Of these four families, only members of the MATE family have been shown to function mechanistically as secondary carriers, and no member of the MVF family has been shown to function as a
Trang 1R E V I E W A R T I C L E
The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP)
exporter superfamily
Rikki N Hvorup, Brit Winnen, Abraham B Chang, Yong Jiang, Xiao-Feng Zhou and Milton H Saier Jr Division of Biological Sciences, University of California at San Diego, USA
The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP)
exporter superfamily (TC #2.A.66)consists of four
previ-ously recognized families: (a)the ubiquitous multi-drug and
toxin extrusion (MATE)family; (b)the prokaryotic
poly-saccharide transporter (PST)family; (c)the eukaryotic
oligosaccharidyl-lipid flippase (OLF)family and (d)the
bacterial mouse virulence factor family (MVF) Of these
four families, only members of the MATE family have been
shown to function mechanistically as secondary carriers,
and no member of the MVF family has been shown to
function as a transporter Establishment of a common
ori-gin for the MATE, PST, OLF and MVF families suggests a
common mechanism of action as secondary carriers
cata-lyzing substrate/cation antiport Most protein members of
these four families exhibit 12 putative transmembrane
a-helical segments (TMSs), and several have been shown to
have arisen by an internal gene duplication event;
topo-logical variation is observed for some members of the
superfamily The PST family is more closely related to the
MATE, OLF and MVF families than any of these latter
three families are related to each other This fact leads to the
suggestion that primordial proteins most closely related to
the PST family were the evolutionary precursors of all members of the MOP superfamily Here, phylogenetic trees and average hydropathy, similarity and amphipathicity plots for members of the four families are derived and provide detailed evolutionary and structural information about these proteins We show that each family exhibits unique characteristics For example, the MATE and PST families are characterized by numerous paralogues within a single organism (58 paralogues of the MATE family are present in Arabidopsis thaliana), while the OLF family consists exclusively of orthologues, and the MVF family consists primarily of orthologues Only in the PST family has extensive lateral transfer of the encoding genes occurred, and in this family as well as the MVF family, topological variation is a characteristic feature The results serve to define a large superfamily of transporters that we predict function to export substrates using a monovalent cation antiport mechanism
Keywords: transport; membranes; proton motive force; superfamily; phylogeny; drug resistance; polysaccharides; lipid-linked oligosaccharides
Major families of drug efflux pumps
Bacterial species that have developed clinical resistance to
antimicrobial agents are increasing in numbers and have
become a serious problem in hospitals [1] One of the major
mechanisms of drug resistance in both prokaryotes and
eukaryotes involves drug efflux from cells There are many
drug efflux systems known in bacteria [2–5], and these
belong to five ubiquitous transporter (super)families [6,7]
Four of them (RND, DMT, MFS and MATE; see below)
one, the ATP-binding cassette (ABC), uses ATP hydrolysis Most characterized members of the resistance/nodula-tion/division (RND)superfamily [8] function as drug or heavy metal efflux pumps in Gram-negative bacteria Homologues in Gram-positive bacteria serve as lipid exporters [9] Small multidrug resistance (SMR)family pumps within the drug/metabolite transporter (DMT) superfamily [10] consist of homodimeric or heterodimeric structures with only four TMSs per subunit [11] They export cationic drugs using a simple cation antiport mechanism involving a conserved glutamyl residue [12] Drug exporters of the major facilitator superfamily (MFS)are found within six families, each known to transport a broad range of structurally distinct drugs [13,14]
The ABC superfamily of ATP-driven transporters includes many families that are potentially active in the uptake or efflux of metabolite analogues and other drugs For instance, the oligopeptide uptake transporter of
antibio-tics such as kanamycin and neomycin (reviewed in [7]) Uptake systems segregate from the efflux systems phylo-genetically [15] Members of this superfamily can also act on
Correspondence to: M Saier, Division of Biological Sciences,
University of California at San Diego, La Jolla, CA 92093 0116,
USA Fax: 001 858 534 7108, Tel.: 001 858 534 4084,
E-mail: msaier@ucsd.edu
Abbreviations: ABC, ATP-binding cassette; MATE, multi-drug and
toxin extrusion; MOP,
multidrug/oligosaccharidyl-lipid/polysaccha-ride; MPA, membrane-periplasmic auxiliary; MVF, mouse virulence
factor; OLF, oligosaccharidyl-lipid flippase; PST, prokaryotic
polysaccharide transporter; TMS, transmembrane segment.
(Received 23 July 2002, revised 14 November 2002,
accepted 9 December 2002)
Trang 2many types of macromolecules, a unique characteristic of
the ABC superfamily [16]
The MATE family of drug exporters (TC #2.A.66.1)
Only a few members of the multidrug and toxin extrusion
(MATE)family [3] are characterized functionally (Table 1)
These proteins include: (a)NorM and (b)VmrA in
Vibrio parahaemolyticus, a halophilic marine bacterium
that is one of the major causes of food poisoning in Japan
[17–19]; (c)YdhE from Escherichia coli, a close homologue
of NorM [20]; (d)Alf5 from the plant, A thaliana [21];
(e)VcmA from Vibrio cholerae non-O1, a nonhalophilic
protein, elevates resistance to the methionine analogue,
ethionine [24]
NorM, VmrA and VcmA have been shown to function
antiporters Several members are annotated as DinF
proteins The functions of the DinF proteins are unknown,
but expression of some of these proteins has been shown to
be induced by DNA damage [25,26] A function related to
the export of nucleotides excised from damaged DNA
during photorepair can be postulated
The polysaccharide transporter (PST) family
(TC #2.A.66.2)
Characterized protein members of the PST family are
generally of 400–500 amino acid residues in size and traverse
the membrane 12 times as putative a-helical TMSs
Analyses conducted in 1997 [27] showed that members of
the PST family formed two major clusters, one of which was
concerned putatively with lipopolysaccharide O-antigen
repeat unit export (flipping)in Gram-negative bacteria, the other which was concerned with exopolysaccharide or capsular polysaccharide export in both Gram-negative and Gram-positive bacteria However, numerous archaeal homologues are now recognized, and bacteria use PST systems to export other complex carbohydrates such as teichuronic acids [28] The mechanism of energy coupling for PST exporters is not established
PST transporters may function together with auxiliary proteins that regulate transport and allow passage of complex carbohydrates across both membranes of the negative bacterial envelope [29] Thus, each Gram-negative bacterial PST system specific for an exo- or capsular polysaccharide functions in conjunction with
a cytoplasmic membrane-periplasmic auxiliary (MPA) protein with a cytoplasmic ATP-binding domain (MPA1-C; TC #8.A.3)as well as an outer membrane auxiliary protein (OMA; TC #1.B.18)[27] Each Gram-positive bacterial PST system functions in conjunction with a homologous MPA1 + C pair of proteins (TC #8.A.3) equivalent to an MPA1-C protein of Gram-negative bacteria The C-domain has been shown to possess tyrosine protein kinase activity, suggesting that it functions
in a regulatory capacity [30] The lipopolysaccharide exporters may function specifically in the translocation of the lipid-linked O-antigen side chain precursor from the inner leaflet of the cytoplasmic membrane to the outer leaflet, but this possibility has not been established experimentally
The oligosaccharidyl-lipid flippase (OLF) family (TC #2.A.66.3)
N-Linked glycosylation in eukaryotic cells follows a conserved pathway in which a tetradecasaccharide
Table 1 Functionally characterized members of the multidrug and toxin extrusion (MATE)family.
Gene name
Arabidopsis thaliana Ath57 Alf5 (BAB02774)Tetramethylammonium, PVP (polyvinylpyrrolidone)
pyrrolidinone
[21] Escherichia coli Eco2 NorM (YdhE)
(P37340)
Ciprofloxacin a , berberine, kanamycin b , streptomycin b , acriflavine, tetraphenylphosphonium ion (TPP) chloramphenicol e , norfloxacin a , enoxacin a , fosfomycin, doxorubicin c , trimethoprim d , ethidium bromide, benzalkonium, deoxycholate
[1,25]
Bacteroides
thetaiotaomicron
Bth1 BexA (BAB64566)Norfloxacin1, ciprofloxacina, ethidium bromide [19] Vibrio cholerae Vch1 NorM (VcmA)
(Q9KRU4)
Norfloxacin1, ciprofloxacina, ofloxacin1, daunomycinc, doxorubicinc, streptomycinb, kanamycinbethidium bromide, 4¢6¢-diamidino-2-phenylindole dihydro-chloride (DAPI), Hoechst33342, acriflavine
[10]
Vibrio parahaemolyticus Vpa1 NorM (O82855)Norfloxacin1; ethidium bromide; kanamycinb;
ciprofloxacin1, streptomycin b
[25] Vpa3 VmrA (BAB68204)4¢6¢-diamidino-2-phenylindole (DAPI), (TPP),
acriflavine, ethidium bromide
[17]
a
Quinolones and fluoroquinolones;baminoglycosides;c anthracyclines such as adriamycin hydrochloride;d trimethoprim-sulfamethox-azole;emiscellaneous antibiotics.
Trang 3endoplasmic reticular (ER)membrane as a
dolichylpyro-phosphate (Dol-PP)-linked intermediate before being
trans-ferred to an asparaginyl-residue in a lumenal protein An
cyto-plasmic side of the membrane and translocated across the
membrane so that the oligosaccharide chain faces the ER
lumen where biosynthesis continues to completion [31] The
exporter in Saccharomyces cerevisiae that catalyzes the
translocation step is the 574 amino acid nuclear division
Rft1 protein with 12 putative TMSs [31] Homologues are
found in plants, animals and fungi
The mouse virulence factor (MVF) family (TC #2.A.66.4)
A single member of the MVF family, MviN of
Salmo-nella typhimurium, has been shown to be an important
virulence factor for this organism when infecting the mouse
[32] In several bacteria, genes encoding MviN homologues
occur in operons that also encode the uridylyl transferase,
GlnD, that functions in the regulation of nitrogen
meta-bolism [33] Nothing more is known about the function of
MviN or any other member of the MVF family However,
as will be shown below, these proteins are related to
members of the PST and MATE families with greatest
sequence similarity to members of the PST family It is
therefore possible that MVF family members are related
functionally to PST family members exporting complex
carbohydrates or related substances
The MOP superfamily
In this paper, we show that the MATE family of drug
exporters, the PST family of polysaccharide exporters, the
OLF family of lipid-linked oligosaccharide exporters and the
MVF family of mouse virulence-related proteins are all
homologous and are, therefore, related by common descent
We designate the superfamily that includes these four related
families the MOP (MATE/MVF-OLF-PST)superfamily
Currently sequenced members of these families are identified,
and the distribution of their members in the living world is
determined While MATE family members are found in all
domains of life (bacteria, archaea and eukaryotes), PST
family members are restricted to prokaryotes (both archaea
and bacteria), OLF family members are restricted to
eukaryotes and MVF family members are restricted to
bacteria In contrast to the MATE and PST families that
exhibit multiple paralogues in any one organism, the
eukaryotic OLF family is currently very small, consisting
of only eight sequenced orthologous members with no more
than one homologue per organism, and the MVF family,
while much larger, probably also consists primarily of
orthologues Because at least some members of the
pro-karyotic-specific PST family can flip lipid-linked
oligosac-charides (i.e O-antigen precursors of lipopolysacoligosac-charides in
Gram-negative bacteria), members of this family may serve
as the functional counterpart of the oligosaccharidyl lipid
exporters of eukaryotes The reported sequence analyses lead
us to suggest that prokaryotic oligosaccharidyl-lipid
export-ers were the primordial systems that gave rise to all membexport-ers
of the MOP superfamily We tabulate these proteins
according to family and derive reliable multiple alignments
website:http://www.biology.ucsd.edu/msaier/align.html/)
We also derive average hydropathy, similarity and amphi-pathicity plots which allow us to make transmembrane topological predictions, and these in turn lead to predictions regarding the evolutionary origins of protein topological types found within the MOP superfamily Most importantly, our results allow us to propose that all members of the MOP superfamily function as secondary efflux carriers using a solute/cation antiport mechanism
Computer methods Sequences of the proteins that comprise the MATE, PST, OLF and MVF families were obtained separately by initial
PSI-BLAST searches [10] using the SCREENTRANSPORTER
program without iterations [35] Recognizable members
June 2002
Multiple sequence alignments were constructed using the
CLUSTAL Xprogram [39] The gap penalty and gap extension
respectively, although other combinations were tried Average hydropathy, similarity and amphipathicity plots
program [40] Phylogenetic trees were derived by the neighbor-joining method from alignments generated with the CLUSTAL X program using the BLOSUM62 scoring
Charge bias analyses of membrane protein topology were
andTOPPRED2 [45] programs Motif searches were
of intrafamilial and interfamilial protein sequence similari-ties (i.e between members within each of the four families of the MOP superfamily as well as between members of the
BLOSUM62 scoring matrix, a gap opening penalty of )8,
BLAST2 program [50] was used additionally for comparison
analyses of internally duplicated intraprotein segments Binary comparison scores are expressed in standard devi-ations (SD)[51] A value of 9 SD is deemed sufficient to
to position TMSs in one protein relative to its homologues Thus, the positions of the extra TMSs in 14 TMS proteins relative to their 12 TMS homologues could be determined using this program
The four tables of family members (Tables S1–S4)and the multiple alignments from which the results reported in this paper were derived (Figs S1–S4), as well as additional
Trang 4supplementary supporting data can be found on our
ALIGN website
Results
The four families of the MOP superfamily
The general characteristics of the four currently recognized
families within the MOP superfamily are summarized in
Table 2 Columns 1 and 2 present the family abbreviations
and the TC # while column 3 gives the number of family
members identified The MATE family is the largest with
203 members while the PST, MVF and OLF families are of
decreasing sizes in that order (155, 45 and 8 recognized
members, respectively) As shown by the results presented in
column 4 of Table 2, most of the members of these four
families fall within the same size range However, a few of
the homologues were much larger (Table 2) Thus, in the
MATE family, two plant proteins, Ath10 and Ath8, have
1094 and 746 amino acid residues, respectively The
extended hydrophilic regions in these two proteins did not
show sequence similarity with anything else in the
data-bases Of greater interest were four large MVF family
homologues, all from high G + C Gram-positive bacteria
These four proteins were Mle of Mycobacterium leprae
(1206 amino acid residues), Mtu of M tuberculosis (1184
amino acid residues), Cgl of Corynebacterium glutamicum
(1083 amino acid residues)and Sco of Streptomyces
homologous to regions of eukaryotic-type serine/threonine
kinases Presumably, these C-terminal domains function in
a regulatory capacity, possibly to control the activities of the
N-terminal transporter protein domains
Column 5 in Table 2 summarizes the topological types
identified within each of the four families of the MOP
superfamily MATE and OLF family permeases may all
have 12 (or possibly 13)TMSs, but about one-third of all
PST family members are predicted to have 14 TMSs, and
MVF family members have 10, 12, 13, 14 or 15 putative
TMSs These will be analyzed in greater detail below
Finally, as summarized in column 6 of Table 2, each of
the four families within the MOP superfamily has
a distinctive organismal distribution While the MATE
family is present in all three domains of living organisms (archaea, bacteria and eukaryotes), the PST family is found both in archaea and bacteria but not in eukaryotes, while the OLF and MVF family members are restricted to eukaryotes and bacteria, respectively
The MATE family Our searches revealed that the MATE family contains 203 currently sequenced proteins, including representatives from all three domains of life (Table S1) In this sense, the family
is ubiquitous The family could be divided into 15 subfamilies (see phylogenetic tree displayed in Fig 1) Most
of the members are of about 450–550 amino acid residues in length and possess 12 putative TMSs The yeast proteins are larger (up to about 700 residues)whereas the archaeal proteins are generally smaller Large transporter size is
a characteristic of the eukaryotic domain while small size is
a characteristic of the archaeal domain [11]
Table S1 presents a summary of the 15 subfamilies (phylogenetic clusters)of the MATE family The sub-families, some of which include sequence divergent proteins, are as presented in the phylogenetic tree shown in Fig 1 The subfamily numbers as well as the names, organismal sources and abbreviations of the members of the MATE family are presented in columns 1–4 of Table S1 A short description of the proteins (column 5), the gene names,
TREMBLandSWISS-PROT)(columns 6–8)are also provided Finally, columns 9–10, respectively, present the protein sizes
in numbers of amino acid residues and the numbers of putative transmembrane a-helical TMSs per polypeptide chain, based on hydropathy plots The same format of presentation is used for tabulation of the proteins of the PST, OLF and MVF families (see Tables S2–S4)
The functionally characterized members of the family are found in subfamilies 1 (Sce3), 3 (Ath57), 4 (Eco2, Vch1, Vpa1), 7 (Vpa3) and 9 (Bth1) Subfamily 1 consists exclusively of yeast proteins; subfamily 2 includes only mammalian proteins, and subfamily 3 contains only plant proteins, mostly from A thaliana Most of the other subfamilies consist exclusively of bacterial and/or archaeal proteins Of them, only subfamilies 6, 7 and 10 include proteins from both of these prokaryotic domains In
Table 2 Characteristics of the families of the MOP superfamily For the MATE family, two larger homologues were found in Arabidopsis thaliana: Ath10, 1094 aas, and Ath8, 746 aas (see web table S1, Results section) No sequence similarity was observed for the extra portions of these proteins For the OLF family, two homologues in Kluyveromyces lactis, Kla, 417 aas, and A thaliana, Ath, 401 aas (see web table S3, Results section)are believed to be fragments For the MVF family, four larger homologues, all from high G + C Gram-positive bacteria, were found in Mycobac-terium leprae, Mle, 1206 aas, MycobacMycobac-terium tuberculosis, Mtu, 1184 aas, CorynebacMycobac-terium glutamicum, Cgl, 1083 aas and Streptomyces coelicolor, Sco, 811 aas Mle, Mtu and Cgl all include C-terminal domains (residues 720–950)that are homologous to each other and to domains in eukaryotic-type serine/threonine protein kinases A, archaea; B, bacteria; E, eukaryotes; aas, amino acid residues per polypeptide chain; TMSs, transmembrane a-helical segments.
Number
of members
Size range (number aas)
Number TMSs
Distribution among organisms
Trang 5subfamily 14, plant and bacterial proteins cluster very
loosely together Thus, seven subfamilies are bacterial
specific, three include both archaeal and bacterial proteins,
one is archaeal specific, one includes bacterial and plant
proteins, and three are eukaryotic specific The three
eukaryotic subfamilies consist of yeast, animal and plant
proteins, respectively (Fig 1)
Many organisms exhibit multiple MATE family
para-logues For example, among the bacteria, E coli and
and L monocytogenes each have six and Clostridium
both S cerevisiae and S pombe have three paralogues, but
these are not all orthologous to each other Most
impres-sively, A thaliana has 58 MATE family paralogues No
archaeon has more than four MATE family paralogues
Individual paralogues from a single species may either be
closely related, presumably arising from a recent gene
duplication event, or distantly related, arising from an earlier gene duplication event Extensive phylogenetic studies of more than 70 transporter families have shown that substrate specificity typically correlates with phylogeny [54–56] although exceptions have been reported [57] This fact allows functional predictions for many uncharacterized transporters
In Subfamily 7, the Thermotoga maritima homologue clusters loosely together with three archaeal proteins
small-subunit ribosomal RNA phylogeny has suggested that this bacterium is one of the deepest and most slowly evolving bacterial lineages [58] By using whole-genome similarity comparisons, T maritima appears to be the most archaeal-like of all sequenced bacteria It has been suggested that much of the similarity between T maritima and the archaea
is due to a shared ancestry of portions of their genomes as
a result of lateral gene transfer [59] In subfamilies 6 and 10,
Fig 1 Phylogenetic tree for the multidrug and toxin extrusion (MATE)family The tree was derived using the CLUSTAL X program The 15 subfamilies are labeled 1–15 together with the class of organisms from which the included proteins were derived; B, bacteria; Ar, archaea;
An, animals; Y, yeast; Pl, plants The arrow indicates the probable root of the tree as determined with outlying sequences.
Trang 6the archaeal proteins are so distant from the bacterial
homologues that the results are probably consistent with
vertical transmission from a common ancestor without
lateral transfer
Drug resistances demonstrated for characterized MATE
proteins mediate resistance to a wide range of cationic dyes,
fluoroquinolones, aminoglycosides and other structurally
diverse antibiotics and drugs It is interesting to note that
while cationic dyes are generally amphipathic and positively
charged, aminoglycosides are strongly hydrophilic, and
norfloxacin is amphiphilic Thus, MATE family transporter
substrates are diverse in nature
Average hydropathy and similarity plots for the MATE
family are shown in Fig 2A All 12 peaks of hydrophobicity
are well conserved Two additional peaks that are very
poorly conserved are found just preceding and following
conserved peak 12 The peak of hydrophobicity preceding
TMS 12 is due to an inserted sequence in just one protein,
C-terminal peak following putative TMS 12 is due to
extension of the animal homologues These few proteins
may have 13 rather than the usual 12 TMSs that
charac-terize the MATE family The extra regions in these few
proteins are presumably nonessential for transport function
Figure 3 shows an alignment of the first half of a MATE
family protein with the second half of the same protein
(PAB0243 from Pyrococcus abyssi) The two halves exhibit
40–50% similarity, 30% identity and a comparison score of 14.5 SD These values are sufficient to establish homology [52] Homology between the two halves of members of the PST and MVF families could also be established but not for the two halves of members of the OLF family
The PST family The sequenced proteins of the PST family are tabulated in Table S2 These proteins are derived exclusively from bacteria and archaea However, many diverse groups of these organisms are represented The transport functions of these systems are indicated when gene position or bio-chemical evidence allows postulation of their substrates The format of presentation for Table S2 (as well as Tables S3 and S4)is as for Table S1 Interestingly, and in contrast
to MATE family members, many PST family members are predicted to exhibit 14 rather than 12 TMSs However, few proteins are predicted to have odd numbers of TMSs (11 or 13) As will be shown below, the extra two TMSs in the 14 TMS PST family proteins are localized to the C-termini of these proteins
A dendrogram for the PST family is shown in Fig 4 Of the 12 clusters shown, only clusters 1, 2, 6 and 12 are restricted to the bacterial domain All other clusters include both archaeal and bacterial proteins This surprising observation shows that protein phylogeny does not corre-late with organism phylogeny In contrast to most families
Fig 2 Average hydropathy plots (top)and average similarity plots (bottom)for the MATE (A), PST(B), OLF(C) and MVF (D) families The
A VE H AS Program [40] was used to generate the plots with a window size of 19 Alignment position is indicated at the bottom of the figures The numbers above the hydropathy plots indicate the numbers of the putative TMSs In A and B, but not C and D, nonhomologous hydrophilic extensions were removed prior to graph generation.
Trang 7of transporters, including the MATE family of the MOP
superfamily, extensive horizontal transfer may have
occurred during the evolution of the PST family
In some cases we were able to provide convincing
evidence that horizontal transfer had in fact occurred For
example, in subfamily 5, Mth3, from the archaeon
Meth-anobacterium thermoautotrophicum, and Cac1, from the
encoding the Clostridium acetobutylicum protein (but not
that encoding the Methanobacterium thermoautotrophicum
homologue)showed a G + C content that differed
sub-stantially from that of the DNA of this organism overall
(0.31 for the genome and 0.24 for the gene) These results
taken together provide strong evidence for lateral transfer of
PST family genes across the bacterial–archaeal boundary It
should be noted that evidence for lateral transfer of genes
encoding cell surface bacterial polysaccharide biosynthetic
enzymes is extensive [60–62]
Figure 2B shows the average hydropathy (top)and
similarity (bottom)plots for PST family members Fourteen
peaks of hydropathy are evident, and for each of the first 12
such peaks, there is a corresponding peak of average
similarity However, the last two peaks of hydropathy are
not well conserved These two peaks represent the extra
peaks present in a minority of PST family members The
identities of the proteins exhibiting 14 rather than 12
putative TMSs is possible by examining the data presented
in Table S2
About one-third of the PST family members were
predicted to exhibit 14 TMSs Surprisingly, these proteins
were found in most subfamilies although only in subfamily 8
did the 14 TMS homologues predominate In some cases,
fairly close homologues were predicted to differ in topology
For example, Axy1 of Acetobacter xylinum (14 TMSs)and
Pae6 of Pseudomonas aeruginosa (12 TMSs)in cluster 1 had
essentially identical topologies except that Axy1 had
a C-terminal extension including the extra two TMSs that were lacking in Pae6 In fact, all 14 TMS proteins that were checked carefully were homologous throughout their first
12 TMSs to the 12 TMSs of their shorter homologues but had an extra C-terminal 2 TMS segment It was therefore concluded that the 14 TMS topological types arose from the
12 TMS proteins by addition of two TMSs at the C-termini The phylogenetic analyses suggest that this event has occurred repeatedly throughout the evolutionary history
of the PST family
The OLF family The proteins of the OLF family are presented in Table S3 and the corresponding phylogenetic tree is shown in Fig 5 All eukaryotic organisms with a fully sequenced genome have one and only one OLF family member with the notable exception of Plasmodium falciparum, a eukaryotic parasite that lacks N-glycoproteins [63] and also lacks an OLF family homologue These proteins are of 401–547 residues in length and display variable numbers of putative TMSs, from eight to 14 The two proteins with only eight putative TMSs, from Kluyveromyces lactis and A thaliana, may be incomplete sequences, due to incomplete sequencing and to nonrecognition of exons, respectively The other proteins are predicted to have 11–14 TMSs, and this prediction is dependent on the TMS prediction program used The actual numbers of TMSs may be 12 as suggested
by Helenius et al 2002 [31] The phylogeny of these proteins follows that of the organisms with the fungal, plant and animal proteins segregating as expected This fact suggests orthologous relationships for all family members and therefore suggests a common function (Fig 5)
The average hydropathy and average similarity plots for the six full length members of the OLF family are shown in Fig 2C We interpret the results in terms of a 12 TMS topology, the same as the major topological type observed
Fig 3 Binary alignment of the first half of a MATE family protein with its second half This protein is one of the three paralogues from Pyrococcus abyssi (PAB0243) The two halves were aligned using the GAP program with 500 random shuffles using the BLOSUM 62 program as the scoring matrix, a gap opening penalty of )8 and a gap extension penalty of )2 The two halves have a similarity of 40.5%, an identity of 29.8% and a comparison score of 14.5 SD |, an identity; :, a close conservative substitution; Æ, a more distant conservative substitution.
Trang 8Fig 4 Phylogenetic dendrogram for the polysaccharide transporter (PST)family The dendrogram was derived essentially as described in the legend
to Fig 1 The 12 subfamilies are labeled 1–12 together with the class of organisms from which the included proteins were derived; B, bacteria;
A, archaea.
Trang 9for the MATE and PST families However, the first two
putative TMSs are not strongly hydrophobic, and it is
therefore possible that these are localized to the cytoplasmic
side of the membrane as has been shown for members of
the chromate-resistance (CHR)family of transporters
(TC #2.A.51)[64] Although the proteins of the OLF
family can be hypothesized to exhibit a 6 + 6 TMS
topology with a large, well-conserved cytoplasmic loop
between putative TMSs 6 and 7 (Fig 2C), homology
between the two halves of these proteins could not be
demonstrated
As noted above, the first two putative TMSs displayed in
Fig 2C are quite hydrophilic, and they were therefore
examined in greater detail When putative TMSs 1 of the
full-length OLF family members were drawn in an a-helical
wheel, the helices (which lack prolyl and glycyl residues)
proved to be strongly amphipathic with three fully
con-served hydrophilic residues (helix residues Q4, R8 and N15)
tightly clustered on one side of the helix All other residues,
hydrophobic or slightly semipolar (data not shown)
Putative helix 2 was similarly amphipathic with four
well-conserved hydrophilic or semipolar residues (S5, E9, Q12
and S13)localized to one side of the helix These two helices
could provide a partially hydrophilic transmembrane
path-way for passage of lipid-linked oligosaccharides through the
membrane Alternatively, these two putative helices may be
localized to the cytoplasmic surface of the membrane The
remarkable conservation of Q4, R8 and N15 in helix 1
suggests an important function for these residues
The MVF family
The phylogenetic tree for the MVF family is shown in
Fig 6 Only two organisms, Pseudomonas aeruginosa and
Streptomyces coelicolor, both large genome organisms, have
more than a single MVF family member encoded within
their genomes, and they have only two MVF family
paralogues Except for the two extra paralogues, Pae2
and Sco2 (subfamily 8), all a-, b-, c- and d-proteobacterial proteins (23 proteins)are found in the lower half of the tree These fall into three primary clusters: cluster 1 includes only a-proteobacterial homologues, cluster 2 includes only b- and c-proteobacterial homologues, and cluster 3 includes the one d-proteobacterial homologue Within cluster 1, the phylogenies of all a-proteobacterial homologues follow the phylogenies of the 16S rRNAs [65], suggesting that they are orthologues Within cluster 2, the phylogenies of most b- and c-proteobacterial homologues are in accordance with those of the 16S rRNAs except for Vch which clusters with Pmu and Hin but should be between Ype and Pae, and Bap which should be close to Eco [65] Finally, the separate clustering of the d-proteobacterial protein, Bba, distant from all other homologues, is as expected
The upper part of the tree shows sequence divergent proteins from sequence divergent bacteria Only a few clusters are noteworthy Thus, cluster 5 includes all four high G + C Gram-positive bacterial homologues, cluster 6 includes the two cyanobacterial homologues, cluster 8 includes the two low G + C Gram-positive bacterial proteins, cluster 11 includes the two e-proteobacterial proteins, and cluster 15 includes the chlamydial orthologues Thus, with the exception of just four proteins (Bap, Vch, Pae2 and Sco2)the protein phylogenies follow the organi-smal (16S rRNA)phylogenies within experimental error This fact suggests that most of these bacterial proteins are orthologues, possibly serving a single function
The G + C contents and codon usage frequencies for the four anomalous genes were compared with the correspond-ing values for the protein-encodcorrespond-ing regions of the genomes
of the same organisms For Bap, the G + C content was 26% for both the gene and the organism For Vch, both values were 48%; for Sco2, the values were 76% for the gene and 72% for the genome; and for Pae2, the values were 72% for the gene and 67% for the genome In no case was the codon usage frequency for the gene significantly different from that for the organism as a whole These approaches therefore failed to provide further evidence for recent horizontal gene transfer of genes encoding MVF homo-logues
All MVF family proteins fell within the size range 480–
555 amino acid residues except for the high G + C Gram-positive bacterial homologues which were large (811–1184 amino acid residues)exhibiting 15 putative TMSs Three of these four proteins exhibit soluble protein kinase-like domains of about 250 residues in their C-terminal regions Most of the proteobacterial homologues appear to have 13
Fig 2D) Thus, there is probably some topological hetero-geneity in the MVF family
Establishment of homology for the four families
of the MOP superfamily The superfamily principle states that if A is homologous to
B, and B is homologous to C, then A is homologous to C [52] We have previously published the criteria used to establish homology, namely a comparison score in excess of
9 SD for two protein sequences of greater than 60 amino acid residues in length [52] Nine SD corresponds to a
Fig 5 Phylogenetic tree for oligosaccharidyl-lipid flippase (OLF)
family proteins See the legend to Fig 1 for format of presentation.
Trang 10arose by chance [51] In order to establish homology
between two coherent families, it is only necessary to
establish homology between one member of each of these
families Two such representative examples for each
inter-familial comparison are presented in Table 3 although
many more with comparison scores in excess of 9 SD could
have been selected When an equivalent number of
non-homologous proteins are compared (i.e comparing proteins
of the MOP superfamily with proteins of the major
facilitator superfamily (MFS; TC #2.A.1), values never
exceeded 7 SD
Figure S5 shows a binary alignment of an established
MATE family member with an established PST family
member The two proteins exhibit 38% similarity and 22%
identity with a comparison score of 28 SD The
corres-ponding alignment for interconnecting the OLF and PST
families is presented in Fig S6 The two proteins are 37%
similar and 25% identical with a comparison score of
13 SD The corresponding alignment for interconnecting
the MVF and PST families is shown in Fig S7 The two
proteins are 36% similar and 26% identical, yielding a
comparison score of 19 SD These comparisons and at least
one additional representative interfamilial comparison, giving values in excess of 9 SD, are summarized in Table 3 Many other binary comparisons gave comparison scores of greater than 9 SD However, the values reported in Table 3 are more than sufficient to establish homology
No member of the OLF family gave a comparison score
in excess of 6 SD with a member of the MATE or MVF family, and no member of the MVF family gave a score with
a MATE family member as high as the values obtained with PST family members The values recorded in Table 3 suggest a relative degree of relatedness of the four families as indicated in Fig 7 It is clear that the PST family is more closely related to the other three families than any two of the latter are related to each other
Identification of interfamilial conserved motifs
motifs in families of proteins, and these can be used to identify regions of sequence similarity between families [66] We therefore applied this program and selected two interfamilial regions of conservation for the pairs of
Fig 6 Phylogenetic tree for mouse virulence factor (MVF)family proteins See the legend to Fig 1 for format of presentation The 15 subfamilies are indicated 1–15.