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Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae

Bart Nelissen , Rupert De Wachter , André Goffeau
DOI: http://dx.doi.org/10.1111/j.1574-6976.1997.tb00347.x 113-134 First published online: 1 September 1997

Abstract

On the basis of the complete genome sequence of the budding yeast Saccharomyces cerevisiae, a computer-aided analysis was carried out of all members of the major facilitator superfamily (MFS), which typically consists of permeases with 12 transmembrane spans. Analysis of all 5885 predicted open reading frames identified 186 potential MFS proteins. Binary sequence comparison made it possible to cluster 149 of them into 23 families. Putative permease functions could be assigned to 12 families, the largest including sugar, amino acid, and multidrug transport. Phylogenetic clustering of proteins allowed us to predict a possible permease function for a total of 119 proteins. Multiple sequence alignments were made for all families, and evolutionary trees were constructed for families with at least four members. The latter resulted in the identification of 21 subclusters with presumably tightly related permease function. No functional clues were predicted for a total of 41 clustered or unclustered proteins.

Keywords
  • Yeast genome
  • Saccharomyces cerevisiae
  • Major facilitator superfamily
  • Permease
  • Classification

1 Introduction

Recently, the complete genome sequence of the budding yeast Saccharomyces cerevisiae has been determined, which encodes approximately 5885 potential proteins [1]. This is the first eukaryotic organism of which the complete genome sequence has become available, and classification and function assignment of its encoded proteins can serve as a model for other eukaryotes. It has been shown previously [2] that computer-aided analysis provides a powerful tool for the classification and function assignment of permeases. In this review, we present such a computer-aided analysis of all members of the major facilitator superfamily (MFS) that are encoded by the yeast genome.

The MFS has originally been defined [3] as a superfamily of permeases that are characterized by two structural units of six transmembrane spanning α-helical segments, connected by a cytoplasmic loop. This results in proteins with a length of 500–600 amino acids, comprising a total of 12 transmembrane spanning segments. The MFS is present in both bacteria and eukaryotes and includes uniporters, symporters, and antiporters. An earlier study [2] distinguished six different families for all species. A more recent study carried out at our laboratory [4] revealed 17 potential MFS families for S. cerevisiae only.

However, all previous studies of permeases that included the MFS were carried out when only part of the yeast genome sequence was determined [25] or were focussed on a single family [6, 7]. This review is thus the first study of all MFS proteins encoded by the entire yeast genome, and reveals more members and families of the MFS in general, and more specifically all members and families of the MFS of yeast.

2 Computer analysis

2.1 Classification into families

The sequences and other relevant data of the 5885 predicted open reading frames (ORFs) encoded by the complete yeast genome were obtained from the European Bioinformatics Institute (http://www.ebi.ac.uk), the Martinsried Institute for Protein Sequences (http://www.mips.biochem.mpg.de/), the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/) and the Yeast Protein Database (http://www.proteome.com/YPDhome.html). These public servers contain the yeast-related information present in the GenBank, PIR, and SwissProt sequence databases.

The number of transmembrane spans of all proteins was predicted by means of the KKD [8] algorithm, with a threshold value of 15 for the peripheral/integral odds [9]. All proteins with at least eight predicted transmembrane spans were used for further analysis to ensure that all proteins with 12 transmembrane spans were included.

All considered protein sequences were subjected to a BLAST [10] search with the BLAST e-mail server version 1.4 at the National Center for Biotechnology Information (Bethesda, MD, USA). Those protein sequences that produced high-scoring segment pairs with P(N)<10−9 were considered to be closely related. Those closely related protein sequences that were not yet present in the dataset were included.

Starting from this dataset, binary comparisons were carried out with the program PRSS, which tests the significance of protein sequence similarity and which belongs to the FASTA [11] software package version 2.0. The total number of binary comparisons can be calculated using the equation C=N(N−1)/2 with C the total number of binary comparisons and N the total number of proteins. Protein sequences were assigned to a family when the PRSS P-value of at least one binary comparison with a protein of that family was smaller than 10−9 [4, 6].

2.2 Multiple sequence alignment and evolutionary tree construction

Within each family of the MFS, protein sequences were aligned with the multiple sequence alignment program PileUp, which belongs to the Wisconsin Sequence Analysis Package [12], version 8.1.

On the basis of these alignments, dissimilarity matrices were calculated and converted into evolutionary distances, assuming [13, 14] that the rate of amino acid substitution follows the Poisson distribution, using the equation DAB=−ln (1−S) with D the evolutionary distance between two proteins A and B, and S the fraction of different amino acids (dissimilarity) between these two sequences. Evolutionary trees were constructed using the neighbor-joining [15] method. Both distance matrix calculation and evolutionary tree construction were carried out with the software package TREECON for Windows [16] version 1.2.

3 Classification of yeast MFS proteins

3.1 Identification of yeast membrane proteins

Approximately 2330 proteins were predicted to be membrane proteins, which corresponds to 40% of all proteins encoded by the yeast genome. However, this is probably an overestimation, especially with respect to the group of proteins with one or two predicted transmembrane spans, which also contains soluble proteins. This is because the number of transmembrane spans cannot be predicted unambiguously [9]

The considered group of proteins with at least eight predicted transmembrane spans initially comprised 341 proteins. On the basis of BLAST searches of these proteins, 93 related yeast proteins that had fewer than eight predicted transmembrane spans were added resulting in a final dataset of 434 proteins.

3.2 Classification of all yeast membrane proteins with at least eight predicted transmembrane spans

A total of 93 961 binary comparisons were carried out in order to compare all 434 proteins of the final dataset. This resulted in the clustering of 333 proteins into 62 families (results not shown). In spite of the fact that 77% of the proteins could be assigned to a family, only 34% of these families had four or more members. All other families had only two or three members. A total of 101 protein sequences could not be related to any other yeast protein sequence. The families based on the binary comparisons were in general agreement with those based on BLAST.

3.3 Identification and classification of yeast MFS proteins

Because the number of transmembrane spans cannot be predicted unambiguously [9] and because many of the proteins/families identified by the current analyses have an unknown function, our working definition of the candidate MFS screened in this study was less stringent than the original proposed definition [3]. In order to be sure that all MFS proteins were included, the following criteria were used to assign single proteins or protein families to the MFS. A single protein belongs to the MFS if it has 10–14 predicted transmembrane spans and has either a known transport function that is not specific for non-MFS (super)families (e.g. members of P-type transport ATPase or ABC transporter superfamilies) or if it has an unknown transport function. A family of proteins belongs to the MFS if at least one of its members has 10–14 predicted transmembrane spans and if a member of the family has a transport function that is not specific for non-MFS (super)families or if they all have an unknown function.

A consequence of this definition is that some proteins or families with an unknown function may be erroneously assigned to the MFS, but there is very little or no chance that a real MFS protein or family will not be assigned to the MFS.

Using this definition, 186 proteins were assigned to the MFS, of which 149 belonged to 23 families. These 23 families are given in Tables 117, while the remaining 37 single proteins are given in Tables 1820. These tables give for each protein the systematic gene name, synonym(s), GenBank accession number, and a brief description [17]. Most families consist of two or three proteins, and only 10 families contain four or more proteins.

View this table:
1

Sugar permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YBR241cYBR1625Z36110x1Similar to hexose permeases
YBR298cMAL31/MAL3T/YBR2116Z36167x1High affinity maltose permease (maltose/H+ symporter)
YCR098cYCR137/GIT1X59720x203Involved in inositol metabolism
YDL138wRGT2/D2160Z74186x1Regulator of glucose transport
YDL194wSNF3/D1234Z74242x1Similar to high affinity glucose permease
YDL199cD1209Z74247x1Similar to hexose permeases
YDL245cHXT15/D0230Z74293x1Similar to YJR158w, YNR072w, YEL069c and YDR342c
YDL247wD0220Z74295x1Similar to maltose permeases including YJR160c, YBR298c, and MAL61
YDR342cHXT7/D9651.11U51032x10High affinity hexose permease, expression is dependent on YDL194w (YDR342c and YDR343c differ by 2 bp)
YDR343cHXT6/D9651.12U51032x11High affinity hexose permease, expression is dependent on YDL194w (YDR342c and YDR343c differ by 2 bp)
YDR345cHXT3/D9651.14U51032x13Low affinity hexose permease
YDR387cD9509.7U32274x7Similar to YDR497c and YOL103w
YDR497cITR1/D9719.3U33057x3myo-Inositol permease (major), similar to YOL103w
YDR536wSTL1/D9719.39U33057x39Similar to hexose permeases
YEL069cHXT13U18795x8Similar to hexose permeases
YFL011wHXT10D50617x58Hexose permease
YFL040wD50617x29Similar to hexose permeases
YGL104cG3090Z72626x1Similar to glucose permeases
YGR289cAGT1/G9585Z73074x1General α-glucoside permease
YHR092cHXT4/LGT1/RAG1M81960x1Moderate to low affinity hexose permease
YHR094cHXT1U00060x9Low affinity hexose permease
YHR096cHXT5U00060x11Similar to hexose permeases
YIL171w/YIL170wHXT12/YI9402.06B/YI9402.06AZ47047x8Similar to hexose permeases (YIL170w and YIL171w are both homologous to HXT genes and are separated by a frameshift)
YJL214wHXT8/J0232/HRA569Z49489x1Similar to hexose permeases
YJL219wHXT9/HRC567/J0222Z49494x1Hexose permease
YJR158wHXT16/J2260Z49658x1Similar to hexose permeases
YJR160cJ2400Z49660x1Similar to maltose permeases (maltose/H+ symporters) YBR298c and MAL61
YLR081wGAL2/(IMP1)/L9449.6/L2373Z73253x1Galactose (and glucose) permease (facilitated diffusion permease), similar to YMR011w
YML123cPHO84/YM7056.03Z49218x3High affinity phosphate permease (phosphate/H+ symporter)
YMR011wHXT2/YM8270.15Z48613x15High affinity hexose permease
YNL318cHXT14/N0345/N0344Z71595x2Similar to hexose permeases
YNR072wHXT17/N3615Z71687x1Similar to hexose permeases
YOL103wITR2/HRB612/O0775Z74845x3myo-Inositol permease (minor), similar to YDR497c
YOL156wHXT11/LGT3/AOB567/O0414Z74898x1Low affinity glucose permease
  • Gene names, synonyms, and the use of the term ‘similar’ are according to the YPD database (Garrels [17]).

View this table:
2

Amino acid permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YBR068cBAP2/YBR0629Z35937x1Branched chain amino acid permease for leucine, valine, and isoleucine
YBR069cVAP1/TAT1/(TAP1)/YBR0710Z35938x1Amino acid permease for valine, leucine, isoleucine, tyrosine, and tryptophan
YBR132cYBR1007Z36001x1Similar to amino acid permeases
YCL025cYCC5X59720x57Similar to YDR508c and other permeases
YDL210wUGA4/D1037Z74258x1GABA specific high affinity permease
YDR046cYD9609.02/(PAP1)/D4180Z49209x2Similar to amino acid permeases
YDR160wYD8358.14Z50046x14Similar to amino acid permeases
YDR508cGNP1/D9719.14U33057x14High affinity glutamine permease
YEL063cCAN1U18795x14Permease for arginine, lysine, ornithine, and canavanine
YFL055wD50617x14Similar to YKR039w and other amino acid permeases
YGL077cHNM1/(CTR1)/CTR/G3213Z72599x1Choline permease
YGR055wMUP1/G4340Z72840x1High affinity methionine permease
YGR191wHIP1/G7572Z72975x2Histidine permease
YHL036wMUP3U11583x15Low affinity methionine permease
YKL174cYKL639Z28174x1Similar to YGL077c and other amino acid permeases
YKR039wGAP1Z28264x1General amino acid permease, transports all naturally occurring l-amino acids, GABA, ornithine, citrulline, some d-amino acids, and some toxic analogues
YLL061wL0555Z73166x1Similar to YKR039w and other amino acid permeases
YNL268wLYP1/N0790Z71544x1High affinity lysine specific permease
YNL270cAPL1/ALP1/N0660Z71546x1Similar to YEL063c and YNL268w, basic amino acids permeases
YNR056cN3502Z71671x1Similar to choline permease YGL077c and GABA specific permease YDL210w
YOL020wTAT2/SCM2/TAP2/LTG3/O2301Z74762x1High affinity tryptophan permease
YOR348cPUT4/O6345Z75256x1High affinity proline and γ-aminobutyrate permease
YPL265wDIP5/P0370Z73621x1Dicarboxylic amino acid permease
YPL274wP0335Z73630x1Similar to YKR039w and other amino acid permeases
View this table:
3

Multidrug permease homologues, family 1

Gene nameSynonym(s)GenBank accession numberDescription
YBR008cYBR0120Z35877x1Similar to multidrug permeases
YBR043cYBR0413Z35912x1Similar to multidrug permeases
YBR180wYBR1242Z36049x1Similar to multidrug permeases
YGR138cG6417Z72923x1Similar to Candida albicans benomyl/methotrexate resistance
YHR048wU00062x19Similar to multidrug permeases
YIL120wYI8277.09Z47047x58Similar to multidrug permeases
YIL121wYI8277.08Z47047x57Similar to multidrug permeases
YLL028wL0939Z73133x1Similar to YPR156c, YBR008c, and YHR048w
YNL065wYNL1613/YNL2417/N2417Z71341x1Similar to multidrug permeases
YNR055cHOL1/N3494Z71670x1Similar to multidrug permeases
YOR273cO5440Z75181x1Similar to YBR008c
YPR156cP9584.7U28371x3Similar to multidrug permeases
View this table:
4

Multidrug permease homologues, family 2

Gene nameSynonym(s)GenBank accession numberDescription
YBR293wYBR2109Z36162x1Similar to multidrug permeases
YCL069wX59720x5Similar to multidrug permeases
YCL070-73cYCL070c/YCL071c/YCL073cX59720x4Similar to multidrug permeases
YDR119wYD9727.14Z48758x14Similar to multidrug permeases
YEL065wU18795x12Similar to multidrug permeases
YGR224wORF_886916/G8537Z73009x1Similar to multidrug permeases
YHL040cU11583x11Similar to multidrug permeases
YHL047cU11583x4Similar to multidrug permeases
YKR105cZ28330x1Similar to multidrug permeases
YKR106wZ28202x1Similar to multidrug permeases
YML116wATR1/SNQ1/M_C542/YM8339.03Z49210x3Aminotriazole and 4-nitroquinoline resistance protein
YMR088CYM9582.13Z49259x14Similar to multidrug permeases
YMR279CYM8021.05Z49704x5Similar to multidrug permeases
YOL158CO0270Z74900x1Similar to multidrug permeases
YOR378wO6745Z75286x1Similar to aminotriazole resistance proteins
YPR198wSGE1/NOR1/P9677.3U25841x12Crystal violet resistance protein
View this table:
5

Allantoate permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YAL067cGEO1U12980x1Suppressor of sulfoxide ethionine resistance
YCR028cFEN2X59720x113Involved fenpropimorph resistance
YGR065cG4539Z72850x1Similar to allantoate permease
YGR260wG9328Z73044x2Similar to YJR152w
YIL166cYI9402.09Z47047x12Similar to YJR152w
YJR152wDAL5/UREP1/J2230Z49652x1Allantoate and ureidosuccinate permeases
YLL055wL0578Z73160x1Similar to YJR152w
YLR004cL1515Z73176x1Similar to YJR152w
View this table:
6

Uracil/uridine/allantoin permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YBL042cYBL0406Z35803x1Uridine permease
YBR021wFUR4/YBR0303Z35890x1Uracil permease
YIR028wDAL4Z47047x205Allantoin permease
YLR237wL8083.2U19027x14Similar to uracil/allantoin permeases
YOR071cO2935Z74979x1Similar to uracil/allantoin permeases
YOR192cO4759Z75100x1Similar to uracil/allantoin permeases
View this table:
7

Monocarboxylate permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YKL221wZ28221x1Similar to mammalian monocarboxylate permeases MCT1 and MCT2
YNL125cESBP6/N1223/N1882Z71401x1Similar to mammalian monocarboxylate permeases MCT1 and MCT2
YOL119cO0569Z74861x1Similar to mammalian monocarboxylate permeases
YOR306cO5658Z75214x1Similar to human X-linked PEST-containing permease
View this table:
8

Sulfate permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YBR294wSUL1/SFP/YBR2110Z36163x1High affinity sulfate permease
YGR125wG6362Z72910x1Unknown function
YLR092wSEL2/L9449.1/L2528Z73264x1Similar to YBR294w
YPR003cYP9723.03/LPZ3CZ48951x3Similar to YBR294w and heterologous sulfate permeases
View this table:
9

Ammonia permease homologues

GeneSynonym(s)GenBank accession numberDescription
YGR121cMEP1/(AAT1)/AMT1/G6331Z72906x1Ammonia permease of high capacity and moderate affinity
YNL142wMEP2/N1207/N1820Z71418x1Ammonia permease of low capacity and high affinity
YPR138cP9659.14U40829x9Similar to YGR121c
View this table:
10

Phosphate permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YCR037cPHO87/YCR524X59720x125Phosphate permease
YJL198wJ0336Z49473x1Similar to YCR037c
YNR013cN2052Z71628x1Similar to YCR037c and YJL198w
View this table:
11

Purine/cytosine permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YER056cFCY2U18813x3Cytosine/purine permease
YER060wFCY21/FCY22U18813x8Similar to YER056c
YGL186cG1370Z72708x1Similar to YER056c and YER060c
View this table:
12

Calcium permease homologues

Gene nameSynonym(s)GenBank accession numberDescription
YDL128wVCX1/HUM1/D2218Z74176x1Calcium permease (H+/Ca2+ antiporter) of the vacuoles
YNL321wN0339Z71597x1Unknown function
View this table:
13

ER protein translocation homologues

Gene nameSynonym(s)GenBank accession numberDescription
YBR283cYBR2020/SSH1Z36152x1Involved in protein translocation into the endoplasmic reticulum
YLR378cSEC61/L3502.5U19104x1Involved in protein translocation into the endoplasmic reticulum
View this table:
14

Vanadate resistance protein homologues

Gene nameSynonym(s)GenBank accession numberDescription
YER039cU18796x7Similar to YGL225w
YGL225wGOG5/VRG4/VAN2/G1001Z72747x1Vanadate resistance protein required for Golgi function
View this table:
15

Spore formation protein homologues

Gene nameSynonym(s)GenBank accession numberDescription
YJL062wHRC830/J1132Z49337x1Unknown function
YKL165cYKL619Z28165x1Possibly involved in spore formation
YLL031cL0929Z73136x1Similar to YJL062w
View this table:
16

Sexual differentiation protein homologues

Gene nameSynonym(s)GenBank accession numberDescription
YJL212cHRD799/J0236Z49487x1Similar to Schizosaccharomyces pombe ISP4+ which is induced by sexual differentiation
YPR194cP9677.13U25841x9Unknown function
View this table:
17

Unknown function

Gene nameSynonym(s)GenBank accession numberFamily
YBL089wYBL0703Z35850x1unknown 1
YEL064cU18795x13unknown 1
YER119cU18916x12unknown 1
YIL088cYI9910.08Z47047x90unknown 1
YJR001wYJR83.4/J1409Z49501x1unknown 1
YKL146wYKL600Z28146x2unknown 1
YNL101wN2185Z71377x1unknown 1
YLL005cL1361Z73110x1unknown 2
YLR241wL9672.9U20865x2unknown 2
YMR266wYM8156.8Z49260x8unknown 2
YOL084wO0953Z74826x1unknown 2
YDR338cD9651.8U51032x6unknown 3
YHR032wU00062x3unknown 3
YMR253cYM9920.07Z48639x7unknown 4
YPL264cP0373Z73620x1unknown 4
YDL231cD0810Z74279x1unknown 5
YGL140cG2550Z72662x1unknown 5
YDL206wD1053Z74254x1unknown 6
YJR106wJ1978Z49606x1unknown 6
YGL084cG3195Z72606x1unknown 7
YPL189wP2201Z73545x1unknown 7
View this table:
18

Single proteins with transport related functions

Gene nameSynonym(s)GenBank accession numberDescription
Multidrug resistance
YCR023cYCR241X59720x108Similar to multidrug permeases from family 2
YJR124cJ2046Z49624x1Similar to multidrug resistance proteins
Na+-H+ permeases
YDR456wD9461.40/NHX1U33007x40Similar to Na+/H+ permeases
YJL094cJ0909Z49369x1Similar to Enterococcus hirae Na+/H+ permease NapA
YLR138wL9606.4/L3149/NHA1Z73310x1Na+/H+ permease
Other permeases
YBR036cCSG2/CLS2/YBR0404Z35905x1Protein required for growth in high calcium concentration
YBR235wYBR1601Z36104x1Similar to vertebrate cation/chloride permeases
YGR227wDIE2/G8547Z73012x1Protein that promotes expression of ITR1
YKL217wJEN1Z28217x1Similar to E. coli osmoregulatory proP proline/betaine and KgtP α-ketoglutarate permeases
YNL275wN0626Z71551x1Similar to human band 3 anion transport protein
YPL092wSSU1/LPG16U43281x16Sulfite sensitivity protein
View this table:
19

Single proteins with an unknown permease function

Gene nameSynonym(s)GenBank accession numberDescription
YGL142cG2535Z72664x1Similar to YOR149c
YNR030wN3265Z71645x1Similar to YOR149c
YBL004wYBL0101Z35765x3Unknown function
YBL020wRFT1/YBL442Z35781x1Protein involved in nuclear division
YCL038cX59720x40Unknown function
YDR141cYD9302.17Z48179x17Unknown function
YDR335wMSN5/D9651.5U51032x3Unknown function
YFL007wD50617x62Unknown function
YGL114wG2950Z72636x1Similar to S. pombe ISP4+ involved in sexual differentiation
YJL091cJ0916Z49366x1Similar to YEL063c
YJL039cJ1216Z49314x1Similar to members of the Hsp70 family
YJL108cJ0811Z49383x1Unknown function
YJL163cJ0544Z49438x1Unknown function
YJL207cJ0312/HRD550Z49482x1Unknown function
YLR459wCDC91/L9122.2U22383x5Unknown function
YMR155wYM8520.04Z49705x4Unknown function
YMR221cYM9959.03Z49939x3Unknown function
YOL137wO0497Z74879x1Unknown function
YOR161cO3568Z75069x1Unknown function
YPL006wYP8132.07/LPA11U33335x11Similar to human PTC (NBCC disease)
View this table:
20

Single proteins with a potential permease function associated with other enzymatic functions

Gene nameSynonym(s)GenBank accession numberDescription
YBL082cALG3/RHK1/YBL0720Z35844x2Mannosyltransferase involved in N-glycosylation
YBR243cALG7/TUR1/YBR1628Z36112x1UDP-N-acetyl-glucosamine-1-P transferase
YGL022wSTT3/G3683Z72544x1Protein required for oligosaccharyltransferase activity
YGR157wCHO2/PEM1/G6673Z72942x1Phosphatidylethanolamine N-methyltransferase
YFL025cD50617x44Similar to mosquito NADH-ubiquinone oxidoreductase
YNL219cN1295/ALG9Z71495x1Mannosyl transferase

4 Evolutionary relationships

4.1 Paralogous vs. orthologous relationships

In general, two different kinds of evolutionary gene relationships can be distinguished [18, 19]: orthologous and paralogous relationships. Orthologous relationships are relationships between genes encoded by different organisms, called orthologues, while paralogous relationships are relationships of genes encoded by the same organism, called paralogues [19]. Homology between orthologues is a result of speciation (multiplication of species), while homology between paralogues is a result of gene duplication [18].

The evolutionary relationships studied in this review are relationships between proteins encoded by a single organism, viz. the budding yeast S. cerevisiae, and can thus be considered paralogous relationships. However, it should be noted that some genes may have been acquired by horizontal gene transfer, and that paralogues could actually have descended from orthologues, and vice versa. Moreover, it cannot be excluded that some proteins are related as a result of convergent evolution.

4.2 Sequence alignments and evolutionary trees

Multiple sequence alignments were made for all 23 families. Based on alignments of families with four or more members, evolutionary trees were constructed which are given in Figs. 110.

1

Evolutionary tree illustrating the relationships between the 34 sugar permease homologues. The distance scale represents the evolutionary distance, expressed as the number of substitutions per amino acid. Synonyms of the gene names, and gene descriptions are given in Table 1.

2

Evolutionary tree illustrating the relationships between the 24 amino acid permease homologues. Synonyms of the gene names, and gene descriptions are given in Table 2.

3

Evolutionary tree illustrating the relationships between the 12 multidrug permease homologues of family 1. Synonyms of the gene names, and gene descriptions are given in Table 3.

4

Evolutionary tree illustrating the relationships between the 16 multidrug permease homologues of family 2. Synonyms of the gene names, and gene descriptions are given in Table 4.

5

Evolutionary tree illustrating the relationships between the eight allantoate permease homologues. Synonyms of the gene names, and gene descriptions are given in Table 5.

6

Evolutionary tree illustrating the relationships between the six uracil/uridine/allantoin permease homologues. Synonyms of the gene names, and gene descriptions are given in Table 6.

7

Evolutionary tree illustrating the relationships between the four monocarboxylate permease homologues. Synonyms of the gene names, and gene descriptions are given in Table 7.

8

Evolutionary tree illustrating the relationships between the four sulfate permease homologues. Synonyms of the gene names, and gene descriptions are given in Table 8.

9

Evolutionary tree illustrating the relationships between the proteins of family 1 with unknown function. Synonyms of the gene names, and gene descriptions are given in Table 17.

10

Evolutionary tree illustrating the relationships between the proteins of family 2 with unknown function. Synonyms of the gene names, and gene descriptions are given in Table 17.

4.3 Permeases

The term ‘permease’ is used throughout this study instead of ‘facilitator’, ‘transport protein’, ‘transporter’ or related terms which all describe proteins with multiple transmembrane spans (usually 12) that exhibit a transport function including mechanisms such as uniport, symport, antiport and facilitated diffusion. The use of ‘permease’ avoids any reference to given mechanisms while remaining consistent with the existing nomenclature.

5 The MFS families

5.1 Sugar permease homologues

The family of sugar permease homologues has 34 members and is the largest MFS family of yeast. All members are given in Table 1 and a graphical representation of their evolutionary relationships is given in Fig. 1, in which five clusters can be distinguished.

Cluster I is a tight cluster that contains 16 hexose permeases (Fig. 1, Table 1). These hexose permeases have been discussed elsewhere in detail [7]. This cluster of hexose permeases includes half of all sugar permeases. This abundance might be explained by the fact that glucose is the primary substrate of S. cerevisiae. Apart from the hexose permeases, this cluster also contains the galactose permease YLR081w (GAL2). Cluster II contains two proteins YBR241c and YGL104c, of which the exact substrate is not known. Cluster III contains four sugar permeases, including the maltose permease YBR298c (MAL31) and the general α-glucoside permease YGR289c (AGT1). The two remaining proteins of this cluster, YDL247c and YJR160c, are closely related to the former, and are thus probably also maltose permeases. Cluster IV consists of two myo-inositol permeases, YOL103w (ITR2) and YDR497c (ITR1), which are very closely related. Finally, cluster V contains YDL138w (RGT2) and YDL194w (SNF3), which appear to be glucose receptors [20]. The remaining six unclustered proteins do not have a known substrate with the exception of YML123c (PHO84), which is an inorganic phosphate permease, and YCR098c (GIT1), which is involved in inositol metabolism.

5.2 Amino acid permease homologues

The family of the amino acid permease homologues contains 24 proteins given in Table 2, and is the second largest family of yeast MFS proteins. The evolutionary relationships of these proteins are given in Fig. 2.

Cluster I seems to consist entirely of amino acid permeases that have a specificity towards neutral amino acids (Fig. 2, Table 2). Proteins YDR508c (GNP1) and YOL020w (TAT2/SCM2/TAP2/LGT3) are specific for neutral polar amino acids, YBR068c (BAP2) is specific for neutral hydrophobic amino acids, and YBR069c (VAP1/TAT1) is specific for neutral polar and hydrophobic amino acids. The specific substrates of YDR046c and YCL025c are not known, but they are probably related to those of their closest homologues, YBR068c (BAP2) (neutral hydrophobic amino acids) and YDR508c (GNP1) (neutral polar amino acids). Cluster II contains four proteins, of which YGR191w (HIP1) is a histidine-specific permease, YKR039w (GAP1) is a general amino acid permease, and YLL061w and YPL274w have no known substrate. Cluster III contains the three basic amino acid permeases YEL063c (CAN1), YNL268w (LYP1), and YNL270c (APL1/ALP1). Clusters I and II are more closely related to each other than to cluster III. The methionine permeases YGR055w (MUP1) and YHL036w (MUP3) are more related to each other than to the other amino acid permease homologues, reflecting their functional relationship. The remaining nine proteins are only distantly related to each other and comprise the proline/γ-aminobutyrate permease YOR348c (PUT4), the dicarboxylic amino acid permease YPL265w (DIP5), an amino acid permease YBR132c with no known specific substrate, the choline permease YGL077c (HNM1/CTR1), the GABA (4-aminobutyric acid) permease YDL210w (UGA4), and finally the proteins YKL174c, YNR056c, YHL036w, YDR160w, and YFL055w, which have no known specific substrate.

5.3 Multidrug permease homologues

The multidrug permeases homologues, which confer a multidrug resistance, comprise a total of 28 proteins, which are divided into two distinct families with 12 and 16 members respectively. This division is in contrast with a previous study in which all multidrug permease homologues were presented in a single family [6], but is in general agreement with other previous studies that divided the multidrug permease homologues into two distinct families [4] or subfamilies [5]. Moreover, this division into two different families is supported by the fact that the members of the multidrug permease homologues families 1 and 2 have 12 and 14 predicted transmembrane spans respectively. Since a detailed description of these proteins can be found elsewhere [6], only a brief discussion is given.

The 12 members of family 1 of the multidrug permease homologues are given in Table 3, while the evolutionary relationships are illustrated in Fig. 3. Two distinct clusters I and II can be distinguished, but the only protein of which a function is known, YNR055c (HOL1), involved in the uptake of histinol, does not seem to be closely related to any of these clusters. However, the members of cluster I are related to the orthologous multidrug resistance proteins from Candida maltosa, C. albicans and Schizosaccharomyces pombe [6].

Table 4 lists all 18 members of family 2 of the multidrug permease homologues. The evolutionary tree given in Fig. 4 shows four clusters. Cluster I contains six proteins with no known substrate, cluster II contains three proteins which includes the aminotriazole and 4-nitroquinoline resistance protein YML116w (ATR1), cluster III contains two proteins with no known substrate, and cluster IV contains four proteins among which the crystal violet resistance protein YPR198w (SGE1). Protein YDR119w does not seem to be closely related to any of these clusters.

5.4 Allantoate permease homologues

The allantoate permease homologues family consists of eight proteins that are given in Table 5. The allantoate permease YJR152w (DAL5) has given its name to the family, the other members of which might transport other weak acids and include the proteins YAl067c (GEO1) and YCR028c (FEN2), which are both involved in some kind of resistance. An evolutionary tree is given in Fig. 5, which shows the presence of two clusters of each three proteins. The allantoate permease YJR152w (DAL5) belongs to cluster II.

5.5 Uracil/uridine/allantoin permease homologues

The family of uracil/uridine/allantoin permease homologues contains six members, given in Table 6. The evolutionary tree, given in Fig. 6, clearly shows two distinct clusters. The permeases of cluster I are very closely related to each other, suggesting a very similar but unknown substrate. Cluster II contains all permeases with known substrate: the uridine permease YBL042c, the uracil permease YBR021w (FUR4), and the allantoin permease YIR028w (DAL4).

5.6 Monocarboxylate permease homologues

The monocarboxylate permease homologues are a family of four members, given in Table 7. The evolutionary tree in Fig. 7 shows that YOR306c and YOL119c are more closely related to each other than to YKL221w and YNL125c, and vice versa. These genes are weakly related to mammalian monocarboxylate permeases.

5.7 Sulfate permease homologues

The four members of the sulfate permease homologues family are given in Table 8. The evolutionary relationships are illustrated in Fig. 8, clearly showing that the sulfate permease YBR294w (SUL1/SFP) and YLR092w are closely related to each other. In contrast, YGR125w and YPR003c are only distantly related to all other members of this family.

5.8 Ammonia permease homologues

This family contains three members, given in Table 9. These are the high capacity ammonia permease YGR121c (MEP1/AMT1) which has a low affinity for ammonia, the low capacity ammonia permease YNL142w (MEP2) which has a high affinity for ammonia, and the permease YPR138c, of which no further details are known.

5.9 Phosphate permease homologues

The three members of the phosphate permease homologue family are given in Table 10. They are the phosphate permease YCR037c (PHO87) and the two permeases YJL198w and YNR013c which have no known substrate. Remarkably, the phosphate permease YML123c (PHO84) does not belong to this family but to the family of sugar permease homologues.

5.10 Purine/cytosine permease homologues

Table 11 lists the three members of the purine/cytosine permease homologue family. These are the purine/cytosine permease YER056c (FCY2) and its two relatives YER060w (FCY21) and YGL186c.

5.11 Calcium permease homologues

The calcium permease homologue family contains two members, the calcium permease YDL128w (VCX1/HUM1) and YNL321w, which are given in Table 12. YDL128w is located in the vacuolar membrane and uses a H+/Ca2+ antiporter mechanism.

6 Families with an unknown permease function

Two families (ER protein translocation and vanadate resistance) are characterized by biochemical parameters not obviously related to transport. Two other families have a member which is involved in global physiological functions such as spore formation or sexual differentiation which could involve several putative transport steps. Seven other families have totally unknown functions even though they are classified as putative permeases according to our criteria. As stated above, rather non-stringent criteria were used to ensure that all real MFS members were comprised in this study.

6.1 ER protein translocation homologues

This family has two members, given in Table 13, of which YLR378c (SEC61) is a component of the Sec61p-Sss1p-Sbh1p complex, and YBR283c (SSH1) is a component of the Ssh1p-Sss1p-Sbh2p complex, both involved in protein translocation into the endoplasmic reticulum. The oligomeric ring of the YLR378c (SEC61) complex may function as a protein conducting channel of the endoplasmic reticulum membrane [21].

6.2 Vanadate resistance protein homologues

The two members of this family are the vanadate resistance protein YGL225w (GOG5/VRG4/VAN2) and the related protein YER039c, given in Table 14. YGL225w is required for normal Golgi functioning. Its mutant has a severe glycosylation defect and abnormal retention of soluble endoplasmic reticulum proteins [22].

6.3 Spore formation protein homologues

The function of the family with three members, given in Table 15, is unknown but one of its members, YKL165c, is involved in spore formation.

6.4 Sexual differentiation protein homologues

The family of sexual differentiation protein homologues contains the two proteins YJL212c and YPR194c, given in Table 16, of which the former is similar to Schizosaccharomyces pombe ISP4+ which is induced by sexual differentiation

6.5 Unknown families 1–7

A total of 17 proteins of unknown function could be clustered in the seven families given in Table 17. The evolutionary relationship and subclustering of the two largest families are given in Figs. 9 and 10. That the seven members of unknown family 1 are indeed permeases is indicated by their weak homology with plant and bacterial amino acid permeases. Also notice that the two members of unknown family 5 are twice as long (1125 and 1219 amino acids) as most MFS proteins.

7 Single MFS proteins

All potential MFS proteins that did not have close relatives in S. cerevisiae on the basis of BLAST or binary comparisons are listed in Tables 18, 19 and 20. Of these 37 proteins, 20 have an unknown function.

Proteins with transport related functions are given in Table 18 and comprise the multidrug resistance protein homologues YCR023c and YJR124c, the cation/Cl symporter homologue YBR235w, the Na+/H+ antiporter homologues YDR456w, YJL094c, and YLR318w (NHA1), the proline/betaine/α-ketoglutarate permease homologue YKL217w (JEN1), and the anion permease homologue YNL275w. Protein YBR036w (CSG2/CLS2) is required for growth in high calcium concentration (>25 mM) (Table 18) and might be a calcium permease. Protein YGR227w (DIE2) promotes the expression of the myo-inositol permease YDR497c (ITR1), which belongs to cluster II of the sugar permease homologues (Fig. 1). The sulfite sensitivity protein YPL092w (SSU1) [23] might be a sulfite permease.

Proteins with an unknown permease function are given in Table 19. A weak similarity between protein YJL091c and the basic amino acid permease YEL063c (CAN1) was described earlier [24], but was not strong enough to be reflected in our computer analyses. The two proteins YGL142c and YNR030w exhibit a weak similarity towards YOR149c (SMP3/LAS2/SAP2), which is required for plasmid maintenance. Remarkably, YGL114w seems to be related to ISP4+, involved in sexual differentiation of S. pombe but not to YJL212c (Table 16).

Finally, proteins with a potential permease function associated with other enzymatic functions are given in Table 20 and include the glucosamine, oligosaccharyl and two mannosyl transferases YBR243c (ALG7/TUR1), YGL022w (STT3), YBL082c (ALG3/RHK1) and YNL219c (ALG9). YGR157w (CHO2/PEM1) is a phosphatidylethanolamine N-methyltransferase whereas YFL025c is similar to subunit 4 from complex I of the respiratory chain. No permease function has been allocated experimentally to any of these membrane proteins. However, our computer analysis raises the possibility that they might have derived from ancestor permeases and possibly that they might still carry out, in addition to their membrane bound metabolic activity, remnant permease functions.

8 Conclusions

This study has shown that computer-aided analysis of protein sequences is a powerful method for the classification and function assignment of yeast membrane proteins. Starting from a dataset of 5885 putative protein sequences encoded by the yeast genome, 186 potential yeast permeases of the MFS were identified. A classification of these proteins resulted in the division of 149 proteins into 23 families, of which 12 could be assigned a (potential) permease function. The group of single proteins contained 20 proteins with an unknown function, while the remaining proteins include transport, drug resistance, transferase proteins and a variety of other functional proteins.

An earlier study of the MFS carried out at our laboratory [4] revealed 100 potential MFS proteins, which could be divided into 17 families, including 13 for which a potential function could be assigned. However, those MFS proteins that were not assigned to a family were not studied. The number of families has thus increased from 17 to 23, while the number of proteins that belong to those families has increased from 100 to 149.

The classification of MFS proteins into families on the basis of public database queries and binary comparisons, and the subsequent alignment and evolutionary tree construction, has allowed us to predict a putative function for proteins with a previously unknown function. The presence of a protein with an unknown function in a family with a known general function, which is based on the presence in that family of permeases with a known substrate, suggests a general function for this protein. Moreover, the close relationship of such a protein with proteins with a known substrate, which can easily be observed in the evolutionary trees, even suggests a specific class of substrates for that protein. It is important to point out that each of the predictions must be tested experimentally before final conclusions are reached on the function of the proteins analyzed. Taking into account our lax definition of candidate MFS, it might turn out in fine that several proteins and families analyzed here have no transport function. On the other hand, it is unlikely that we missed any real permeases.

It is obvious that the approach discussed above can be used for the analysis of other yeast membrane proteins, possibly also for soluble proteins, and for the analysis of proteins from other organisms of which the full genome sequences are becoming increasingly available.

9 Note added in proof

The following open reading frames with reported permease function were not included in our analysis because they do not belong to the major facilitator superfamily according to our definition.

View this table:

Acknowledgements

The authors would like to thank P. Mordant, J.-L. Jonniaux and J. Dupuis for retrieving and maintaining the sequences from public databases, and Y. Van de Peer for implementing the possibility of unrooted tree topologies in TREECON for Windows. This work was supported by the Services Fédéraux des Affaires Scientifiques, Techniques et Culturelles: Poles d’Attraction Inter Universitaires.

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View Abstract