while excluding ions ulatory responses, while the other one preserved the ancestral Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 and other solutes from passing through. Maintenance of cellu- function; (2) sub-functionalization, in which the functions of the lar volume and internal osmotic pressure, through aquaporin ancestral gene were distributed between the paralogs (Botstein gating, are regulated by various factors, such as pH, phospho- and Fink 2011). Expression analysis suggests that both yeast rylation, pressure, tension, solute gradients and temperature Aqy1 and Aqy2 are involved in similar but specific functions and (Chaumont, Moshelion and Daniels 2005; Leitao˜ et al. 2012; are somehow differently regulated (Ahmadpour et al. 2014). Soveral et al. 2006, 2008). The present review focuses on the occurrence of MIPs in var- Aquaporins belong to a highly conserved group of the major ious yeast species as well as in different strains of S. cerevisiae. intrinsic proteins (MIP) family, comprising more than 1700 in- Various polymorphisms in these sequences leading to inacti- tegral membrane proteins (Abascal, Irisarri and Zardoya 2014). vating mutations and non-functional alleles are explored. Their MIP-related proteins configure channels across the biological topology and regulatory domains are also analyzed. membrane to facilitate the bidirectional flux of water and small solutes like glycerol (Zardoya et al. 2002). MIP family members are divided in three groups: (1) aquaporins (orthodox, ordinary, GENES ENCODING MIPs SEQUENCES IN conventional or classical aquaporins); (2) aquaglyceroporins (un- YEASTS conventional or heterodox aquaporins); and (3) aquaporins with Genome sequencing studies have revealed that yeasts have unusual NPA (Asn-Pro-Ala) boxes (unorthodox, superaquaporins two putative orthodox aquaporins encoding genes, AQY1 (ORF or subcellular aquaporins). Members of aquaporin group specif- YPR192w)andAQY2 (ORF YLL052c-YLL053c) and two aquaglyc- ically facilitate water transport (Takata, Matsuzaki and Tajika eroporin encoding genes, FPS1 (ORF YLL043W)andYFL054C 2004), whereas aquaglyceroporins besides water majorly trans- (Bonhivers et al. 1998;Carbreyet al. 2001). For analyses of port glycerol (Luyten et al. 1995). Many lines of evidence sug- aquaglyceroporin sequences in the present study, we focused on gest that apart from glycerol, other small solutes, such as ar- Fps1, which is considered the main glycerol transporter (Nehls senite and antimony (Wysocki et al. 2001), acetic acid (Mol- and Dietz 2014). lapour and Piper 2007), ethanol (Teixeira et al. 2009), polyols Protein sequences of orthodox aquaporins (Aqy1 and Aqy2) (Karlgren et al. 2004) and boron (Nozawa et al. 2006), can also be and of aquaglyceroporin (Fps1) within the yeasts genome transported through aquaglyceroporins in yeasts. The third sub- were searched by BLASTp and tBLASTn tools from the avail- family, known as superaquaporins has been recently described able genome databases at Saccharomyces Genome Database and has been only found in multicellular organisms includ- (SGD, http://www.yeastgenome.org), Genolevures´ (http://www. ing nematodes, insects and vertebrates (Ishibashi, Tanaka and genolevures.org) and NCBI (http://www.ncbi.nlm.nih.gov). Ob- Morishita 2014). In mammals they have been found in kidney, tained sequences were compared with the sequences of func- liver, sperm, thymus and brain cells at the membranes of in- tional versions of orthodox aquaporins and aquaglyceroporin tracellular organelles like lysosome, mitochondria and endo- proteins of S. cerevisiae 1278B strain. Detailed compilations of plasmic reticulum. Although their exact function is still un- MIP sequences obtained in the genomes of 47 yeast species and known, studies indicate their role in intracellular water trans- of 38 strains of S. cerevisiae identified from various niches are port (Ishibashi, Tanaka and Morishita 2014). presented in Tables 1 and 2, respectively. Sequences annotated Yeasts are important life forms for humans; their association in this study were designated according to their higher percent- to mankind is as ancient as the history of bread, beer and wine. age identity with ScAqy1, ScAqy2 or ScFps1. All the complete or- They have enormous economical impact through the commer- thodox aquaporins and aquaglyceroporin sequences were used cial production of fermented foods and of bioethanol as well as to construct phylogenetic trees by MEGA5.1 software using various pharmaceutical compounds. In last few decades, yeasts neighbor-joining method (Tamura et al. 2011). Phylogenetic re- have drawn attention of researchers as model system and tools lations among the MIP sequences of yeast species are presented to study numerous biotechnological areas (Botstein and Fink in Fig. 1, whereas distances among MIP sequences of S. cerevisiae 2011). Advancements in full genome sequencing of yeasts may strains are presented in Fig. 2. provide great opportunity to look deeper into the molecular ba- sis of water transport in these important life forms. Four MIP sequences, two orthodox aquaporins (ScAQY1 and ScAQY2)and MIPs sequences in yeast species two aquaglyceroporins (ScFPS1 and ScYFL054C), have been identified in the Saccharomyces cerevisiae genome. Aqy and Fps1 pro- Within all available yeast species genomes, 68 MIP sequences teins have been localized in the plasma membrane, whereas were identified, out of which, 43 sequences have resemblance Yfl054c (recently named as Aqy3) is supposed to be localized in with orthodox aquaporins while the other 25 sequences were the vacuolar membrane (Patel 2013). more similar to aquaglyceroporins. The phylogenetic tree (Fig. 1) Sabir et al. 3 ) ) et al. 2011 2011 ), reanalyzed ) ) ) ), Tanghe ) ) ), reanalyzed in ) ) ) ) ) ) ) ) 2005 ) 2011 ( ) ( 2004 2004 2001 ( ( 2011 2011 2011 ( ( ( ( 2012 2004 2004 2004 2004 2004 2004 2009 ) and were designated ( 2005 ( ( ( ( ( ( ( ( 2014 et al. et al. ( et al. et al. t proteins and transcripts et al. et al. et al. et al. 1994 et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. ) sequences as subject sequence 2005 Gibson this work ( in this work Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 S. cerevisiae ScFps1 References % identity with ScAqy2 % identity with 53 52 2156 Carbrey 5953 32 52 NCBI 21 Butler ScAqy1 % identity with c a c ––––Soveral 444824.1445420.1 72 71 69 68 24 22 Dujon Dujon 445318.1 21 25 51461517.2 Dujon 39 41 21 Dujon 453974.1 19 23 47 Dujon 715831.1 number 003645093.1 52 53 23 Wendland and Walther ( 003865862.1 52 55 24 NCBI 002421855.1 53 52006685162.1 20 54 NCBI 52 22003956520.1003959066.1 Wohlbach 64 22 66 26 22 57 NCBI NCBI 003648500.1 18 20 45 Wendland and Walther ( 002619875.1 Accession CpAqy2 147 CCE41463.1 AgFps1 476 AAS51160.2 20 22 45 Dietrich CgAqy2.2CgFps1 290 652 XP XP CmFps1Notfound––––Analyzedinthiswork DhFps1Notfound––––Prista DbFpNotfound––––Analyzedinthiswork CdFps1Notfound––––Analyzedinthiswork CoFps1Notfound––––Analyzedinthiswork KaFps1 638 XP CdAqy1CgAqy2.1 273 293 XP XP CtAqy1 268 XP DhAqy2 310KaAqy2KnAqy1 XP 295 291 XP CCK71445.1 70 69 20 Gordon Sequence designation Length (aa) CpFps1Notfound––––Analyzedinthiswork EcFps1 516 XP CtFps1CtrFps1Notfound––––Analyzedinthiswork Not found – – Analyzed in this work KnFps1 651 CCK72370.1 20 23 52 Gordon ClFps1Notfound––––Analyzedinthiswork KlFps1 563 XP CaFps1Notfound––––Pettersson CfFps1Notfound––––Analyzedinthiswork T T T T T T ATCC10895 AgAqy2 307 NP˙986401.2 51 52 16 Dietrich Co 90–125 CoAqy1 272 XP AWRI1499DBVPG#7215 DbAqy2 EcAqy2 273 278 EIF49326.1 XP 56 57 22 Curtin CBS138 CATCC CBS 8797 SC5314 CaAqy1 273 XP ATCC10573 YJS4271 CfAqy1 232 CDR39718.1 57 55 22 Freel Xu316 CmAqy2 269 EMG48197.1 50 51 23 NCBI MYA-4646 MYA-3404 CtrAqyATCC42720 ClAqy1 Not found 202 XP NRRL Y-1140 KlAqy1 273 CAH02367.2 54 53 23 Dujon CBS7987 CBS767 fabianii ) ) CBS 2517 naganishii gossypii ) ) Hansenula ( lusitaniae K. africanus ) ( Yeast aquaporins. Yeast aquaporin sequences searched by NCBI BLASTp and tBLASTn alignments with ScAqy1, ScAqy2 and ScFps1 protein sequences agains Eremothecium Candida Saccharomyces ( ( ( according to their higher percent ofwith identity threshold with E-value them. of A 5e- Blastp 112. was performed using sequences identified in thepresent studyquerry as sequence with Table 1. of the available genome database, respectively. Obtained sequences were compared with ScAqy1, ScAqy2 and ScFps1 on CLUSTAL W (Thompson, Higgins and C. orthopsilosis E.cymbalariae C. glabrata C. parapsilosis K. africana SpeciesA. C. albicans Strain C. tenuis C. maltosa Dekkera bruxellensis C. tropicalis C. K. Cyberlindnera K. lactis C. dubliniensis D. hansenii 4 FEMS Yeast Research, 2016, Vol. 16, No. 3 . . et al. et al et al ´ e ´ e ) ) ) ), Sabir ), reanalyzed ), Laiz 2006 ), Laiz ( ), Daniels, 2006 ) ) ) ) ) ) ), reanalyzed in ), reanalyzed in ) 2009 ) ( 2005 2005 1998 ( ( ( 1998 2003 et al. ( ) ) 2001 2000 2011 2011 2011 2011 2011 2011 2011 ( 2007 ( ( ( ( ( ( ( ( ( et al. et al. et al. et al. et al. 2013 2013 et al. ( ( et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. ), Soveral ) ) et al. et al. 2000 2016 2000 ( this work this work Wood and Yeager ( ( in this work ( Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 ScFps1 References % identity with ScAqy2 % identity with 5518 5692 2121 20 88 36 25 Karlsson 87 De Schutter 20 2487 92 10060 Souciet 25 Pettersson 22 99 25 58 100 Bonhivers 24 25 Pettersson 20 Carbrey 54 Soveral Analyzed in this work 100 87 20 Bonhivers ScAqy1 b b b % identity with b c a a b a a number 001383665.1 53 52 21 Jeffries 003677997.1 68 78 23 Gordon 001527487.1004205422.1 54003668934.1003671440.1 41 68 18 56 42 70 20 24 21 NCBI 24 NCBI 62 Gordon Gordon 003673249.1 22 24 54 Gordon 002553728.1 21 25 46 NCBI 002492992.1 Accession 002494229.1 AACE03000006 AACG02000085.1 ACVY01000031.1 AACE03000008.2 | | | | gb gb gb gb ScaFps1 679 XP LeFps1MfFps1 Not found Not found – – – – – – – – Analyzed in this work Analyzed in this work Sequence designation Length (aa) KmFps1KcAqy2KcFps1LtAqy 572 269LtFps1 Not foundLeAqy2 Not foundMfAqy2 614 BAO42722.1 275NdAqy2 CDK29538.1 – 291OpAqy1 – 19 302PgAqy 55 XP XP 260PgFps1 XP – Not found 20 – XP 56 Not found ESX00840.1SaAqy1 – – 44SbAqy1 – 20 58 – 305 NCBI ScaAqy2 274 NCBI – – – 245 57 – EJS41248.1 Analyzed in this work Soveral AL401051.1 SchAqy2 – XP 91 22SkAqy1 – 289 NCBI 277 – 89 – AAG53971.1 Soveral Analyzed in this work 23 Liti SbFps1 661 ScFps1 669 SkFps1 572 NdFps1 722 XP PsFps1SaFps1 Not found 663 – EJS42811.1 – 21 – 26 94 – Liti Analyzed in this work PpFps1 326 XP SchFps1 Not found – – – – Analyzed in this work ScAqy2 289 P0CD89 T T T T T T T T T T T T 1278B ScAqy1 327 P0CD92 CBS 6340 CBS 1993 NRRL YB-4239 ATCC 20864 PpAqy1 279 XP CBS 421 DL-1 DMKU3-1042 KmAqy2 309 BAO39643.1 51CBS 566 55 23 NCBI CBS 6054 PsAqy1 267 XP CBS 10644 CBS 4309 CBS 3082 ´ OpFps1 Not foundCBS 7002 – – – – Analyzed in this work CBS 400 ) CBS 7064 parapolymorpha ). ) stipitis castellii thermotolerans ) ) capsulata ) pastoris guilliermondii ) ) ) kluyveri ) P. Sorbitophila ( Pichia Continued ( ( Hansenula ( Naumovozyma Lachancea Kluyveromyces Komagataella Meyerozyma Scheffersomyces ( ( ( ( ( ( Table 1. M. farinosa N. dairenensis SpeciesK. marxianus Kuraishia L. Lodderomyces elongisporus Strain P. Ogataea S. cerevisiae P. S. arboricolus P. S. bayanus S. S. chevalieri S. Sabir et al. 5 et al. ), reanalyzed ), reanalyzed ), reanalyzed ), reanalyzed ), Sabir ), reanalyzed ) ) ) ) ), reanalyzed in ) ) ), reanalyzed in ), reanalyzed in ) ) ) 2005 2005 2005 2005 2005 2005 2011 ( ( ( ( ( ( ) ( 2007 ( 2011 2011 2011 2011 2011 2011 2011 ( ( 2003 2003 ( ( ( ( ( 2004 ( ( ( 2016 et al. et al. et al. et al. et al. et al. ( et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. ) 2016 in this work this work in this work in this work in this work ( this work this work in this work Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 ScFps1 References % identity with ScAqy2 % identity with 159819 1498 8820 2586 89 8920 21 25 Pettersson 95 91 Pettersson 21 25 Analyzed in this work 98 Soveral 27 Pettersson 99 Soveral Sabir 19 20 35 NCBI ScAqy1 b b b b % identity with b,c b b c ––––Soveral 504854.1 48 47 26 Dujon number 007373966.1004179361.1004182287.1 51 49 61 52 51 62 24 20 Wohlbach 23 NCBI NCBI 004180716.1003681451.1 19 66001642816.1 19 22 65 20 60 20 NCBI Gordon 54 Scannell 002498330.1 20 21 52 NCBI Accession AABY01000011 AACI03001126.1 AABZ01000399.1 AACH01001150.1 AABY01000032.1 ABPO01000065 AZAA01000411.1 | | | | | | | gb gb gb gb gb gb gb Type strain. T Incomplete sequence and c YlFps1Notfound––––Pettersson SppaFps1Notfound––––Analyzedinthiswork TbAqy2.2TbFps1 295 603 XP XP SkuAqy1SkuAqy2SkuFps1 306SmAqy1 289 259 305 EJT42783.1 EJT44804.1 91 87 86 94SppaAqy2 24 24TbAqy2.1 271 Cliften Cliften 344TdAqy1VpAqyNotfound––––Soveral XP 311WcAqy1 XP WcFps1 278 XP 422ZrAqyNotfound––––Analyzedinthiswork ZrFps1ZbAqyNotfound––––Pettersson CCH44986.1 692 CCH44822.1 55 XP 54 23 NCBI Sequence designation Length (aa) TdFps1 643 CCE93548.1 19 23 61 Gordon VpFps1 602 XP SmFps1 623 SpFps1SpaAqy2 673 SpaFps1 289 669 SchjFps1SchpFps1Notfound––––Pettersson Not found – Analyzed in this work T Genomic sequence, T T T T T b T T T CBS 6284 CBS 111 CBS 8840 CBS 432 SpAqy1 278 CLIB122 YlAqy1 284 XP CBS 1146 CBS 2163 CBS 732 yFS275SchjAqyNotfound––––Soveral ATCC 24843 SchpAqyCBS 10155 Not found CBS 680 CBS 8839 Weihenstephan 34/70 ). polyspora ) ciferrii ) Continued ( Hansenula ( Kluyveromyces ( Protein characterised for water transport activity, Table 1. a T. blattae SpeciesS. kudriavzevi Strain S. paradoxus Y. lipolytica T. delbrueckii V. W. Z. rouxii Z. bailii Sch. japonicus Sch. pombe S. mikatae S. pastorianus Spathaspora passalidarum 6 FEMS Yeast Research, 2016, Vol. 16, No. 3 ), ), ) ) ) ) 1998 1998 ), Soveral ) ( ( 1997 1997 ) ) ) ) ) ) ) ) ) ) ) ( ( 1997 ( 2012 ( 2000 2000 et al. et al. 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2007 ( ( ( ( ( ( ( ( ( ( .( .( ) et al. et al. ( et al. et al. et al et al 2007 et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. ´ ´ e e et al. Bonhivers Laiz References for sequences Bussey Will Will Bonhivers Laiz Will Wei a , originated from different niches. Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 insertion insertion insertion insertion NCBI insertion insertion Will insertion 789 789 789 789 789 789 789 P255T substitutions, A substitution, A N47D, R42K, V53A substitutions, A P255T substitutions, A P255T substitutions, A water transport water transport S. cerevisiae ScFps1 Observations 1278B % identity with ScAqy2 1278B % Identity with ScAqy1 20 2520 9987 2520 100 99 Analyzed 25 in this work Contig109 25 Analyzed in this work Functional for 100 Analyzed in this work 100 87 20 Functional for b b 1278B % identity with b c c 015518.1 96 87 21 N47D, V121M, 013048.1 85 99 23 11-bp deletion Johnston 013057.1 20 25 99 Johnston number Accession ACVY01000031.1 AEHG01000091.1 | | gb gb . The table lists the aquaporins and aquaglyceroporin sequences detected in various strains of ScAqy1 327 P0CD92 ScAqy2 289ScFps1 669 P0CD89 ScAqy1 327 GQ848562.1 99 87 20 Will ScAqy1 305 ADC55546.1ScAqy1 96 305ScAqy1 305 ADC55547.1 87 A6ZX66.1 97 21 95 87 V121M 86 21 A 21 V121M, P255T, ScAqy2ScFps1NA–––– 149ScAqy2ScFps1 289ScAqy2 ADC55263.1ScFps1NA–––– 667 288 ADC55259.1 69 AJV79976 ADC55258.1 86 20 79 86 99 25 24 99 11-bp 25 deletion Will 99 25 Will NCBI Will ScAqy1 305 ADC55541.1 95 87 21 N47D, V121M, ScAqy2ScFps1 149 669 ADC55272.1 JRIS01000073.1 69 79 24 11-bp deletion Will ScAqy1 305 NP ScAqy2ScFps1 149 669 NP NP ScAqy1 305 ADC55556.1 96 87 21 N47D, V121M, ScAqy2ScFps1 149 669 ADC55274.1 ALAV01000217.1 20 69 25 80 100 24 11-bp deletion Will Ralser ScAqy1 258 EIW08659.1 84 99 21 A ScFps1 669 1278B 1278B 1278B S. cerevisiae Sequence designation Length (aa) W303 S288c CEN YJM454 YJM627 YJM436 YJM627 YJM454 BY4741 S288c BY4741 W303 YJM436 Aquaporins in different strains of 1278B Laboratory strain Table 2. StrainS288c Provenience Laboratory strain S288c YJM627YJM454 Clinical isolate France YJM627 YJM789 Clinical isolate USA YJM454 Clinical isolate USA YJM789 BY4741 Laboratory strain BY4741 CEN.PK113-7D Laboratory strain CEN W303 Laboratory strain W303 YJM436 Clinical isolate USA YJM436 Sabir et al. 7 ) ) 2011 2011 ( ( ) ) ) ) ) ) ) ) ) ) ) ) ) ) 2009 2009 2009 2016 et al. et al. ( ( ( 2014 2014 2014 2014 ( 2010 2010 2010 2010 ( ( ( ( 2007 2007 ( ( ( ( ( ( et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Will Will Borneman Novo Treu Borneman References for sequences Treu Analyzed in this work Sabir 40 a Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 Insertions insertion Analyzed in this work insertion, 789 1079 1286 P308S, G226S substitutions P308S substitutions P308S substitutions P308S substitutions P308S, G226S substitutions R42K, V53A substitutions P308S substitutions bp) deletion, G T P308S substitutions ScFps1 Observations 1278B % identity with ScAqy2 1278B % Identity with ScAqy1 20 2515 99 Contig00539 17 Treu 99 9020 86 N-terminal (208 25 20 R42K, V53A, 14 9920 17 NCBI 25 95 T 99 Analyzed in this work 1278B b b % identity with b b b b number EGA77792.1 Accession | AAEG01000111.1 AEJS01000049.1 JRIP01000058.1 ABPC01000928.1 | | | | gb gb gb gb ScAqy1 327 ScAqy2 149 B3LTC5.1 69 79 25 11-bp deletion NCBI ScFps1 669 ScAqy2ScFps1 149 667 A7A0K8.1 EDN59506 70 20 80 25 24 99 11-bp deletion Wei Wei ScAqy1 327 C8ZJM1.1 98 86 20 R42K, V53A, ScAqy2ScFps1 149 669 C8ZCS3.1 CAY81197.1 69 20 79 25 24 100 11-bp deletion Novo Novo ScAqy1ScFps1 305 669 EGA76526.1 gb 96 87 21 A ScAqy1 327 EWG82962.1 99 86 20 R42K, V53A, ScFps1 669 APIP01000176.1 20 25 99 Contig00176 Treu ScAqy1 327 EWG88294.1 98 86 19 R42K, V53A, ScFps1 669 APIQ01000539.1 ScAqy1 327 GQ848555.1 98 86 19 R42K, V53A, ScAqy2ScFps1 149 384 ADC55261.1 69 79 24 11-bp deletion Will ScAqy1ScFps1 327 453 EGA84321.1 98 86 19 R42K, V53A, ScAqy1 327 GQ848553.1 99 86 20 R42K, V53A, ScAqy2ScFps1NA–––– 149 ADC55260.1 69 79 24 11-bp deletion Will M22 I4 Sequence designation Length (aa) YJM789 M22 VL3 RM11-1a Vin13 VL3 YJM789 EC1118 I4 P301 EC1118 Vin13 R008 RM11-1a ). strain, France strain, South Africa Continued ( Table 2. Strain Provenience RM11-1a Vinyard, California RM11-1a M22 Vinyard, Italy M22 VL3 Commercial wine R008 Vinyard, Italy R008 I4 Vinyard, Italy I4 EC1118 Commercial wine strain EC1118 Vin13 Commercial wine P301 Vinyard, Italy P301 8 FEMS Yeast Research, 2016, Vol. 16, No. 3 ) ) 2011 2011 ( ( ) ) ) ) ) ) ) ) ) ) ) ) et al. et al. 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 ( ( ( ( ( ( ( ( ( ( ( ( et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Borneman Borneman References for sequences Analyzed in this work Will Will 789 789 Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 a 1287 insertion Analyzed in this work ,T insertion insertion insertion Will insertion Will insertion Will insertion Will deletion Analyzed in this work deletion Will 1277 789 789 789 789 789 789 197 25 1277 substitutions, A substitution, A substitutions, A substitution, A insertions insertion insertion ScFps1 Observations 1278B % identity with ScAqy2 1278B % Identity with ScAqy1 1515 19 19 95 W 97 T 17 19 79 A 1278B b b b % identity with b number Accession AEWK01002276.1 AEHH01000049.1 AEEZ01000062.1 AEWK01002277.1 | | | | gb gb gb gb 306 EGA60170.1 95 86 20 N47D, V121M 294 EGA56339.1 98 86 21 N47D, V121M ScAaqy1 ScFps1 444 ScAaqy1 ScFps1 453 ScAqy1ScAqy2 305ScFps1NA–––– ScAqy1 289ScAqy2 ADC55548.1 305ScFps1NA–––– ADC55255.1 289 97 ADC55545.1 86 ADC55253.1 97 88 86 99 88 21 99 25 A 21 24 A Will Will ScAqy1ScAQy2 305ScFps1 289 ADC55543.1 645 ADC55254.1 JRIC01000243.1 97 86 20 88 99 25 21 A 25 99 Will Analyzed in this work ScAqy1ScAqy2ScFps1NA–––– ScAqy1 305 289ScAqy2 ADC55557.1ScFps1NA–––– 305 ADC55265.1 289 97 ADC55558.1 86 ADC55266.1 96 87 86 99 87 21 99 25 A 21 V121M 25 Will Will ScAqy1ScAqy2ScFps1 305 43 ADC55549.1 99 ADC55257.1 96 16 87 33 21 V121M 21 G YPS1000 YPS1009 YPS1000 YPS1009 YPS163 DY8 YPS163 Y10 Sequence designationFoster’sB Length (aa) YPS1009 YPS163 Foster’sO DY8 DY8 DY9 DY9 Y10 Foster’sB Foster’sO YPS1000 DY9 Y10 ). brewing strain (ale) (USA) Pennsylvania (USA) brewing strain (ale) Wisconsin (USA) Wisconsin (USA) Philippines (USA) Continued ( Table 2. StrainFoster’sB Provenience Commercial YPS1009 Oak, New Jersey YPS163 Oak, Foster’sO Commercial DY8 Soil beneath oak, DY9 OakY10 bark, Cocconut, YPS1000 Oak, New Jersey Sabir et al. 9 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) 2011 2011 2011 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 ( ( ( ( ( ( ( ( ( ( ( ( et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Will References for sequences Will (Akao 789 Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 a 789 insertion Will insertion Will insertion Will insertion Will deletion Will deletion Will deletion Will deletion Will 789 789 789 789 25 528 528 528 substitution, A substitution insertion insertion deletion, A ScFps1 Observations 1278B % identity with ScAqy2 1278B % Identity with ScAqy1 18 25 98 Scaffold-1110 Analyzed in this work 1278B % identity with b number Accession JRIM01001102.1 | gb NA–––– NA–––– NA–––– NA–––– 305289 ADC55551.1 ADC55268.1305 97289 86 ADC55552.1 ADC55271.1283 88 97 96188 86 ADC55554.1 21 ADC55267.1 88283 93 25 99 A 188 82 ADC55553.1639 21 ADC55264.1 86283 25 93 A 188 95 83 ADC55555.1 Will 22 ADC55269.1 86 93 N-terminal 25 95 83 Will G 22 86 24 95 A G 25 24 A G ScAqy1 ScAqy2 ScFps1 ScAqy1 ScAqy2 ScFps1 ScAqy1 ScAqy2 ScFps1 ScAqy1 ScAqy2 ScFps1 ScAqy1 ScAqy2 ScFps1 ScAqy1ScAqy2ScFps1NA–––– 327 43 ADC55257.1 ADC55273.1 99 16 87 33 20 21 V121M, P308S G ScAqy1 294 GAA27146.1 96 89 21 V121M ScAqy2ScFps1 240 667 GAA22751.1 BABQ01000117.1 20 98 25 87 99 22 (Akao (Akao UWOPS03- 461.4 UWOPS05- 217.3 UWOPS05- 227.2 UWOPS83- 787.3 UWOPS87- 2421 K7 Y1 Sequence designationY1 Length (aa) UWOPS83- 787.3 UWOPS87- 2421 UWOPS05- 217.3 227.2 UWOPS05- 227.2 K7 2421 UWOPS03- 461.4 UWOPS05- 217.3 Y1 787.3 UWOPS03- 461.4 ). North America Malaysia Malaysia Continued ( Table 2. StrainY1 Provenience Mushroom, UWOPS05-227.2 Palm, Malaysia UWOPS05- UWOPS87-2421 Cladode, Hawaii UWOPS87- UWOPS03-461.4 Palm nector, UWOPS05-217.3 Palm nector, K7 Sake, Japan K7 UWOPS83-787.3 Fruit, Bahamas UWOPS83- 10 FEMS Yeast Research, 2016, Vol. 16, No. 3 ) ) ) ) ) ) ) ) ) ) ) 2009 2009 ( ( 2016 2016 2016 ( ( ( 2010 2010 2010 2010 2010 2010 ( ( ( ( ( ( et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Sabir Argueso References for sequences Will Will Analyzed in this work 789 789 789 Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 a insertion Sabir deletion Will deletion Will 219 25 25 substitution, A substitution, A substitutions P308S substitutions substitution, A insertion insertion insertion ScFps1 Observations 1278B % identity with ScAqy2 1278B % Identity with ScAqy1 209919 25 86 22 99 25 Scaffold-14899 V53A, Sabir 96 P308S 20 A 87 25 21 V121M 99 Analyzed in this work 1278B b b b b b % identity with b number Accession AEWM01001183.1 AEWM01002257.1 AMDD01000033.1 AMDD01000023.1 AEWM01002256.1 Protein characterised for water transport activity, NA- Not available. | | | | | c gb gb gb gb gb 327289 GQ848566.1667 ADC55270.1 JRIG01000141.1 99 86 87 99 20 25 Will Will Genomic sequences, b ScAqy1 ScAqy2 ScFps1 ScAqy1ScFps1 327 73 ScAqy1 327ScFps1 EEU07393.1 669 98 EEU06572.1 20 86 25 20 R42K, V53A, 99 Argueso ScAqy1ScFps1 293 669 ScAqy1ScAqy2ScFps1NA–––– 305 43 ADC55544.1 ADC55262.1 96 16 87 33 21 V121M 21 G ScAqy1 305 ADC55542.1 96 87 21 V121M ScAqy2ScFps1NA–––– 43 ADC55256.1 16 33 21 G Sequence designation Length (aa) Y12 Jay291 DBVPG6044 DBVPG6044 CLIB324 CLIB324 Jay291 ZTW1 ZTW1 K1 Y12 K1 Y12 ). isolate, West Africa bioethanol strain PE-2, Brazil West Africa DBVPG6044 isolate, Vietnam bioethanol isolate, China Continued ( References of observations are cited and listed in the text, Table 2. a StrainK1 Provenience Y12 Sake, Japan K1 Palm wine Jay291 industrial DBVPG6044 Bili wine, CLIB324 Bakery ZTW1 Corn mash Sabir et al. 11 Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021

Figure 1. Phylogenetic tree based on the protein sequences of orthodox aquaporins (Aqy1 and Aqy2) and aquaglyceroporin (Fps1) within genome of yeast species. Dendogram was generated by neighbor-joining method (applied to 1000 bootstrap data sets) using the MEGA5.1 programme (Tamura et al. 2011). Dendogram depicting the relationship between aquaporins (highlighted with red) and aquaglyceroporin (highlighted with orange) sequences detected in yeasts. Sequences of S. cerevisiae (ScAqy1, ScAqy2 and ScFps1) are marked with arrows. MIPs of Saccharomyces genus clustered together (highlighted with darker shades in each MIP group), except SkAqy1 and ScaAqy2 in aquaporins and SkFps1 in aquaglyceroporins. A detailed description of terminology used and accession numbers are listed in Table 1. consists of two branches: one for orthodox aquaporins and an- and S. kluyveri (SkAqy1, 60% and SkFps1, 74%) which displayed other for aquaglyceroporins, which are grouped together and more divergence with lower identity (Fig. 1;Table1). Among presented with red and orange colors, respectively. Protein se- Aqy1 sequences, Yarrowia lipolytica (YlAqy1) demonstrated the quences of ScAqy1 and ScAqy2 were highly similar (87% iden- lowest sequence identity (48%), whereas in the Aqy2 group, tical), supporting that their origin is the probable event of gene Debaryomyces hansenii (DhAqy2, 41%) and Millerozyma farinose duplication (Wolfe and Shields 1997). Contrarily, ScFps1 showed (MfAqy2, 42%) exhibited the lowest similarity and clustered very low similarity with ScAqy1 (20%) and ScAqy2 (25%), sug- in separate sub-branch in phylogenetic tree (Fig. 1). Fps1 se- gesting its origin from a distinct subfamily (aquaglyceroporin) quences with lower similarity (<47%) (AgFps1, EcFps1, KlFps1, of MIPs (Abascal, Irisarri and Zardoya 2014). In both aquaporins KmFps1 and LtFps1) were clustered together in separate sub- and aquaglyceroporin groups, MIPs of the different species of the branch and displayed highest divergence (Fig. 1). Among all Fps1 Saccharomyces genus clustered together (highlighted with darker Kluyveromyces marxianus (KmFps1) showed lowest similarity shades in each MIP group), showing high percentage identity (44%) (Table 1). The longest Fps1 sequence was observed in Nau- (91%–99%) with S. cerevisiae MIPs (marked with arrow), except- movozyma dairenensis (722 amino acids). We assume that Fps1 ing sequences of S. castellii (ScaAqy2, 78% and ScaFps1, 54%) in Pichia pastoris (326 amino acids) is truncated, since it showed 12 FEMS Yeast Research, 2016, Vol. 16, No. 3 Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021

Figure 2. Phylogenetic tree based on the protein sequences of orthodox aquaporins (Aqy1 and Aqy2) and aquaglyceroporin (Fps1) within genomes of various strains of S. cerevisiae. Dendogram was generated by neighbor-joining method (applied to 1000 bootstrap data sets) using the MEGA5.1 programme (Tamura et al. 2011). Dendogram depicting the relationship between Aqy1 (purple), Aqy2 (green) and Fps1 (yellow) within various S. cerevisiae strains from different niches. All the MIP subgroups are clustered together, except CEN ScAqy1, which clustered with Aqy2 instead of Aqy1 group. MIPs of the reference strain (1278B) are highlighted in blue. Detailed description of terminology used and accession numbers are listed in Table 2. abbreviated topology (which will be discussed in subsequent water/solute channel. Additionally, orthodox aquaporins have topic). been also reported to be involved in transport of solutes other An interesting pattern for the number of aquapor- than water (Pettersson et al. 2005). On the other hand, neither ins/aquaglyceroporins genes present in yeasts was observed. orthodox aquaporins nor aquaglyceroporin (Fps1) was found in Almost all yeast species possess at least one water/solute the genomes of C. tropicalis, P. guilliermondii and two species of channel protein (either Aqy1, Aqy2 or Fps1). Existence of Schizosaccharomyces (Sch. japonicas and Sch. pombe), but Yfl054c more than one orthodox aquaporin gene in yeast is rare, and was found in some of these yeasts (not shown). is only constrained to those yeasts, which have undergone whole-genome duplication (Tanghe, Van Dijck and Thevelein MIPs sequences in S. cerevisiae strains 2006). For instance, S. cerevisiae and the closely related C. glabrata have both AQY1 and AQY2 genes (Table 1). On the A detailed compilation of MIP proteins within 38 strains of other hand, L. thermotolerans, Vanderwaltozyma polyspora and two S. cerevisiae from different niches is presented in Table 2.In species of the Zygosaccharomyces genus (Z. rouxii and Z. bailii) these strains, 89 MIP sequences (65 orthodox aquaporins and do not possess any orthodox aquaporin, but present at least 24 aquaglyceroporins) were identified in databases. Phyloge- one aquaglyceroporin, suggesting its putative versatile role as netic distances among all proteins are depicted in Fig. 2.Two Sabir et al. 13

initial branches of the tree clearly separate orthodox aquaporins these two substitutions were assumed to be more critical than from aquaglyceroporins. Further, orthodox aquaporins are di- N47D. vided into two subbranches, one for Aqy1 and another for Aqy2. A single nucleotide insertion (A789) resulting in a frame-shift While the second subbranch majorly comprised Aqy2, it is fur- mutation with stop codon after 305 amino acids was found in ther divided and comprises a few sequences previously anno- some laboratory, clinic, fruits and oak isolates (Table 2). The re- tated as Aqy1 in some strains (Malaysian, fermentation, sake, sulting proteins have shorter C-terminal as compared to the bakery and bioethanol strains). All the clusters of MIP proteins functional Aqy1 (327 amino acid) of 1278B strain. This inser- are displayed with different colors (Aqy1: purple, Aqy2: green tion was also observed, particularly, in laboratory strains S288c and Fps1: orange), and MIPs of the reference strain (1278B) are and derivatives, which possess non-functional Aqy1 (Laize´ et al. highlighted with blue. 1999). To confirm the suspected negative effect of a shorter C- Alignment of each MIP protein group demonstrated an obvi- terminal in laboratory strains, water permeability of Aqy1 in ous high identity within the strains. High sequence identities of 1278B strain without C-terminal as well as of a chimeric Aqy1 all Aqy1 with that of the 1278B strain were found (93%–99%). of a wild-type strain with the C-terminal from a laboratory strain Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 Exceptionally, sequence of CEN.PK113-7D strain (CEN ScAqy1) was measured (Bonhivers et al. 1998; Laize´ et al. 1999). Surpris- showed lower similarity (84%). CEN ScAqy1 was grouped with ingly, loss of C-terminal led to higher water permeability with Aqy2 cluster and has higher (99%) identity with 1278B ScAqy2 enhanced membrane protein accumulation. It was anticipated (Fig. 2,Table2). Probably, this aquaporin may belong to Aqy2 that rather than adversely affecting the water transport activity, group and has been mistakenly annotated as Aqy1 in databases. the C-terminal negatively controls the number of aquaporins be- Fps1 also showed high sequence identity (90%–100%) within ing targeted to the membrane (Bonhivers et al. 1998; Laize´ et al. the strains, except Y10 strain (Y10 ScFps1) that showed lower 1999). On the contrary, recently this A789 insertion is considered similarity (79%). As compared to Aqy1 and Fps1, Aqy2 showed equivalent to A881 deletion in strains having longer C-terminal higher divergence (33%–99% identity) within strains from vari- Aqy1 (Will et al. 2010). ous niches (laboratory, clinic, vineyard, oak and fruit isolates). In native Malaysian palm strains, another non-functional AQY allele caused by deletion of the N-terminal (106 bp) (Will et al. 2010) was found (Table 2). In vineyard and commercial wine POLYMORPHISM IN MIPs OF S. CEREVISIAE strains, four substitutions R42K, V53A, P308S and G226S were identified (Karpel and Bisson 2006)(Table2). The exact effect of STRAINS the first three substitutions on water transport activity was not AQY1 polymorphism evaluated, although it was presumed that the first two substitutions might have an insignificant effect on water flux but P308S In laboratory strains, two alleles (AQY1-1 and AQY1-2)forAQY1 might have a major effect due to its close proximity to the C- have been reported, so far. AQY1-1 allele was found only in terminal (Laize´ et al. 1999). On the other hand, G226S located 1278 strain, while most of the other strains harbor AQY1-2 near the second NPA motif demonstrated positive effect on wa- (Laize´ et al. 2000). Water transport studies suggest that only ter transport activity (Karpel and Bisson 2006), and was found AQY1-1 has the ability to encode functional aquaporins, when only in two Italian vineyard strains (P301 and M22) in the present expressed in Xenopus laevis oocytes as well as in spheroplasts study (Table 2). prepared from 1278 strain (Laize´ et al. 1999). Whereas AQY1-2 allele carries three point mutations encoding a non-functional AQY2 polymorphism protein. This mutant protein was unable to be properly expressed in the membrane of oocytes and yeast cells, and did not AQY2 gene also has two alleles in laboratory strains: functional enhance water permeability in Xenopus oocytes (Bonhivers et al. (AQY2-1) and non-functional (AQY2-2). Like AQY1, the functional 1998). allele AQY2-1 was only found in 1278 strain and its derivatives, Many different point mutations (substitution, deletion and while all other laboratory strains possess the non-functional al- insertion) have been described in Aqy1 sequences (Bonhivers lele. AQY2-1 encodes a non-disrupted protein functional for wa- et al. 1998; Laize´ et al. 1999; Ahmed Khan et al. 2000;Carbrey ter transport, although neither its expression in the membrane et al. 2001; Karpel and Bisson 2006). Among all the observed mu- nor water transport activity were observed in Xenopus oocytes tations, three different substitutions (V121M, P255T and N47D) heterologous system (Laize´ et al. 2000). Later, with a specific an- were scrutinized in the present study. Substitutions of V121M tibody, AQY2-1 was localized in the plasma membrane as well as and P255T were observed in all laboratorial and in some clini- in the endoplasmic reticulum of 1278 strain. Additionally, en- cal strains (YJM436 and YJM789) (Table 2). Substitution of N47D hanced water permeability of vesicles derived from yeast con- was observed in laboratory as well as in commercial brewing taining AQY2-1 was also demonstrated (Meyrial et al. 2001). strains. The first two residues have been found highly conserved The AQY2-2 allele harbors an 11-bp deletion, causing a and are considered crucial for water transport activity in animal frame-shift mutation with premature stop codon. This dele- and plant aquaporins (Bonhivers et al. 1998;Carbreyet al. 2001; tion splits the coding region in two open reading frames, which Will et al. 2010). To explore the significance of these residues, encode truncated proteins unable to mediate water transport a reverse mutation approach was attempted in strains express- (Laize´ et al. 2000). In the present study, all the laboratory and ing non-functional aquaporins (Bonhivers et al. 1998). Enhanced vineyard strains as well as some clinical isolates (YJM436 and water permeability and higher expression of mutated proteins YJM789) possess the 11-bp deletion in AQY2 sequences result- was observed due to double (M121V/T255P) and triple (D47N/ ing in truncated proteins (149 amino acids) (Table 2). Besides M121V/T255P) reverse substitutions, while single and other dou- this well-known 11-bp deletion, two different alleles caused by ble substitutions had not this positive effect (Bonhivers et al. deletions at G25 and G528 were found in AQY2 of natural iso- 1998). Since structural positions of these residues suggest that lates (Table 2) (Laize´ et al. 2000; Will et al. 2010). These dele- V121M (in loop B) may block the pore and P255T (in sixth trans- tions also caused frame-shift with premature stop codon, result- membrane helix) may change the conformation of the protein, ing in truncated proteins (43 and 188 amino acids, respectively). 14 FEMS Yeast Research, 2016, Vol. 16, No. 3

Several other non-functional AQY2 alleles showing polymor- frequency of occurrence among the whole sequences (Fig. 3B). phism through different restriction patterns were also reported Extremely different sequences (incomplete or too long) were in industrial strains (Laize´ et al. 2000), suggesting that their func- discarded in some of the web logo creation. N-terminal se- tional presence is not favorable for yeasts at least in the condi- quences of aquaporins were not considered for web logo be- tions tested so far. cause of highly dissimilar size, and their sequence alignment is presented in Fig. S1A (Supporting Information). Sequences of S. FPS1 polymorphism cerevisiae and P. pastoris were used as reference sequences to compare and predict the important residues. Sequences of S. Many new mutations in aquaglyceroporins were revealed by the cerevisiae are the best studied and annotated, while the Aqy1 of analysis performed in this study (Table 2). These polymorphisms P. pastoris is the only yeast aquaporin whose crystal structure in aquaglyceroporins have not been previously recognized. In has been established (Fischer et al. 2009) and the transmembrane the nucleotide sequence of M22 strain, a frame-shift caused by protein analyzed at highest resolution, so far (Kosinska Eriksson

deletion of the initial 208 bp was found, but this mutation was et al. 2013). Positions of amino acids at x-axis of sequence logos Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 nullified by G40 insertion (Table 2). An insertion of T1079 in nu- for aquaporins are corresponding to P. pastoris Aqy1; whereas for cleotide sequences of strains M22, VL3 (corresponding to T1286) aquaglyceroporins, S. cerevisiae Fps1 was considered as reference and Foster´s O (corresponding to T1287) was observed. This in- (Fig. 3B). sertion caused frame-shift with stop codon, resulting in a truncated protein (Table 2). Other insertions of a nucleotide at 1277 Orthodox aquaporins (Aqy1 and Aqy2) position in commercial brewing strains, (Foster’s B (W1277)and Foster’s O (T1277)) were found, which again shifted the frame, Aqy1 sequences of all yeasts showed slightly longer N-terminal resulting in a different length of truncated proteins (453 and than C-terminal (Fig. 3A). Sequence analysis have revealed that 444 amino acids, respectively) (Table 2). Furthermore, two differ- both terminal halves of aquaporins have similar sequences and ent mutations in nucleotide sequences of strains Y10 (A197 dele- are arranged in tandem repeats, suggesting possible intragenic tion) and CLIB324 (A219 insertion) were also observed, resulting duplication of half-sized gene encoding only one half of the pro- in frame-shift with premature stop codon and truncated protein (Reizer, Reizer and Saier 1993). Each half of the protein con- teins (Table 2). tains a loop (LB and LE) with a highly conserved Asn-Pro-Ala We assume that the mutations identified, here, in aquaglyc- (NPA) motif. Both functional loops (LB and LE) fold back into the eroporins may putatively result in loss of their functions. Sim- plane of the membrane and form short helices embedded in the ilarly to aquaporins, functional loss of aquaglyceroporins may membrane (Jung et al. 1994), which further arranged to form the be related with required adaptation according to habitat of the water selective channel. These motifs are highly conserved in strains. Under hyperosmotic conditions, yeasts produce glyc- all yeast species, except a replacement of the first NPA to NPC in erol as an osmolyte and closure/inactivation of Fps1 is essen- loop LB of Y. lipolytica (Pettersson et al. 2005) (Fig. S1A, Supporting tial to maintain their intracellular high concentration (Tamas´ Informarion). Moreover, only one NPA was found in truncated et al. 1999). Probably, the loss of aquaglyceroporin function is aquaporins of Clavispora lusitaniae and C. parapsilosis in loop LB majorly related with the maintenance of high concentration of and loop LE, respectively (Fig. S1A, Supporting Informarion). Be- intracellular glycerol, required in various hyper-osmotic condi- sides NPA, other important residues at ar/R constriction (filter-2) tions in nature. Under high salt concentration, in several yeast are also important for substrate selectivity (Beitz et al. 2006). Se- species, including the highly osmotolerant D. hansenii and M. lectivity filter of PpAqy1 is formed92 ofPhe (Fig. S1A, Supporting + farinosa (or P. sorbitophila), active glycerol uptake (H /glycerol or Informarion, in LB) and Arg227,His212 and Ala221 (Fig. S1A, Sup- + Na /glycerol symporters) have been shown important for their porting Informarion, in loop LE), defining its specificity for wa- halotolerance (Lages, Silva-Grac¸a and Lucas 1999; Prista et al. ter (Cui and Bastien 2012). All these residues, except Ala221,are 2005). Interestingly, these two yeast species do not possess Fps1 highly conserved within all yeast species (Fig. 3B in loop LE). In- (Table 1), suggesting that their absence was beneficial under terestingly, all species of the Saccharomyces genus possess thre- such conditions. onine instead of Ala221 (Fig. S1A, Supporting Informarion). A mechano-sensitive mechanism for gating was suggested TOPOLOGY AND IMPORTANT RESIDUES by crystal structure of PpAqy1 (Fischer et al. 2009; Tornroth- Horsefield et al. 2010). Tyr31 plays an important role in gating Topology of all obtained sequences was predicted by ExPASy of PpAqy1 by blocking the pore with the help of Pro29,Gly108, topology prediction tools e.g. TMHMM (Krogh et al. 2001), HMM- Gly109 and Ala190, which hence are expected to be highly con- TOP(TusnadyandSimon2001) and TMPred (Hofmann 1993). All served in fungal water channels (Fischer et al. 2009). Sequence the yeast aquaporins and aquaglyceroporins exhibit the typical alignment of yeasts aquaporins showed that Gly108,Gly109 and topology of the MIP family, consisting of six transmembrane he- Ala190 (Fig. 3B; Fig. S1A, Supporting Informarion, in loop LB) are lices (TMH1–TMH6) connected with five loops (LA–LE) and cy- conserved in all yeast species. On the other hand, Pro29 and Tyr31 tosolic N and C termini (Fig. 3A) (Nehls and Dietz 2014). are not highly conserved (Fig. S1A, Supporting Informarion, in To display the conservation of important residues in a par- N-terminal). Tyr31 was only found in some species of Candida (C. ticular region, Web logos of sequence alignment in impor- albicans, C. dubliniensis, C. maltosa, C. orthopsilosis), also in K. marx- tant regions of aquaporins (loops LB and LE) and aquaglyc- ianus, Kuraishia capsulate, Lodderomyces elongisporus, S. kluyveri, eroporins (loops LB and LE, and N- and C-terminals) were Spathaspora passalidarum and Wickerhamomyces ciferrii, whereas constructed according to (Schneider and Stephens 1990). Se- we found that Pro29 was conserved in most yeast species (C. al- quences of particular regions with equal length were aligned bicans, C. dubliniensis, C. maltosa, C. orthopsilosis, C. tenuis, Cl. lusi- by ClustalX (Thompson et al. 1997) and BioEdit (Hall 1999) taniae, K. marxianus, K. capsulate, L. elongisporus, P. stipitis, Spa. (Fig. S1, Supporting Information) and were analyzed by Web passalidarum, W. ciferrii, Y. lipolytica and in all species of Saccha- logo site (http://weblogo.threeplusone.com/create.cgi). Height of romyces) (Fig. S1A, Supporting Informarion, in N-terminal). Other residues presented in a particular region is proportional to their gating mechanisms may probably exist in species which do not Sabir et al. 15 Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021

Figure 3. Schematic representation of predicted topology and conserved residues in yeast aquaporins and aquaglyceroporins. Left panel refers to aquaporins and right panel present aquaglyceroporins. (A) Topology prediction of both sequences showed typical characteristics of MIP family: cytosolic N- and C- terminals and six transmembrane helices (TMH1–TMH6) connected with five loops (LA–LE). N-terminal of both sequences is longer than C-terminal. Aquaglyceroporinsve ha very long N-terminals. Conserved NPA motifs in aquaporins, and their homolog NPS and NLA motifs in aquaglyceroporins are represented by yellow circles. ar/R constriction residues in aquaporins for selectivity filter (Cui and Bastien 2012) are shown in purple ovals. Residues important for gating (P29 and Y31,G108 and G109 in PpAqy1) in aquaporins, while N228 P229 Q230 in aquaglyceroporins are shown in red circles. Putative phosphorylation site at S107 (aquaporins) and T231 (aquaglyceroporins) are shown in green stars. Regulatory domains in aquaglyceroporins are represented in orange circles. (B) Sequence logos were created to identify critical residues in the sequences. Multiple alignments of sequences are presented as graphical view of sequence logos. Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position (Schneider and Stephens 1990). In aquaporins, 42 sequences for highly conserved loop B and loop E were aligned. For aquaglyceroporins, 23 sequences for all selected regions (loop B, loop E, regulatory domains of N-and C-terminals) were aligned to identify the conservation pattern. Sequence alignments of the conserved regions are presented in supplementary data (Fig. S1, Supporting Information). The color scheme of amino acids is: green-polar, purple-neutral, blue-basic, red-acidic and black-hydrophobic. posses these two residues (Cui and Bastien 2012). For example, (Pettersson et al. 2005). Their first prominent distinct charac- membrane tension was suggested for regulation of yeast aqua- teristic is their larger size due to significantly longer N- and porins gating (Soveral et al. 2008). Besides, a putative phospho- C-terminals (Fig. 3A). In all sequences, usually 128–266 amino rylation site at Ser107 was also considered important for activity acid long N-terminals were observed. C-terminals of all yeast of aquaporins (Fischer et al. 2009), and was conserved in all se- aquaglyceroporins are 82–183 amino acids long. Contrary to all quences (Fig. 3B; Fig. S1A, Supporting Information, in loop LB). aquaglyceroporin sequences, topology prediction of P. pastoris In sum, yeast orthodox aquaporins represent a vastly diverse Fps1, strongly suggests only five transmembrane helices instead group of proteins, ranging the similarity between 34% and 98% of six. We considered this as being truncated/incomplete (Ta- and possess highly conserved regulatory amino acid residues for ble 1). gating, phosphorylation and selectivity. Yeast orthodox aqua- The second distinct feature of aquaglyceroporins is the less porins are more close to plant aquaporins in comparison to an- rigorously conserved NPA motifs of loop LB and loop LE. In loop imal water channels (Pettersson et al. 2005). LB, the first two residues (Asn352 and Pro353) are highly conserved in all available sequences, but the third (Ala354)isnotconserved in most species and it is replaced by Ile in C. glabrata,ValinEr- Aquaglyceroporin (Fps1) emothecium cymbalariae and Gly in W. ciferrii, while in all other Typical transmembrane topology of yeast aquaglyceroporins yeasts it is replaced by Ser (Fig. 3B; Fig. S1B, Supporting Informa- categorized them for MIPs family members, but major struc- tion). On the other hand, in loop LE the first (Asn480) and the third tural differences and alterations in highly conserved amino (Ala482) residues are highly conserved, while the second residue acid residues distinguish this group from orthodox aquaporins (Pro481) is mainly replaced by Leu in most yeasts or by Met in C. 16 FEMS Yeast Research, 2016, Vol. 16, No. 3

glabrata, Kazachstania africana, N. dairenensis, Tetrapisispora blat- ally, we also observed several inactivating mutations in the se- tae, Torulaspora delbrueckii, Z. rouxii and Z. bailii (Fig. 3B; Fig. S1B, quences of aquaglyceroporins, opening new perspectives on Supporting Informarion). their functional specification. Two regulatory domains located in N- and C-terminals for gating of aquaglyceroporins have been identified (Tamas´ et al. SUPPLEMENTARY DATA 2003; Pettersson et al. 2005). Both domains are 12 amino acids long and are located just before and after the first and the Supplementary data are available at FEMSYR online. sixth transmembrane helices, respectively. The N-terminal sequence 225LYQNPQTPTVLP236 is strictly conserved among all yeasts species, except in Ashbya gossypii and Z. rouxi,inwhich FUNDING His was found in the place of Tyr226. While in W. ciferrii, both Ty r 226 and P236 are replaced by Glu (Fig. 3B; Fig. S1B, Supporting This work was supported by Fundac¸ao˜ para a Cienciaˆ e Tecnolo- gia (FCT), Portugal (SFRH/BPD/89427/2012 to Farzana Sabir and

Informarion). Besides amino acid composition, proximity of this Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 domain to the first transmembrane helix is also important for PTDC/AGR/AAM/099154/2008). proper glycerol transport and regulation through Fps1 (Tamas´ Conflict of interest. et al. 2003). In the C-terminal, the 535HESPVNWSLPVY546 se- None declared. quence is also considered important for regulation of aquaglyceroporins (Hedfalk et al. 2004). In this domain, the first half (HES- REFERENCES PVN) is comparatively more conserved than the second half (WSLPVY), except for a replacement of Val539 to Leu in K. lac- Abascal F, Irisarri I, Zardoya R. Diversity and evolution of tis, K. marxianus, L. thermotolerans and W. ciferrii. The second half membrane intrinsic proteins. Biochim Biophys Acta 2014;1840: sequence was absent with a replacement of Glu536 to Asp was 1468–81. found in W. ciferrii (Fig. 3B; Fig. S1B, Supporting Information). Ahmadpour D, Geijer C, Tamas MJ et al. Yeast reveals unexpected Mutational analysis revealed that the strains expressing roles and regulatory features of aquaporins and aquaglycero- truncated N- and C-terminals with deleted regulatory domains porins. Biochim Biophys Acta 2014;1840:1482–91. were inefficient for glycerol accumulation and thus were sen- Ahmed Khan S, Zhang N, Ismail T et al. Functional analysis of sitive to hyperosmotic stress (Tamas´ et al. 2003; Hedfalk et al. eight open reading frames on chromosomes XII and XIV of 2004), arsenite (Wysocki et al. 2001) and acetic acid (Mollapour Saccharomyces cerevisiae. Yeast 2000;16:1457–68. and Piper 2007). Although exchanging these domains between Akao T, Yashiro I, Hosoyama A et al. Whole-Genome Sequencing ScFps1 and AgFps1 could not demonstrate the expected regu- of Sake Yeast Saccharomyces cerevisiae Kyokai no. 7. DNA Res lation, suggesting that not only the existence of terminal do- 2011;18:423–34. mains but also the interplay between termini and transmem- Argueso JL, Carazzolle MF, Mieczkowski PA et al. Genome struc- brane core is crucial for regulation of glycerol flux (Geijer et al. ture of a Saccharomyces cerevisiae strain widely used in 2012). Besides, other factors and their compatibility in heterol- bioethanol production. Genome Res 2009;19:2258–70. ogous systems may also be important for gating. Random mu- Beitz E. Aquaporins from pathogenic protozoan parasites: struc- tation studies in different genetic screenings for Fps1 identified ture, function and potential for chemotherapy. Biol Cell various other important residues in the first transmembrane he- 2005;97:373–83. lix and in loop LB (Karlgren et al. 2004). Beitz E, Wu B, Holm LM et al. Point mutations in the aro- Similar to PpAqy1 gating, a mechanosensitive mechanism of matic/arginine region in aquaporin 1 allow passage of gating in Fps1 was proposed (Tamas´ et al. 2003;Karlgrenet al. urea, glycerol, ammonia, and protons. P Natl Acad Sci USA 2004). The highly conserved 228NPQ230 present in the regulatory 2006;103:269–74. domain of the N-terminal of the aquaglyceroporin is assumed Bonhivers M, Carbrey JM, Gould SJ et al. Aquaporins in Saccha- to fold back into the membrane, like NPA of loop B and loop E romyces: genetic and functional distinctions between labora- of aquaporins, and tend to block the pore with the help of im- tory and wild-type strains. J Biol Chem 1998;273:27565–72. portant residues found in loop B. Phosphorylation of Thr231 in Borneman AR, Desany BA, Riches D et al. Whole-genome com- the N-terminal domain is another regulatory mechanism con- parison reveals novel genetic elements that characterize the sidered for gating in glycerol channels (Thorsen et al. 2006). In genome of industrial strains of Saccharomyces cerevisiae. PLoS the present study, we found that this residue is conserved in all Genet 2011;7:e1001287. yeasts, except in W. ciferrii (Fig. 3B; Fig. S1B, Supporting Infor- Botstein D, Fink GR. Yeast: an experimental organism for 21st mation). Studies suggest that Hog1 (High Osmolarity Glycerol)- Century biology. Genetics 2011;189:695–704. dependent putative phosphorylation at Thr231 leads to unreg- Bussey H, Storms RK, Ahmed A et al. The nucleotide se- ulated channel activity, which in turn raises the sensitivity of quence of Saccharomyces cerevisiae chromosome XVI. Nature yeasts to arsenite (Thorsen et al. 2006) and acetic acid (Mollapour 1997;387:103–5. and Piper 2007). Butler G, Rasmussen MD, Lin MF et al. Evolution of pathogenic- ity and sexual reproduction in eight Candida genomes. Nature 2009;459:657–62. CONCLUDING REMARKS Carbrey JM, Bonhivers M, Boeke JD et al. Aquaporins in Saccha- romyces: characterization of a second functional water chan- Most laboratory strains of S. cerevisiae have mutations in both nel protein. P Natl Acad Sci USA 2001;98:1000–5. AQY1 and AQY2, resulting in truncated proteins, unable to trans- Chaumont F, Moshelion M, Daniels MJ. Regulation of plant aqua- port water across the plasma membrane. On the other hand, porin activity. Biol Cell 2005;97:749–64. wild and less domesticated strains possess at least one gene Cliften P, Sudarsanam P, Desikan A et al. Finding functional fea- coding for a functional aquaporin, suggesting the advantage of tures in Saccharomyces genomes by phylogenetic footprint- functional aquaporins in harsh natural conditions. Addition- ing. Science 2003;301:71–6. Sabir et al. 17

Cui Y, Bastien DA. Molecular dynamics simulation and bioinfor- Krogh A, Larsson B, Von Heijne G et al. Predicting trans- matics study on yeast aquaporin Aqy1 from Pichia pastoris. membrane protein topology with a hidden Markov model: Int J Biol Sci 2012;8:1026–35. application to complete genomes. J Mol Biol 2001;305: Curtin CD, Borneman AR, Chambers PJ et al. De-novo assem- 567–80. bly and analysis of the heterozygous triploid genome of the Kruse E, Uehlein N, Kaldenhoff R. The aquaporins. Genome Biol wine spoilage yeast Dekkera bruxellensis AWRI1499. PloS One 2006;7:206. 2012;7:e33840. Lages F, Silva-Grac¸a M, Lucas C. Active glycerol uptake is a Daniels MJ, Wood MR, Yeager M. In vivo functional assay of a re- mechanism underlying halotolerance in yeasts: a study of 42 combinant aquaporin in Pichia pastoris. Appl Environ Microb species. Microbiology 1999;145:2577–85. 2006;72:1507–14. Laize´ V, Gobin R, Rousselet G et al. Molecular and func- De Schutter K, Lin Y-C, Tiels P et al. Genome sequence of tional study of AQY1 from Saccharomyces cerevisiae: role the recombinant protein production host Pichia pastoris. Nat of the C-terminal domain. Biochem Bioph Res Co 1999;257:

Biotechnol 2009;27:561–6. 139–44. Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 Dietrich FS, Voegeli S, Brachat S et al. The Ashbya gossypii genome LaizeV,TacnetF,RipocheP´ et al. Polymorphism of Saccharomyces as a tool for mapping the ancient Saccharomyces cerevisiae cerevisiae aquaporins. Yeast 2000;16:897–903. genome. Science 2004;304:304–7. Leitao˜ L, Prista C, Moura TF et al. Grapevine aquaporins: gating Dujon B, Sherman D, Fischer G et al. Genome evolution in yeasts. of a tonoplast intrinsic protein (TIP2;1) by cytosolic pH. PloS Nature 2004;430:35–44. One 2012;7:e33219. Fischer G, Kosinska-Eriksson U, Aponte-Santamaria C et al. Crys- Liti G, Nguyen Ba AN, Blythe M et al. High quality de novo tal structure of a yeast aquaporin at 1.15 angstrom reveals a sequencing and assembly of the Saccharomyces arboricolus novel gating mechanism. PLoS Biol 2009;7:e1000130. genome. BMC Genomics 2013;14:69. Freel KC, Sarilar V, Neuveglise C et al. Genome sequence of the Luyten K, Albertyn J, Skibbe WF et al. Fps1, a yeast member of yeast Cyberlindnera fabianii (Hansenula fabianii). Genome An- the MIP family of channel proteins, is a facilitator for glycerol nounc 2014;2:e00638-14. uptake and efflux and is inactive under osmotic stress. EMBO Geijer C, Ahmadpour D, Palmgren M et al. Yeast aquaglycero- J 1995;14:1360. porins use the transmembrane core to restrict glycerol trans- Maurel C, Chrispeels MJ. Aquaporins. A molecular entry into port. J Biol Chem 2012;287:23562–70. plant water relations. Plant Physiol 2001;125:135–8. Gordon JL, Armisen´ D, Proux-Wera´ E et al. Evolutionary erosion of Meyrial V, Laize´ V, Gobin R et al. Existence of a tightly regu- yeast sex chromosomes by mating-type switching accidents. lated water channel in Saccharomyces cerevisiae. Eur J Biochem P Natl Acad Sci USA 2011;108:20024–9. 2001;268:334–43. Hall TA. BioEdit: a user-friendly biological sequence alignment Mollapour M, Piper PW. Hog1 mitogen-activated protein kinase editor and analysis program for Windows 95/98/NT. Nucleic phosphorylation targets the yeast Fps1 aquaglyceroporin for Acid S 1999;41:95–8. endocytosis, thereby rendering cells resistant to acetic acid. Hedfalk K, Bill RM, Mullins JGL et al. A regulatory domain in the Mol Cell Biol 2007;27:6446–56. C-terminal extension of the yeast glycerol channel Fps1p. J Nehls U, Dietz S. Fungal aquaporins: cellular functions and Biol Chem 2004;279:14954–60. ecophysiological perspectives. Appl Microbiol Biot 2014;98: Hofmann KWS. TMbase—a database of membrane spanning 8835–51. proteins segments. Biol Chem H-S 1993;374:166. Novo M, Bigey F, Beyne E et al. Eukaryote-to-eukaryote gene Ishibashi K, Tanaka Y, Morishita Y. The role of mam- transfer events revealed by the genome sequence of the wine malian superaquaporins inside the cell. Biochim Biophys Acta yeast Saccharomyces cerevisiae EC1118. P Natl Acad Sci USA 2014;1840:1507–12. 2009;106:16333–8. Jeffries TW, Grigoriev IV, Grimwood J et al. Genome sequence Nozawa A, Takano J, Kobayashi M et al. Roles of BOR1, DUR3,and of the lignocellulose-bioconverting and xylose-fermenting FPS1 in boron transport and tolerance in Saccharomyces cere- yeast Pichia stipitis. Nat Biotechnol 2007;25:319–26. visiae. FEMS Microbiol Lett 2006;262:216–22. Johnston M, Hillier L, Riles L et al. The nucleotide sequence Paganelli CV, Solomon AK. The rate of exchange of tritiated of Saccharomyces cerevisiae chromosome XII. Nature 1997;387: water across the human red cell membrane. J Gen Physiol 87–90. 1957;41:259–77. Jung JS, Preston GM, Smith BL et al. Molecular structure of the wa- Patel D. Characterization of vacuole aquaporin function and ter channel through aquaporin CHIP. The hourglass model. J its implication in membrane fission. Masters Thesis.Concor- Biol Chem 1994;269:14648–54. dia University, Faculty of Arts and Science (Biology). 2013. Kaldenhoff R, Fischer M. Aquaporins in plants. Acta Physiol http://spectrum.library.concordia.ca/977797/1/Patel MSc 2006;187:169–76. F2013.pdf. Karlgren S, Filipsson C, Mullins JGL et al. Identification of Pettersson N, Filipsson C, Becit E et al. Aquaporins in yeasts and residues controlling transport through the yeast aquaglyc- filamentous fungi. Biol Cell 2005;97:487–500. eroporin Fps1 using a genetic screen. Eur J Biochem Preston GM, Carroll TP, Guggino WB et al. Appearance of water 2004;271:771–9. channels in Xenopus oocytes expressing red cell CHIP28 pro- Karlsson M, Fotiadis D, Sjovall¨ S et al. Reconstitution of water tein. Science 1992;256:385–7. channel function of an aquaporin overexpressed and puri- Prista C, Loureiro-Dias MC, Montiel V et al. Mechanisms underly- fied from Pichia pastoris. FEBS Lett 2003;537:68–72. ing the halotolerant way of Debaryomyces hansenii. FEMS Yeast Karpel JE, Bisson LF. Aquaporins in Saccharomyces cerevisiae wine Res 2005;5:693–701. yeast. FEMS Microbiol Lett 2006;257:117–23. Ralser M, Kuhl H, Ralser M et al. The Saccharomyces cerevisiae Kosinska Eriksson U, Fischer G, Friemann R et al. Subangstrom W303-K6001 cross-platform genome sequence: insights into resolution X-Ray structure details aquaporin-water interac- ancestry and physiology of a laboratory mutt. Open Biol tions. Science 2013;340:1346–9. 2012;2:120093. 18 FEMS Yeast Research, 2016, Vol. 16, No. 3

Reizer J, Reizer A, Saier MH. The MIP family of integral Tanghe A, Van Dijck P, Thevelein JM. Why do microorganisms membrane channel proteins: sequence comparisons, evo- have aquaporins? Trends Microbiol 2006;14:78–85. lutionary relationships, reconstructed pathway of evolu- Teixeira MC, Raposo LR, Mira NP et al. Genome-wide identifi- tion, and proposed functional differentiation of the two re- cation of Saccharomyces cerevisiae genes required for max- peated halves of the proteins. Crit Rev Biochem Mol 1993;28: imal tolerance to ethanol. Appl Environ Microb 2009;75: 235–57. 5761–72. Sabir F, Prista C, Madeira A et al. Water transport in yeasts. Adv Thompson JD, Gibson TJ, Plewniak F et al. The CLUSTAL X Exp Med Biol 2016;892:107–24. windows interface: flexible strategies for multiple sequence Scannell DR, Frank AC, Conant GC et al. Independent sorting-out alignment aided by quality analysis tools. Nucleic Acids Res of thousands of duplicated gene pairs in two yeast species 1997;25:4876–82. descended from a whole-genome duplication. P Natl Acad Sci Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving USA 2007;104:8397–402. the sensitivity of progressive multiple sequence alignment

Schneider TD, Stephens RM. Sequence logos: a new way to through sequence weighting, position-specific gap penalties Downloaded from https://academic.oup.com/femsyr/article/16/3/fow025/2467777 by guest on 30 September 2021 display consensus sequences. Nucleic Acids Res 1990;18: and weight matrix choice. Nucleic Acids Res 1994;22:4673–80. 6097–100. Thorsen M, Di Y, Tangemo¨ C et al. The MAPK Hog1p modulates Souciet J, Aigle M, Artiguenave F et al. Genomic exploration of Fps1p-dependent arsenite uptake and tolerance in yeast. Mol the hemiascomycetous yeasts: 1. A set of yeast species for Biol Cell 2006;17:4400–10. molecular evolution studies. FEBS Lett 2000;487:3–12. Tornroth-Horsefield S, Hedfalk K, Fischer G et al. Structural Soveral G, Madeira A, Loureiro-Dias MC et al. Membrane ten- insights into eukaryotic aquaporin regulation. FEBS Lett sion regulates water transport in yeast. Biochim Biophys Acta 2010;584:2580–8. 2008;1778:2573–9. Treu L, Toniolo C, Nadai C et al. The impact of genomic variability Soveral G, Madeira A, Loureiro-Dias MC et al. Water transport in on gene expression in environmental Saccharomyces cerevisiae intact yeast cells as assessed by fluorescence self-quenching. strains. Environ Microbiol 2014;16:1378–97. Appl Environ Microbiol 2007;73:2341–3. Tusnady GE, Simon I. The HMMTOP transmembrane topology Soveral G, Prista C, Moura TF et al. Yeast water channels: an prediction server. Bioinformatics 2001;17:849–50. overview of orthodox aquaporins. Biol Cell 2011;103:35–54. Wei W, McCusker JH, Hyman RW et al. Genome sequencing Soveral G, Veiga A, Loureiro-Dias MC et al. Water channels are and comparative analysis of Saccharomyces cerevisiae strain important for osmotic adjustments of yeast cells at low tem- YJM789. P Natl Acad Sci USA 2007;104:12825–30. perature. Microbiology 2006;152:1515–21. Wendland J, Walther A. Genome evolution in the eremothecium Takata K, Matsuzaki T, Tajika Y. Aquaporins: water channel pro- cladeoftheSaccharomyces complex revealed by comparative teins of the cell membrane. Prog Histochem Cyto 2004;39:1–83. genomics. G3 2011;1:539–48. Tamas´ MJ, Karlgren S, Bill RM et al. A short regulatory domain Will JL, Kim HS, Clarke J et al. Incipient balancing selection restricts glycerol transport through yeast Fps1p. J Biol Chem through adaptive loss of aquaporins in natural Saccharomyces 2003;278:6337–45. cerevisiae populations. PLoS Genet 2010;6:e1000893. Tamas´ MJ, Luyten K, Sutherland FCW et al. Fps1p controls Wohlbach DJ, Kuo A, Sato TK et al. Comparative genomics of the accumulation and release of the compatible solute xylose-fermenting fungi for enhanced biofuel production. P glycerol in yeast osmoregulation. Mol Microbiol 1999;31: Natl Acad Sci USA 2011;108:13212–7. 1087–104. Wolfe KH, Shields DC. Molecular evidence for an ancient dupli- Tamura K, Peterson D, Peterson N et al. MEGA5: molecular evo- cation of the entire yeast genome. Nature 1997;387:708–12. lutionary genetics analysis using maximum likelihood, evo- WysockiR,Chery´ CC, Wawrzycka D et al. The glycerol channel lutionary distance, and maximum parsimony methods. Mol Fps1p mediates the uptake of arsenite and antimonite in Sac- Biol Evol 2011;28:2731–9. charomyces cerevisiae. Mol Microbiol 2001;40:1391–401. Tanghe A, Carbrey JM, Agre P et al. Aquaporin expression and Zardoya R, Ding X, Kitagawa Y et al. Origin of plant glycerol trans- freeze tolerance in Candida albicans. Appl Environ Microb porters by horizontal gene transfer and functional recruit- 2005;71:6434–7. ment. P Natl Acad Sci USA 2002;99:14893–6.