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.

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 [2–5] 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.

Computer analysis

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].

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.

Classification of yeast MFS proteins

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.

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.

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 1–17, while the remaining 37 single proteins are given in Tables 18–20. 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.

1

Sugar permease homologues

Gene name Synonym(s) GenBank accession number Description 
YBR241c YBR1625 Z36110x1 Similar to hexose permeases 
YBR298c MAL31/MAL3T/YBR2116 Z36167x1 High affinity maltose permease (maltose/H+ symporter) 
YCR098c YCR137/GIT1 X59720x203 Involved in inositol metabolism 
YDL138w RGT2/D2160 Z74186x1 Regulator of glucose transport 
YDL194w SNF3/D1234 Z74242x1 Similar to high affinity glucose permease 
YDL199c D1209 Z74247x1 Similar to hexose permeases 
YDL245c HXT15/D0230 Z74293x1 Similar to YJR158w, YNR072w, YEL069c and YDR342c 
YDL247w D0220 Z74295x1 Similar to maltose permeases including YJR160c, YBR298c, and MAL61 
YDR342c HXT7/D9651.11 U51032x10 High affinity hexose permease, expression is dependent on YDL194w (YDR342c and YDR343c differ by 2 bp) 
YDR343c HXT6/D9651.12 U51032x11 High affinity hexose permease, expression is dependent on YDL194w (YDR342c and YDR343c differ by 2 bp) 
YDR345c HXT3/D9651.14 U51032x13 Low affinity hexose permease 
YDR387c D9509.7 U32274x7 Similar to YDR497c and YOL103w 
YDR497c ITR1/D9719.3 U33057x3 myo-Inositol permease (major), similar to YOL103w 
YDR536w STL1/D9719.39 U33057x39 Similar to hexose permeases 
YEL069c HXT13 U18795x8 Similar to hexose permeases 
YFL011w HXT10 D50617x58 Hexose permease 
YFL040w  D50617x29 Similar to hexose permeases 
YGL104c G3090 Z72626x1 Similar to glucose permeases 
YGR289c AGT1/G9585 Z73074x1 General α-glucoside permease 
YHR092c HXT4/LGT1/RAG1 M81960x1 Moderate to low affinity hexose permease 
YHR094c HXT1 U00060x9 Low affinity hexose permease 
YHR096c HXT5 U00060x11 Similar to hexose permeases 
YIL171w/YIL170w HXT12/YI9402.06B/YI9402.06A Z47047x8 Similar to hexose permeases (YIL170w and YIL171w are both homologous to HXT genes and are separated by a frameshift) 
YJL214w HXT8/J0232/HRA569 Z49489x1 Similar to hexose permeases 
YJL219w HXT9/HRC567/J0222 Z49494x1 Hexose permease 
YJR158w HXT16/J2260 Z49658x1 Similar to hexose permeases 
YJR160c J2400 Z49660x1 Similar to maltose permeases (maltose/H+ symporters) YBR298c and MAL61 
YLR081w GAL2/(IMP1)/L9449.6/L2373 Z73253x1 Galactose (and glucose) permease (facilitated diffusion permease), similar to YMR011w 
YML123c PHO84/YM7056.03 Z49218x3 High affinity phosphate permease (phosphate/H+ symporter) 
YMR011w HXT2/YM8270.15 Z48613x15 High affinity hexose permease 
YNL318c HXT14/N0345/N0344 Z71595x2 Similar to hexose permeases 
YNR072w HXT17/N3615 Z71687x1 Similar to hexose permeases 
YOL103w ITR2/HRB612/O0775 Z74845x3 myo-Inositol permease (minor), similar to YDR497c 
YOL156w HXT11/LGT3/AOB567/O0414 Z74898x1 Low affinity glucose permease 
Gene name Synonym(s) GenBank accession number Description 
YBR241c YBR1625 Z36110x1 Similar to hexose permeases 
YBR298c MAL31/MAL3T/YBR2116 Z36167x1 High affinity maltose permease (maltose/H+ symporter) 
YCR098c YCR137/GIT1 X59720x203 Involved in inositol metabolism 
YDL138w RGT2/D2160 Z74186x1 Regulator of glucose transport 
YDL194w SNF3/D1234 Z74242x1 Similar to high affinity glucose permease 
YDL199c D1209 Z74247x1 Similar to hexose permeases 
YDL245c HXT15/D0230 Z74293x1 Similar to YJR158w, YNR072w, YEL069c and YDR342c 
YDL247w D0220 Z74295x1 Similar to maltose permeases including YJR160c, YBR298c, and MAL61 
YDR342c HXT7/D9651.11 U51032x10 High affinity hexose permease, expression is dependent on YDL194w (YDR342c and YDR343c differ by 2 bp) 
YDR343c HXT6/D9651.12 U51032x11 High affinity hexose permease, expression is dependent on YDL194w (YDR342c and YDR343c differ by 2 bp) 
YDR345c HXT3/D9651.14 U51032x13 Low affinity hexose permease 
YDR387c D9509.7 U32274x7 Similar to YDR497c and YOL103w 
YDR497c ITR1/D9719.3 U33057x3 myo-Inositol permease (major), similar to YOL103w 
YDR536w STL1/D9719.39 U33057x39 Similar to hexose permeases 
YEL069c HXT13 U18795x8 Similar to hexose permeases 
YFL011w HXT10 D50617x58 Hexose permease 
YFL040w  D50617x29 Similar to hexose permeases 
YGL104c G3090 Z72626x1 Similar to glucose permeases 
YGR289c AGT1/G9585 Z73074x1 General α-glucoside permease 
YHR092c HXT4/LGT1/RAG1 M81960x1 Moderate to low affinity hexose permease 
YHR094c HXT1 U00060x9 Low affinity hexose permease 
YHR096c HXT5 U00060x11 Similar to hexose permeases 
YIL171w/YIL170w HXT12/YI9402.06B/YI9402.06A Z47047x8 Similar to hexose permeases (YIL170w and YIL171w are both homologous to HXT genes and are separated by a frameshift) 
YJL214w HXT8/J0232/HRA569 Z49489x1 Similar to hexose permeases 
YJL219w HXT9/HRC567/J0222 Z49494x1 Hexose permease 
YJR158w HXT16/J2260 Z49658x1 Similar to hexose permeases 
YJR160c J2400 Z49660x1 Similar to maltose permeases (maltose/H+ symporters) YBR298c and MAL61 
YLR081w GAL2/(IMP1)/L9449.6/L2373 Z73253x1 Galactose (and glucose) permease (facilitated diffusion permease), similar to YMR011w 
YML123c PHO84/YM7056.03 Z49218x3 High affinity phosphate permease (phosphate/H+ symporter) 
YMR011w HXT2/YM8270.15 Z48613x15 High affinity hexose permease 
YNL318c HXT14/N0345/N0344 Z71595x2 Similar to hexose permeases 
YNR072w HXT17/N3615 Z71687x1 Similar to hexose permeases 
YOL103w ITR2/HRB612/O0775 Z74845x3 myo-Inositol permease (minor), similar to YDR497c 
YOL156w HXT11/LGT3/AOB567/O0414 Z74898x1 Low affinity glucose permease 

Gene names, synonyms, and the use of the term ‘similar’ are according to the YPD database (Garrels [17]).

2

Amino acid permease homologues

Gene name Synonym(s) GenBank accession number Description 
YBR068c BAP2/YBR0629 Z35937x1 Branched chain amino acid permease for leucine, valine, and isoleucine 
YBR069c VAP1/TAT1/(TAP1)/YBR0710 Z35938x1 Amino acid permease for valine, leucine, isoleucine, tyrosine, and tryptophan 
YBR132c YBR1007 Z36001x1 Similar to amino acid permeases 
YCL025c YCC5 X59720x57 Similar to YDR508c and other permeases 
YDL210w UGA4/D1037 Z74258x1 GABA specific high affinity permease 
YDR046c YD9609.02/(PAP1)/D4180 Z49209x2 Similar to amino acid permeases 
YDR160w YD8358.14 Z50046x14 Similar to amino acid permeases 
YDR508c GNP1/D9719.14 U33057x14 High affinity glutamine permease 
YEL063c CAN1 U18795x14 Permease for arginine, lysine, ornithine, and canavanine 
YFL055w  D50617x14 Similar to YKR039w and other amino acid permeases 
YGL077c HNM1/(CTR1)/CTR/G3213 Z72599x1 Choline permease 
YGR055w MUP1/G4340 Z72840x1 High affinity methionine permease 
YGR191w HIP1/G7572 Z72975x2 Histidine permease 
YHL036w MUP3 U11583x15 Low affinity methionine permease 
YKL174c YKL639 Z28174x1 Similar to YGL077c and other amino acid permeases 
YKR039w GAP1 Z28264x1 General amino acid permease, transports all naturally occurring l-amino acids, GABA, ornithine, citrulline, some d-amino acids, and some toxic analogues 
YLL061w L0555 Z73166x1 Similar to YKR039w and other amino acid permeases 
YNL268w LYP1/N0790 Z71544x1 High affinity lysine specific permease 
YNL270c APL1/ALP1/N0660 Z71546x1 Similar to YEL063c and YNL268w, basic amino acids permeases 
YNR056c N3502 Z71671x1 Similar to choline permease YGL077c and GABA specific permease YDL210w 
    
YOL020w TAT2/SCM2/TAP2/LTG3/O2301 Z74762x1 High affinity tryptophan permease 
YOR348c PUT4/O6345 Z75256x1 High affinity proline and γ-aminobutyrate permease 
YPL265w DIP5/P0370 Z73621x1 Dicarboxylic amino acid permease 
YPL274w P0335 Z73630x1 Similar to YKR039w and other amino acid permeases 
Gene name Synonym(s) GenBank accession number Description 
YBR068c BAP2/YBR0629 Z35937x1 Branched chain amino acid permease for leucine, valine, and isoleucine 
YBR069c VAP1/TAT1/(TAP1)/YBR0710 Z35938x1 Amino acid permease for valine, leucine, isoleucine, tyrosine, and tryptophan 
YBR132c YBR1007 Z36001x1 Similar to amino acid permeases 
YCL025c YCC5 X59720x57 Similar to YDR508c and other permeases 
YDL210w UGA4/D1037 Z74258x1 GABA specific high affinity permease 
YDR046c YD9609.02/(PAP1)/D4180 Z49209x2 Similar to amino acid permeases 
YDR160w YD8358.14 Z50046x14 Similar to amino acid permeases 
YDR508c GNP1/D9719.14 U33057x14 High affinity glutamine permease 
YEL063c CAN1 U18795x14 Permease for arginine, lysine, ornithine, and canavanine 
YFL055w  D50617x14 Similar to YKR039w and other amino acid permeases 
YGL077c HNM1/(CTR1)/CTR/G3213 Z72599x1 Choline permease 
YGR055w MUP1/G4340 Z72840x1 High affinity methionine permease 
YGR191w HIP1/G7572 Z72975x2 Histidine permease 
YHL036w MUP3 U11583x15 Low affinity methionine permease 
YKL174c YKL639 Z28174x1 Similar to YGL077c and other amino acid permeases 
YKR039w GAP1 Z28264x1 General amino acid permease, transports all naturally occurring l-amino acids, GABA, ornithine, citrulline, some d-amino acids, and some toxic analogues 
YLL061w L0555 Z73166x1 Similar to YKR039w and other amino acid permeases 
YNL268w LYP1/N0790 Z71544x1 High affinity lysine specific permease 
YNL270c APL1/ALP1/N0660 Z71546x1 Similar to YEL063c and YNL268w, basic amino acids permeases 
YNR056c N3502 Z71671x1 Similar to choline permease YGL077c and GABA specific permease YDL210w 
    
YOL020w TAT2/SCM2/TAP2/LTG3/O2301 Z74762x1 High affinity tryptophan permease 
YOR348c PUT4/O6345 Z75256x1 High affinity proline and γ-aminobutyrate permease 
YPL265w DIP5/P0370 Z73621x1 Dicarboxylic amino acid permease 
YPL274w P0335 Z73630x1 Similar to YKR039w and other amino acid permeases 
3

Multidrug permease homologues, family 1

Gene name Synonym(s) GenBank accession number Description 
YBR008c YBR0120 Z35877x1 Similar to multidrug permeases 
YBR043c YBR0413 Z35912x1 Similar to multidrug permeases 
YBR180w YBR1242 Z36049x1 Similar to multidrug permeases 
YGR138c G6417 Z72923x1 Similar to Candida albicans benomyl/methotrexate resistance 
YHR048w  U00062x19 Similar to multidrug permeases 
YIL120w YI8277.09 Z47047x58 Similar to multidrug permeases 
YIL121w YI8277.08 Z47047x57 Similar to multidrug permeases 
YLL028w L0939 Z73133x1 Similar to YPR156c, YBR008c, and YHR048w 
YNL065w YNL1613/YNL2417/N2417 Z71341x1 Similar to multidrug permeases 
YNR055c HOL1/N3494 Z71670x1 Similar to multidrug permeases 
YOR273c O5440 Z75181x1 Similar to YBR008c 
YPR156c P9584.7 U28371x3 Similar to multidrug permeases 
Gene name Synonym(s) GenBank accession number Description 
YBR008c YBR0120 Z35877x1 Similar to multidrug permeases 
YBR043c YBR0413 Z35912x1 Similar to multidrug permeases 
YBR180w YBR1242 Z36049x1 Similar to multidrug permeases 
YGR138c G6417 Z72923x1 Similar to Candida albicans benomyl/methotrexate resistance 
YHR048w  U00062x19 Similar to multidrug permeases 
YIL120w YI8277.09 Z47047x58 Similar to multidrug permeases 
YIL121w YI8277.08 Z47047x57 Similar to multidrug permeases 
YLL028w L0939 Z73133x1 Similar to YPR156c, YBR008c, and YHR048w 
YNL065w YNL1613/YNL2417/N2417 Z71341x1 Similar to multidrug permeases 
YNR055c HOL1/N3494 Z71670x1 Similar to multidrug permeases 
YOR273c O5440 Z75181x1 Similar to YBR008c 
YPR156c P9584.7 U28371x3 Similar to multidrug permeases 
4

Multidrug permease homologues, family 2

Gene name Synonym(s) GenBank accession number Description 
YBR293w YBR2109 Z36162x1 Similar to multidrug permeases 
YCL069w  X59720x5 Similar to multidrug permeases 
YCL070-73c YCL070c/YCL071c/YCL073c X59720x4 Similar to multidrug permeases 
YDR119w YD9727.14 Z48758x14 Similar to multidrug permeases 
YEL065w  U18795x12 Similar to multidrug permeases 
YGR224w ORF_886916/G8537 Z73009x1 Similar to multidrug permeases 
YHL040c  U11583x11 Similar to multidrug permeases 
YHL047c  U11583x4 Similar to multidrug permeases 
YKR105c  Z28330x1 Similar to multidrug permeases 
YKR106w  Z28202x1 Similar to multidrug permeases 
YML116w ATR1/SNQ1/M_C542/YM8339.03 Z49210x3 Aminotriazole and 4-nitroquinoline resistance protein 
YMR088C YM9582.13 Z49259x14 Similar to multidrug permeases 
YMR279C YM8021.05 Z49704x5 Similar to multidrug permeases 
YOL158C O0270 Z74900x1 Similar to multidrug permeases 
YOR378w O6745 Z75286x1 Similar to aminotriazole resistance proteins 
YPR198w SGE1/NOR1/P9677.3 U25841x12 Crystal violet resistance protein 
Gene name Synonym(s) GenBank accession number Description 
YBR293w YBR2109 Z36162x1 Similar to multidrug permeases 
YCL069w  X59720x5 Similar to multidrug permeases 
YCL070-73c YCL070c/YCL071c/YCL073c X59720x4 Similar to multidrug permeases 
YDR119w YD9727.14 Z48758x14 Similar to multidrug permeases 
YEL065w  U18795x12 Similar to multidrug permeases 
YGR224w ORF_886916/G8537 Z73009x1 Similar to multidrug permeases 
YHL040c  U11583x11 Similar to multidrug permeases 
YHL047c  U11583x4 Similar to multidrug permeases 
YKR105c  Z28330x1 Similar to multidrug permeases 
YKR106w  Z28202x1 Similar to multidrug permeases 
YML116w ATR1/SNQ1/M_C542/YM8339.03 Z49210x3 Aminotriazole and 4-nitroquinoline resistance protein 
YMR088C YM9582.13 Z49259x14 Similar to multidrug permeases 
YMR279C YM8021.05 Z49704x5 Similar to multidrug permeases 
YOL158C O0270 Z74900x1 Similar to multidrug permeases 
YOR378w O6745 Z75286x1 Similar to aminotriazole resistance proteins 
YPR198w SGE1/NOR1/P9677.3 U25841x12 Crystal violet resistance protein 
5

Allantoate permease homologues

Gene name Synonym(s) GenBank accession number Description 
YAL067c GEO1 U12980x1 Suppressor of sulfoxide ethionine resistance 
YCR028c FEN2 X59720x113 Involved fenpropimorph resistance 
YGR065c G4539 Z72850x1 Similar to allantoate permease 
YGR260w G9328 Z73044x2 Similar to YJR152w 
YIL166c YI9402.09 Z47047x12 Similar to YJR152w 
YJR152w DAL5/UREP1/J2230 Z49652x1 Allantoate and ureidosuccinate permeases 
YLL055w L0578 Z73160x1 Similar to YJR152w 
YLR004c L1515 Z73176x1 Similar to YJR152w 
Gene name Synonym(s) GenBank accession number Description 
YAL067c GEO1 U12980x1 Suppressor of sulfoxide ethionine resistance 
YCR028c FEN2 X59720x113 Involved fenpropimorph resistance 
YGR065c G4539 Z72850x1 Similar to allantoate permease 
YGR260w G9328 Z73044x2 Similar to YJR152w 
YIL166c YI9402.09 Z47047x12 Similar to YJR152w 
YJR152w DAL5/UREP1/J2230 Z49652x1 Allantoate and ureidosuccinate permeases 
YLL055w L0578 Z73160x1 Similar to YJR152w 
YLR004c L1515 Z73176x1 Similar to YJR152w 
6

Uracil/uridine/allantoin permease homologues

Gene name Synonym(s) GenBank accession number Description 
YBL042c YBL0406 Z35803x1 Uridine permease 
YBR021w FUR4/YBR0303 Z35890x1 Uracil permease 
YIR028w DAL4 Z47047x205 Allantoin permease 
YLR237w L8083.2 U19027x14 Similar to uracil/allantoin permeases 
YOR071c O2935 Z74979x1 Similar to uracil/allantoin permeases 
YOR192c O4759 Z75100x1 Similar to uracil/allantoin permeases 
Gene name Synonym(s) GenBank accession number Description 
YBL042c YBL0406 Z35803x1 Uridine permease 
YBR021w FUR4/YBR0303 Z35890x1 Uracil permease 
YIR028w DAL4 Z47047x205 Allantoin permease 
YLR237w L8083.2 U19027x14 Similar to uracil/allantoin permeases 
YOR071c O2935 Z74979x1 Similar to uracil/allantoin permeases 
YOR192c O4759 Z75100x1 Similar to uracil/allantoin permeases 
7

Monocarboxylate permease homologues

Gene name Synonym(s) GenBank accession number Description 
YKL221w  Z28221x1 Similar to mammalian monocarboxylate permeases MCT1 and MCT2 
YNL125c ESBP6/N1223/N1882 Z71401x1 Similar to mammalian monocarboxylate permeases MCT1 and MCT2 
YOL119c O0569 Z74861x1 Similar to mammalian monocarboxylate permeases 
YOR306c O5658 Z75214x1 Similar to human X-linked PEST-containing permease 
Gene name Synonym(s) GenBank accession number Description 
YKL221w  Z28221x1 Similar to mammalian monocarboxylate permeases MCT1 and MCT2 
YNL125c ESBP6/N1223/N1882 Z71401x1 Similar to mammalian monocarboxylate permeases MCT1 and MCT2 
YOL119c O0569 Z74861x1 Similar to mammalian monocarboxylate permeases 
YOR306c O5658 Z75214x1 Similar to human X-linked PEST-containing permease 
8

Sulfate permease homologues

Gene name Synonym(s) GenBank accession number Description 
YBR294w SUL1/SFP/YBR2110 Z36163x1 High affinity sulfate permease 
YGR125w G6362 Z72910x1 Unknown function 
YLR092w SEL2/L9449.1/L2528 Z73264x1 Similar to YBR294w 
YPR003c YP9723.03/LPZ3C Z48951x3 Similar to YBR294w and heterologous sulfate permeases 
Gene name Synonym(s) GenBank accession number Description 
YBR294w SUL1/SFP/YBR2110 Z36163x1 High affinity sulfate permease 
YGR125w G6362 Z72910x1 Unknown function 
YLR092w SEL2/L9449.1/L2528 Z73264x1 Similar to YBR294w 
YPR003c YP9723.03/LPZ3C Z48951x3 Similar to YBR294w and heterologous sulfate permeases 
9

Ammonia permease homologues

Gene Synonym(s) GenBank accession number Description 
YGR121c MEP1/(AAT1)/AMT1/G6331 Z72906x1 Ammonia permease of high capacity and moderate affinity 
YNL142w MEP2/N1207/N1820 Z71418x1 Ammonia permease of low capacity and high affinity 
YPR138c P9659.14 U40829x9 Similar to YGR121c 
Gene Synonym(s) GenBank accession number Description 
YGR121c MEP1/(AAT1)/AMT1/G6331 Z72906x1 Ammonia permease of high capacity and moderate affinity 
YNL142w MEP2/N1207/N1820 Z71418x1 Ammonia permease of low capacity and high affinity 
YPR138c P9659.14 U40829x9 Similar to YGR121c 
10

Phosphate permease homologues

Gene name Synonym(s) GenBank accession number Description 
YCR037c PHO87/YCR524 X59720x125 Phosphate permease 
YJL198w J0336 Z49473x1 Similar to YCR037c 
YNR013c N2052 Z71628x1 Similar to YCR037c and YJL198w 
Gene name Synonym(s) GenBank accession number Description 
YCR037c PHO87/YCR524 X59720x125 Phosphate permease 
YJL198w J0336 Z49473x1 Similar to YCR037c 
YNR013c N2052 Z71628x1 Similar to YCR037c and YJL198w 
11

Purine/cytosine permease homologues

Gene name Synonym(s) GenBank accession number Description 
YER056c FCY2 U18813x3 Cytosine/purine permease 
YER060w FCY21/FCY22 U18813x8 Similar to YER056c 
YGL186c G1370 Z72708x1 Similar to YER056c and YER060c 
Gene name Synonym(s) GenBank accession number Description 
YER056c FCY2 U18813x3 Cytosine/purine permease 
YER060w FCY21/FCY22 U18813x8 Similar to YER056c 
YGL186c G1370 Z72708x1 Similar to YER056c and YER060c 
12

Calcium permease homologues

Gene name Synonym(s) GenBank accession number Description 
YDL128w VCX1/HUM1/D2218 Z74176x1 Calcium permease (H+/Ca2+ antiporter) of the vacuoles 
YNL321w N0339 Z71597x1 Unknown function 
Gene name Synonym(s) GenBank accession number Description 
YDL128w VCX1/HUM1/D2218 Z74176x1 Calcium permease (H+/Ca2+ antiporter) of the vacuoles 
YNL321w N0339 Z71597x1 Unknown function 
13

ER protein translocation homologues

Gene name Synonym(s) GenBank accession number Description 
YBR283c YBR2020/SSH1 Z36152x1 Involved in protein translocation into the endoplasmic reticulum 
YLR378c SEC61/L3502.5 U19104x1 Involved in protein translocation into the endoplasmic reticulum 
Gene name Synonym(s) GenBank accession number Description 
YBR283c YBR2020/SSH1 Z36152x1 Involved in protein translocation into the endoplasmic reticulum 
YLR378c SEC61/L3502.5 U19104x1 Involved in protein translocation into the endoplasmic reticulum 
14

Vanadate resistance protein homologues

Gene name Synonym(s) GenBank accession number Description 
YER039c  U18796x7 Similar to YGL225w 
YGL225w GOG5/VRG4/VAN2/G1001 Z72747x1 Vanadate resistance protein required for Golgi function 
Gene name Synonym(s) GenBank accession number Description 
YER039c  U18796x7 Similar to YGL225w 
YGL225w GOG5/VRG4/VAN2/G1001 Z72747x1 Vanadate resistance protein required for Golgi function 
15

Spore formation protein homologues

Gene name Synonym(s) GenBank accession number Description 
YJL062w HRC830/J1132 Z49337x1 Unknown function 
YKL165c YKL619 Z28165x1 Possibly involved in spore formation 
YLL031c L0929 Z73136x1 Similar to YJL062w 
Gene name Synonym(s) GenBank accession number Description 
YJL062w HRC830/J1132 Z49337x1 Unknown function 
YKL165c YKL619 Z28165x1 Possibly involved in spore formation 
YLL031c L0929 Z73136x1 Similar to YJL062w 
16

Sexual differentiation protein homologues

Gene name Synonym(s) GenBank accession number Description 
YJL212c HRD799/J0236 Z49487x1 Similar to Schizosaccharomyces pombe ISP4+ which is induced by sexual differentiation 
YPR194c P9677.13 U25841x9 Unknown function 
Gene name Synonym(s) GenBank accession number Description 
YJL212c HRD799/J0236 Z49487x1 Similar to Schizosaccharomyces pombe ISP4+ which is induced by sexual differentiation 
YPR194c P9677.13 U25841x9 Unknown function 
17

Unknown function

Gene name Synonym(s) GenBank accession number Family 
YBL089w YBL0703 Z35850x1 unknown 1 
YEL064c  U18795x13 unknown 1 
YER119c  U18916x12 unknown 1 
YIL088c YI9910.08 Z47047x90 unknown 1 
YJR001w YJR83.4/J1409 Z49501x1 unknown 1 
YKL146w YKL600 Z28146x2 unknown 1 
YNL101w N2185 Z71377x1 unknown 1 
YLL005c L1361 Z73110x1 unknown 2 
YLR241w L9672.9 U20865x2 unknown 2 
YMR266w YM8156.8 Z49260x8 unknown 2 
YOL084w O0953 Z74826x1 unknown 2 
YDR338c D9651.8 U51032x6 unknown 3 
YHR032w  U00062x3 unknown 3 
YMR253c YM9920.07 Z48639x7 unknown 4 
YPL264c P0373 Z73620x1 unknown 4 
YDL231c D0810 Z74279x1 unknown 5 
YGL140c G2550 Z72662x1 unknown 5 
YDL206w D1053 Z74254x1 unknown 6 
YJR106w J1978 Z49606x1 unknown 6 
YGL084c G3195 Z72606x1 unknown 7 
YPL189w P2201 Z73545x1 unknown 7 
Gene name Synonym(s) GenBank accession number Family 
YBL089w YBL0703 Z35850x1 unknown 1 
YEL064c  U18795x13 unknown 1 
YER119c  U18916x12 unknown 1 
YIL088c YI9910.08 Z47047x90 unknown 1 
YJR001w YJR83.4/J1409 Z49501x1 unknown 1 
YKL146w YKL600 Z28146x2 unknown 1 
YNL101w N2185 Z71377x1 unknown 1 
YLL005c L1361 Z73110x1 unknown 2 
YLR241w L9672.9 U20865x2 unknown 2 
YMR266w YM8156.8 Z49260x8 unknown 2 
YOL084w O0953 Z74826x1 unknown 2 
YDR338c D9651.8 U51032x6 unknown 3 
YHR032w  U00062x3 unknown 3 
YMR253c YM9920.07 Z48639x7 unknown 4 
YPL264c P0373 Z73620x1 unknown 4 
YDL231c D0810 Z74279x1 unknown 5 
YGL140c G2550 Z72662x1 unknown 5 
YDL206w D1053 Z74254x1 unknown 6 
YJR106w J1978 Z49606x1 unknown 6 
YGL084c G3195 Z72606x1 unknown 7 
YPL189w P2201 Z73545x1 unknown 7 
18

Single proteins with transport related functions

Gene name Synonym(s) GenBank accession number Description 
Multidrug resistance 
YCR023c YCR241 X59720x108 Similar to multidrug permeases from family 2 
YJR124c J2046 Z49624x1 Similar to multidrug resistance proteins 
Na+-H+ permeases 
YDR456w D9461.40/NHX1 U33007x40 Similar to Na+/H+ permeases 
YJL094c J0909 Z49369x1 Similar to Enterococcus hirae Na+/H+ permease NapA 
YLR138w L9606.4/L3149/NHA1 Z73310x1 Na+/H+ permease 
Other permeases 
YBR036c CSG2/CLS2/YBR0404 Z35905x1 Protein required for growth in high calcium concentration 
YBR235w YBR1601 Z36104x1 Similar to vertebrate cation/chloride permeases 
YGR227w DIE2/G8547 Z73012x1 Protein that promotes expression of ITR1 
YKL217w JEN1 Z28217x1 Similar to E. coli osmoregulatory proP proline/betaine and KgtP α-ketoglutarate permeases 
YNL275w N0626 Z71551x1 Similar to human band 3 anion transport protein 
YPL092w SSU1/LPG16 U43281x16 Sulfite sensitivity protein 
Gene name Synonym(s) GenBank accession number Description 
Multidrug resistance 
YCR023c YCR241 X59720x108 Similar to multidrug permeases from family 2 
YJR124c J2046 Z49624x1 Similar to multidrug resistance proteins 
Na+-H+ permeases 
YDR456w D9461.40/NHX1 U33007x40 Similar to Na+/H+ permeases 
YJL094c J0909 Z49369x1 Similar to Enterococcus hirae Na+/H+ permease NapA 
YLR138w L9606.4/L3149/NHA1 Z73310x1 Na+/H+ permease 
Other permeases 
YBR036c CSG2/CLS2/YBR0404 Z35905x1 Protein required for growth in high calcium concentration 
YBR235w YBR1601 Z36104x1 Similar to vertebrate cation/chloride permeases 
YGR227w DIE2/G8547 Z73012x1 Protein that promotes expression of ITR1 
YKL217w JEN1 Z28217x1 Similar to E. coli osmoregulatory proP proline/betaine and KgtP α-ketoglutarate permeases 
YNL275w N0626 Z71551x1 Similar to human band 3 anion transport protein 
YPL092w SSU1/LPG16 U43281x16 Sulfite sensitivity protein 
19

Single proteins with an unknown permease function

Gene name Synonym(s) GenBank accession number Description 
YGL142c G2535 Z72664x1 Similar to YOR149c 
YNR030w N3265 Z71645x1 Similar to YOR149c 
YBL004w YBL0101 Z35765x3 Unknown function 
YBL020w RFT1/YBL442 Z35781x1 Protein involved in nuclear division 
YCL038c  X59720x40 Unknown function 
YDR141c YD9302.17 Z48179x17 Unknown function 
YDR335w MSN5/D9651.5 U51032x3 Unknown function 
YFL007w  D50617x62 Unknown function 
YGL114w G2950 Z72636x1 Similar to S. pombe ISP4+ involved in sexual differentiation 
YJL091c J0916 Z49366x1 Similar to YEL063c 
YJL039c J1216 Z49314x1 Similar to members of the Hsp70 family 
YJL108c J0811 Z49383x1 Unknown function 
YJL163c J0544 Z49438x1 Unknown function 
YJL207c J0312/HRD550 Z49482x1 Unknown function 
YLR459w CDC91/L9122.2 U22383x5 Unknown function 
YMR155w YM8520.04 Z49705x4 Unknown function 
YMR221c YM9959.03 Z49939x3 Unknown function 
YOL137w O0497 Z74879x1 Unknown function 
YOR161c O3568 Z75069x1 Unknown function 
YPL006w YP8132.07/LPA11 U33335x11 Similar to human PTC (NBCC disease) 
Gene name Synonym(s) GenBank accession number Description 
YGL142c G2535 Z72664x1 Similar to YOR149c 
YNR030w N3265 Z71645x1 Similar to YOR149c 
YBL004w YBL0101 Z35765x3 Unknown function 
YBL020w RFT1/YBL442 Z35781x1 Protein involved in nuclear division 
YCL038c  X59720x40 Unknown function 
YDR141c YD9302.17 Z48179x17 Unknown function 
YDR335w MSN5/D9651.5 U51032x3 Unknown function 
YFL007w  D50617x62 Unknown function 
YGL114w G2950 Z72636x1 Similar to S. pombe ISP4+ involved in sexual differentiation 
YJL091c J0916 Z49366x1 Similar to YEL063c 
YJL039c J1216 Z49314x1 Similar to members of the Hsp70 family 
YJL108c J0811 Z49383x1 Unknown function 
YJL163c J0544 Z49438x1 Unknown function 
YJL207c J0312/HRD550 Z49482x1 Unknown function 
YLR459w CDC91/L9122.2 U22383x5 Unknown function 
YMR155w YM8520.04 Z49705x4 Unknown function 
YMR221c YM9959.03 Z49939x3 Unknown function 
YOL137w O0497 Z74879x1 Unknown function 
YOR161c O3568 Z75069x1 Unknown function 
YPL006w YP8132.07/LPA11 U33335x11 Similar to human PTC (NBCC disease) 
20

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

Gene name Synonym(s) GenBank accession number Description 
YBL082c ALG3/RHK1/YBL0720 Z35844x2 Mannosyltransferase involved in N-glycosylation 
YBR243c ALG7/TUR1/YBR1628 Z36112x1 UDP-N-acetyl-glucosamine-1-P transferase 
YGL022w STT3/G3683 Z72544x1 Protein required for oligosaccharyltransferase activity 
YGR157w CHO2/PEM1/G6673 Z72942x1 Phosphatidylethanolamine N-methyltransferase 
YFL025c  D50617x44 Similar to mosquito NADH-ubiquinone oxidoreductase 
YNL219c N1295/ALG9 Z71495x1 Mannosyl transferase 
Gene name Synonym(s) GenBank accession number Description 
YBL082c ALG3/RHK1/YBL0720 Z35844x2 Mannosyltransferase involved in N-glycosylation 
YBR243c ALG7/TUR1/YBR1628 Z36112x1 UDP-N-acetyl-glucosamine-1-P transferase 
YGL022w STT3/G3683 Z72544x1 Protein required for oligosaccharyltransferase activity 
YGR157w CHO2/PEM1/G6673 Z72942x1 Phosphatidylethanolamine N-methyltransferase 
YFL025c  D50617x44 Similar to mosquito NADH-ubiquinone oxidoreductase 
YNL219c N1295/ALG9 Z71495x1 Mannosyl transferase 

Evolutionary relationships

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.

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. 1–10.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The MFS families

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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].

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].

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.

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

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.

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.

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.

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.

Gene Synonym GenBank accession number Description 
YJL129c TRK1 Z49404x1 K+ permease (high affinity) 
YKR050w TRK2 Z28275x1 K+ permease (moderate affinity) 
YKR093w PTR2 Z28318x1 Peptide permease 
YPR201w ARC3 U25841x15 Arsenite permease 
YOL122c SMF1 Z74864x1 Mn2+ permease 
YHR050w SMF2 U00062x21 Co2+ permease 
YLR034c SMF3 Z73206x1 Protein similar to SMF2 
Gene Synonym GenBank accession number Description 
YJL129c TRK1 Z49404x1 K+ permease (high affinity) 
YKR050w TRK2 Z28275x1 K+ permease (moderate affinity) 
YKR093w PTR2 Z28318x1 Peptide permease 
YPR201w ARC3 U25841x15 Arsenite permease 
YOL122c SMF1 Z74864x1 Mn2+ permease 
YHR050w SMF2 U00062x21 Co2+ permease 
YLR034c SMF3 Z73206x1 Protein similar to SMF2 

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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Author notes

1
Laboratorium voor Experimentele Genetica en Transgenese, Centrum voor Menselijke Erfelijkheid, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium.