BACKGROUND
the major intrinsic proteins   <cit> , or aquaporins  <cit> , allow for the passive transmembrane transport of water and other small, uncharged polar molecules  <cit> .
glyceroporin  from escherichia coli <cit>  and aquaporin  <dig>  from bovine  <cit>  were the first mips for which the 3d structures were established through x-ray crystallographic analysis.
the similarities between proteins of the mip family suggest that they have a common origin  <cit> .
plant aquaporins comprise a large protein family .
the topology of mips resembles a sandwich, consisting of six transmembrane α-helical strands .
these strands are connected to each other by five loops .
the lb and le loops each consist of a short α-helix connected by highly conserved npa-motifs, and these loops are partly located within the membrane  <cit> .
certain amino acid residues  of the h <dig> and h <dig> strands, together with two a.a. of the le site in the same plane, form the so-called ar/r-filter , which determines the substrate specificity of the protein  <cit> .
certain sub-families of mips contain conserved a.a. within the ar/r-filter  <cit> : e.g.
f <dig>  h <dig>  c <dig>  and r <dig> in the aquaporin hsaqp <dig>  <cit> ; and w <dig>  g <dig>  f <dig>  and r <dig> in the glyceroporin ecglpf  <cit> .
the pore diameter of the latter is larger than the former  <cit> .
it was recently shown that substitution of a.a. within the ar/r-filter results in a change in substrate specificity or in a loss of function  <cit> .
however, the design of the ar/r filter is not the only determinant of specificity.
determinants other than the ar/r filter before experimental studies cannot be identified by theoretical analysis for a majority of mips which are considered in the present study.
diatoms are unicellular, phototrophic, eukaryotic organisms that are present in all marine and freshwater habitats.
they originated as a result of double endosymbiosis followed by long-term  evolution , which resulted in the migration of many genes, such as bacterial genes, into the diatom nuclear genome  <cit> .
a general feature of diatoms is the presence of an intricately ornamented cell wall, known as a frustule, which consists of silica.
the synthesis of the solid and nearly anhydrous elements of the frustule takes place within specialised sub-cellular vesicles   <cit> .
maturation of the frustule requires the removal of water from the sdv.
it has been proposed that this process is mediated by aquaporins  <cit> .
in this study we investigate mips from chromista, phylum  heterokontophyta .
it was proposed that all pigmented heterokonts appeared due to double symbiosis and the simultaneous appearance of the ability to build cell walls with silica  <cit> .
we used taxonomy from algae base  <cit> .
ten mip genes were found in the complete genome sequences for the diatoms thalassiosira pseudonana tpmip <dig>  tpmip <dig>  <cit>  and t. oceanica tomip <dig>  tomip <dig>  <cit>  , phaeodactylum tricornutum ptmip <dig>  ptmip <dig>  ptmip <dig>  ptmip <dig>  ptmip <dig>  <cit>  , and nannochloropsis gaditana ngmip  <cit>  .
a mip gene was also recently discovered in the genome of the freshwater araphid pennate diatom synedra acus subsp.
radians .
the length of the predicted sarmip is  <dig> a.a.  <cit> .
mip genes were found in the genomes of the diatoms pseudo-nitzschia multiseries <cit>  and fragilariopsis cylindrus <cit>  .
the database dedicated to major intrinsic proteins mipdb  <cit>  contains seven mip sequences from ectocarpus siliculosus, esaqp, espip, and esmip , as well as from aureococcus anophagefferens, aamip <dig>  aamip <dig>  aamip <dig>  and aamip <dig> .
the purpose of the present study was to compare the predicted a.a. sequences of  <dig> mips from these algae with mip sequences from a wide variety of organisms.
we show that mips from heterokonts belong to different subfamilies, and nine of them merge into a new large intrinsic protein  subfamily, which is closely related with the sip subfamily  <cit>  and the mipc subfamily  <cit> .
RESULTS
search for homologues in the mipdb
we used the mipdb to find homologues of the  <dig> mips from heterokonts.
this database contains  <dig> mips belonging to  <dig> groups :  <dig> aqpes,  <dig> aqpps,  <dig> glaes,  <dig> glaps,  <dig> glpps,  <dig> nips,  <dig> pips,  <dig> sips, and  <dig> tips, as well as  <dig> predicted mips.
there are  <dig> sequences that are unclassified mips.
mipdb contains  <dig> mips from heterokonts,  <dig> of them belonging to the unclassified group and one of them  belonging to aqpe .
to find closely related sequences, we used the phmmer procedure in the hmmer3-package.
we selected the proteins with the smallest e-values from each of the mipdb groups.
these proteins are hypothesised to be related to the  <dig> mips studied.
phylogenetic analysis
to classify the mips from heterokonts, their a.a. sequences were subjected to a phylogenetic analysis together with proteins selected based on the results of a search for homologues and reference sequences, which included a typical aquaporin , a glyceroporin , and nips from rice and maize .
it has been showed that these four nips are transporters of silicic acid  <cit> .
among the  <dig> analysed mips from green algae, we identified seven different groups, including pips, glpf-like intrinsic proteins, and mips unique to green algae   <cit> .
these proteins have also been analysed in order to compare the results.
the total number of sequences subjected to phylogenetic analysis was  <dig>  alignment was performed using the profile of the mip pf <dig> in the pfam database.
the results of the phylogenetic analysis  demonstrated that the topology of the tree constructed from the selected sequences corresponds to the modern understanding of the phylogeny of mips  <cit> .
mips from heterokonts fall into different clades.figure  <dig>
phylogenetic tree of  <dig> mip sequences constructed using the neighbour joining  method and the jones-taylor-thornton  evolutionary model .
three sequences  from p. tricornutum, and aamip <dig> from the a. anophagefferens clustered within a large clade that includes glap, glae, and glpp with a bootstrap support of 58%.
the sequences of tpmip <dig> from t. pseudonana, tomip <dig> from t. oceanic, and aamip <dig> from a. anophagefferens clustered with unclassified mips from bacteria with bootstrap supports of 52% and 100%, respectively.
two sequences  from the brown alga e. siliculosus clustered with plant pips with a bootstrap support of 79%.
nine sequences  constituted a separate clade with a bootstrap support of 98%.
their closest relatives are the plant sips and mipc from green algae.
aamip <dig> from a. anophagefferens clustered with mipe from green algae with a bootstrap support of 71%.
only one sequence of ptmip <dig> did not reliably belong to any clade.
esaqp and espip clustered with ccpip4; <dig> and ccpip4; <dig> from green algae.
aamip <dig>  ptmip <dig>  ptmip <dig>  and ptmip <dig> clustered with gips from green algae as a sister clade with a bootstrap support of 58%.
peculiarities of the structures of mips from heterokonts
of the  <dig> heterokonts’ mips studied , only eight contain a pair of conserved npa motifs.
it worth noting that the esmip, ngmip, ptmip <dig>  pnmmip, fcmip, sarmip, tpmip <dig>  and tomip <dig> proteins, which are the closest to sips, all have an npm motif in place of the second npa.
however, the second motif of sips is an npa and the first motif is variable in the third a.a. position.
aamip <dig> has nga instead the first npa motif.
the mipc sequences contain the first modified motif np.figure  <dig>
comparison of npa motifs, ar/r filters, and c-terminal a.a. of  <dig> mips from heterokonts with some mips of different subfamilies.
a phylogenetic tree was constructed using the neighbour joining  method and the jones-taylor-thornton  evolutionary model .
the amino acids belonging to the ar/r filters are shown in additional file  <dig> and table  <dig> and figure  <dig>  mips  <dig>   <dig>  and  <dig> from p. tricornutum have a.a. compositions in the ar/r filter that are similar to the glap mip, whereas the ar/r filter of aamip <dig>  which was in the same clade as glap, glae, and glpp, has the same a.a. composition as human aquaporin hsaqp <dig> at positions h <dig>  le <dig>  and le <dig> table  <dig>
selectivity of the mips of heterokonts based on the similarity of their ar/r filter to the ar/r filters of the mips with known selectivity
the amino acid composition of the ar/r filters of ptmip <dig>  aamip <dig>  and espip was identical to that of the ar/r filter of human aquaporin hsaqp <dig>  the ar/r filter of esaqp differs from the ar/r filter of ptmip <dig>  aamip <dig>  and espip by the presence of an alanine  in place of the cysteine  at position le <dig>  the le <dig> position in the filters of esmip, ngmip, ptmip <dig>  pnmmip, fcmip, aamip <dig>  sarmip, tpmip <dig>  and tomip <dig>  as well as a majority of the filters of the sister clade, was occupied by a proline .
unlike the other mips, their le <dig> positions are occupied by leucine  or isoleucine .
hence, esmip, ngmip, ptmip <dig>  pnmmip, fcmip, aamip <dig>  sarmip, tpmip <dig>  and tomip <dig> differed from the other mips in this feature.
the h <dig> strand of the filter includes tryptophan /tyrosine  in these nine proteins, which is a more typical feature of glpp, glap, and nips that are not able to transport silicic acid .
discussion
phylogenetic analysis of mip a.a. sequences predicted from the nucleotide sequences of the respective genes, as well as a comparison of the ar/r filters of  <dig> mips from heterokonts, revealed that some of these proteins have very close homologues among the  <dig> proteins documented in the mipdb.
on the basis of the ar/r filter, substrate specificity could be suggested.
three proteins  from the diatom p. tricornutum were classified as glpf-like intrinsic proteins.
their closest homologues are damip, which is from the sulphate reducing anaerobic proteobacteria desulfuromonas acetoxidans, and ghmip, which is from the gram-positive coccus gemella haemolysans.
the ar/r filters of ptmip <dig>  ptmip <dig>  and ptmip <dig> are identical to those of nips of subgroup i, which have glycerol permease activity   <cit> .
aamip <dig> from a. anophagefferens also belongs to the glpf-like intrinsic proteins, as revealed by the phylogenetic analysis .
however, three a.a. in the ar/r filter in the h <dig>  le <dig>  and le <dig> of aamip <dig> are identical to those found in the human aquaporin hsaqp <dig>
two proteins, esaqp and espip, of the e. siliculosus belong to the pip subfamily, as revealed by the phylogenetic analysis .
unlike esaqp and espip, all other pips are highly conserved  <cit> .
the composition of the ar/r filter of esaqp and espip is different from that of the ar/r filter of pips at some positions .
however, the composition of the ar/r filters of espip and hsaqp <dig> is identical.
we propose that, based on their sequence composition, these two proteins from brown alga e. siliculosus are intermediate forms between the human hsaqp <dig> and plant pips.
aamip <dig> from a. anophagefferens clusters with a specific subfamily mipe from green algae on the phylogenetic tree.
the ar/r filters of aamip <dig> and mipes are identical to those of hsaqp <dig>  this similarity suggests these proteins have specificities for water.
a phylogenetic analysis revealed that ptmip <dig> from the diatom p. tricornutum does not have close relatives.
the sequence of this protein differs from classical aquaporins in that the first npa motif in ptmip <dig> is transformed into npg, but all residues of the ar/r filter are identical to those of the hsaqp <dig> and mipes.
tpmip <dig> and tomip <dig> from diatoms and aamip <dig> from a. anophagefferens cluster with bacterial mips on the phylogenetic tree.
multiple alignments have shown that the ar/r filters of bacterial mips, tpmip <dig>  tomip <dig>  and aamip <dig> differ from those found in the proteins from other subfamilies .
the functions of these bacterial mips are not yet known.
therefore, no function can be proposed at this time for tpmip <dig>  tomip <dig>  or aamip <dig>  interestingly, the aquaporin tcaqpe of the parasitic trypanosome trypanosoma cruzi falls into the same clade, although its ar/r filter  <cit>  is different from that of typical aquaporins at all four positions.
nine proteins  form a separate clade adjacent to the sip clade of plants and the mipc clade of green algae.
ishibashi et al.
<cit>  concluded that during their evolution, sips and xips lost conservation of the npa motifs.
the first motif that replaced npa in sips was np, while in mipc it was np.
the motif that is found in place of the second npa in these nine proteins is npm.
we found four other proteins with npm in place of the second npa in the mipdb.
one of these proteins belongs to the nip subfamily, and the other three belong to an uncharacterised group.
however, arginine  is c–terminal to npm in the le <dig> position in these four proteins, whereas leucine  or isoleucine  are c-terminal to npm in mips from heterokonts.
the le <dig> position in the ar/r filters of esmip, ngmip, ptmip <dig>  pnmmip, fcmip, aamip <dig>  sarmip, tpmip <dig>  and tomip <dig> are occupied by the same a.a. as those in these positions in sips.
however, the sites at positions h <dig> and le <dig> are occupied by different a.a. from those found in the same positions in the sips.
remarkably, a tryptophan  at position h <dig> occurs in all glyceroporins, as well as in nips that are not able to transport silicic acid .
residues of the ar/r filter of mipc do not match any one a.a. position of the ar/r filter of these nine proteins.
similarities were revealed in the terminal a.a. of lysine  in sips and esmip, ngmip, ptmip <dig>  pnmmip, fcmip, sarmip, tpmip <dig>  and tomip <dig> .
of all the mipcs, only one sequence  contains a terminal lysine .
on the basis of the above evidence, we suggest a new phylogenetic clade, lips, which includes nine proteins: esmip, ngmip, ptmip <dig>  pnmmip, fcmip, aamip <dig>  sarmip, tpmip <dig>  and tomip <dig> .
this new clade has high bootstrap support.
indeed, seven of the nine proteins of the subfamily are large , with esmip consisting of  <dig> a.a. and ngmip consisting of  <dig> a.a. .
however, esmip and ngmip are similar to other lips in terms of phylogeny  and the structure of the ar/r filters .
heterokonts are thought to be derived from a secondary endosymbiotic process between a red alga and a heterotrophic eukaryote  <cit> .
recent studies of the genomes of the diatoms have revealed the participation of green algae in the origin of some membrane transporters  <cit> .
we showed that of the mips from heterokonts, one protein  has a relationship with a specific subfamily mipe from green algae.
according to our analysis, none of the  <dig> analysed mips from heterokonts are relatives to nips, which transports silicic acid, on the phylogeny , and have dissimilar ar/r filter .
CONCLUSIONS
heterokonts, like other organisms, contain a variety of mips, which could allow for the transport of substances, such as water, glycerol, urea, carbon dioxide, etc.
we found that heterokonts contain mips that belong to different subfamilies, such as pip, gip, and mipe.
the most surprising finding is that during their evolution, heterokonts acquired unique genes, such as those that encode the mips of the lip subfamily.
these unusual proteins encoded by these genes are only distantly related to typical aqua- or glyceroporins, and are characterised by a specific motif and the composition of the ar/r filter.
notably, none of mips from heterokonts have any similarities with nips that are responsible for transporting silicic acid.

 the major intrinsic proteins, or aquaporins, allow for the passive transmembrane transport of water and other small, uncharged polar molecules. glyceroporin from escherichia coli and aquaporin from bovine were the first mips for which the 3d structures were established through x-ray crystallographic analysis. the similarities between proteins of the mip family suggest that they have a common origin. plant aquaporins comprise a large protein family. the topology of mips resembles a sandwich, consisting of six transmembrane α-helical strands. these strands are connected to each other by five loops. the lb and le loops each consist of a short α-helix connected by highly conserved npa-motifs, and these loops are partly located within the membrane. certain amino acid residues of the h and h strands, together with two a.a. of the le site in the same plane, form the so-called ar/r-filter, which determines the substrate specificity of the protein. certain sub-families of mips contain conserved a.a. within the ar/r-filter: e.g. f h c and r in the aquaporin hsaqp; and w g f and r in the glyceroporin ecglpf. the pore diameter of the latter is larger than the former. it was recently shown that substitution of a.a. within the ar/r-filter results in a change in substrate specificity or in a loss of function. however, the design of the ar/r filter is not the only determinant of specificity. determinants other than the ar/r filter before experimental studies cannot be identified by theoretical analysis for a majority of mips which are considered in the present study. diatoms are unicellular, phototrophic, eukaryotic organisms that are present in all marine and freshwater habitats. they originated as a result of double endosymbiosis followed by long-term evolution, which resulted in the migration of many genes, such as bacterial genes, into the diatom nuclear genome. a general feature of diatoms is the presence of an intricately ornamented cell wall, known as a frustule, which consists of silica. the synthesis of the solid and nearly anhydrous elements of the frustule takes place within specialised sub-cellular vesicles. maturation of the frustule requires the removal of water from the sdv. it has been proposed that this process is mediated by aquaporins. in this study we investigate mips from chromista, phylum heterokontophyta. it was proposed that all pigmented heterokonts appeared due to double symbiosis and the simultaneous appearance of the ability to build cell walls with silica. we used taxonomy from algae base. ten mip genes were found in the complete genome sequences for the diatoms thalassiosira pseudonana tpmip tpmip and t. oceanica tomip tomip, phaeodactylum tricornutum ptmip ptmip ptmip ptmip ptmip, and nannochloropsis gaditana ngmip. a mip gene was also recently discovered in the genome of the freshwater araphid pennate diatom synedra acus subsp. radians. the length of the predicted sarmip is a.a.. mip genes were found in the genomes of the diatoms pseudo-nitzschia multiseries and fragilariopsis cylindrus. the database dedicated to major intrinsic proteins mipdb contains seven mip sequences from ectocarpus siliculosus, esaqp, espip, and esmip, as well as from aureococcus anophagefferens, aamip aamip aamip and aamip. the purpose of the present study was to compare the predicted a.a. sequences of mips from these algae with mip sequences from a wide variety of organisms. we show that mips from heterokonts belong to different subfamilies, and nine of them merge into a new large intrinsic protein subfamily, which is closely related with the sip subfamily and the mipc subfamily.  search for homologues in the mipdb we used the mipdb to find homologues of the mips from heterokonts. this database contains mips belonging to groups: aqpes, aqpps, glaes, glaps, glpps, nips, pips, sips, and tips, as well as predicted mips. there are sequences that are unclassified mips. mipdb contains mips from heterokonts, of them belonging to the unclassified group and one of them belonging to aqpe. to find closely related sequences, we used the phmmer procedure in the hmmer3-package. we selected the proteins with the smallest e-values from each of the mipdb groups. these proteins are hypothesised to be related to the mips studied. phylogenetic analysis to classify the mips from heterokonts, their a.a. sequences were subjected to a phylogenetic analysis together with proteins selected based on the results of a search for homologues and reference sequences, which included a typical aquaporin, a glyceroporin, and nips from rice and maize. it has been showed that these four nips are transporters of silicic acid. among the analysed mips from green algae, we identified seven different groups, including pips, glpf-like intrinsic proteins, and mips unique to green algae. these proteins have also been analysed in order to compare the results. the total number of sequences subjected to phylogenetic analysis was alignment was performed using the profile of the mip pf in the pfam database. the results of the phylogenetic analysis demonstrated that the topology of the tree constructed from the selected sequences corresponds to the modern understanding of the phylogeny of mips. mips from heterokonts fall into different clades.figure phylogenetic tree of mip sequences constructed using the neighbour joining method and the jones-taylor-thornton evolutionary model. three sequences from p. tricornutum, and aamip from the a. anophagefferens clustered within a large clade that includes glap, glae, and glpp with a bootstrap support of 58%. the sequences of tpmip from t. pseudonana, tomip from t. oceanic, and aamip from a. anophagefferens clustered with unclassified mips from bacteria with bootstrap supports of 52% and 100%, respectively. two sequences from the brown alga e. siliculosus clustered with plant pips with a bootstrap support of 79%. nine sequences constituted a separate clade with a bootstrap support of 98%. their closest relatives are the plant sips and mipc from green algae. aamip from a. anophagefferens clustered with mipe from green algae with a bootstrap support of 71%. only one sequence of ptmip did not reliably belong to any clade. esaqp and espip clustered with ccpip4; and ccpip4; from green algae. aamip ptmip ptmip and ptmip clustered with gips from green algae as a sister clade with a bootstrap support of 58%. peculiarities of the structures of mips from heterokonts of the heterokonts’ mips studied, only eight contain a pair of conserved npa motifs. it worth noting that the esmip, ngmip, ptmip pnmmip, fcmip, sarmip, tpmip and tomip proteins, which are the closest to sips, all have an npm motif in place of the second npa. however, the second motif of sips is an npa and the first motif is variable in the third a.a. position. aamip has nga instead the first npa motif. the mipc sequences contain the first modified motif np.figure comparison of npa motifs, ar/r filters, and c-terminal a.a. of mips from heterokonts with some mips of different subfamilies. a phylogenetic tree was constructed using the neighbour joining method and the jones-taylor-thornton evolutionary model. the amino acids belonging to the ar/r filters are shown in additional file and table  and figure  mips and from p. tricornutum have a.a. compositions in the ar/r filter that are similar to the glap mip, whereas the ar/r filter of aamip which was in the same clade as glap, glae, and glpp, has the same a.a. composition as human aquaporin hsaqp at positions h le and le table selectivity of the mips of heterokonts based on the similarity of their ar/r filter to the ar/r filters of the mips with known selectivity the amino acid composition of the ar/r filters of ptmip aamip and espip was identical to that of the ar/r filter of human aquaporin hsaqp the ar/r filter of esaqp differs from the ar/r filter of ptmip aamip and espip by the presence of an alanine in place of the cysteine at position le the le position in the filters of esmip, ngmip, ptmip pnmmip, fcmip, aamip sarmip, tpmip and tomip as well as a majority of the filters of the sister clade, was occupied by a proline. unlike the other mips, their le positions are occupied by leucine or isoleucine. hence, esmip, ngmip, ptmip pnmmip, fcmip, aamip sarmip, tpmip and tomip differed from the other mips in this feature. the h strand of the filter includes tryptophan /tyrosine in these nine proteins, which is a more typical feature of glpp, glap, and nips that are not able to transport silicic acid. discussion phylogenetic analysis of mip a.a. sequences predicted from the nucleotide sequences of the respective genes, as well as a comparison of the ar/r filters of mips from heterokonts, revealed that some of these proteins have very close homologues among the proteins documented in the mipdb. on the basis of the ar/r filter, substrate specificity could be suggested. three proteins from the diatom p. tricornutum were classified as glpf-like intrinsic proteins. their closest homologues are damip, which is from the sulphate reducing anaerobic proteobacteria desulfuromonas acetoxidans, and ghmip, which is from the gram-positive coccus gemella haemolysans. the ar/r filters of ptmip ptmip and ptmip are identical to those of nips of subgroup i, which have glycerol permease activity. aamip from a. anophagefferens also belongs to the glpf-like intrinsic proteins, as revealed by the phylogenetic analysis. however, three a.a. in the ar/r filter in the h le and le of aamip are identical to those found in the human aquaporin hsaqp two proteins, esaqp and espip, of the e. siliculosus belong to the pip subfamily, as revealed by the phylogenetic analysis. unlike esaqp and espip, all other pips are highly conserved. the composition of the ar/r filter of esaqp and espip is different from that of the ar/r filter of pips at some positions. however, the composition of the ar/r filters of espip and hsaqp is identical. we propose that, based on their sequence composition, these two proteins from brown alga e. siliculosus are intermediate forms between the human hsaqp and plant pips. aamip from a. anophagefferens clusters with a specific subfamily mipe from green algae on the phylogenetic tree. the ar/r filters of aamip and mipes are identical to those of hsaqp this similarity suggests these proteins have specificities for water. a phylogenetic analysis revealed that ptmip from the diatom p. tricornutum does not have close relatives. the sequence of this protein differs from classical aquaporins in that the first npa motif in ptmip is transformed into npg, but all residues of the ar/r filter are identical to those of the hsaqp and mipes. tpmip and tomip from diatoms and aamip from a. anophagefferens cluster with bacterial mips on the phylogenetic tree. multiple alignments have shown that the ar/r filters of bacterial mips, tpmip tomip and aamip differ from those found in the proteins from other subfamilies. the functions of these bacterial mips are not yet known. therefore, no function can be proposed at this time for tpmip tomip or aamip interestingly, the aquaporin tcaqpe of the parasitic trypanosome trypanosoma cruzi falls into the same clade, although its ar/r filter is different from that of typical aquaporins at all four positions. nine proteins form a separate clade adjacent to the sip clade of plants and the mipc clade of green algae. ishibashi et al. concluded that during their evolution, sips and xips lost conservation of the npa motifs. the first motif that replaced npa in sips was np, while in mipc it was np. the motif that is found in place of the second npa in these nine proteins is npm. we found four other proteins with npm in place of the second npa in the mipdb. one of these proteins belongs to the nip subfamily, and the other three belong to an uncharacterised group. however, arginine is c–terminal to npm in the le position in these four proteins, whereas leucine or isoleucine are c-terminal to npm in mips from heterokonts. the le position in the ar/r filters of esmip, ngmip, ptmip pnmmip, fcmip, aamip sarmip, tpmip and tomip are occupied by the same a.a. as those in these positions in sips. however, the sites at positions h and le are occupied by different a.a. from those found in the same positions in the sips. remarkably, a tryptophan at position h occurs in all glyceroporins, as well as in nips that are not able to transport silicic acid. residues of the ar/r filter of mipc do not match any one a.a. position of the ar/r filter of these nine proteins. similarities were revealed in the terminal a.a. of lysine in sips and esmip, ngmip, ptmip pnmmip, fcmip, sarmip, tpmip and tomip. of all the mipcs, only one sequence contains a terminal lysine. on the basis of the above evidence, we suggest a new phylogenetic clade, lips, which includes nine proteins: esmip, ngmip, ptmip pnmmip, fcmip, aamip sarmip, tpmip and tomip. this new clade has high bootstrap support. indeed, seven of the nine proteins of the subfamily are large, with esmip consisting of a.a. and ngmip consisting of a.a.. however, esmip and ngmip are similar to other lips in terms of phylogeny and the structure of the ar/r filters. heterokonts are thought to be derived from a secondary endosymbiotic process between a red alga and a heterotrophic eukaryote. recent studies of the genomes of the diatoms have revealed the participation of green algae in the origin of some membrane transporters. we showed that of the mips from heterokonts, one protein has a relationship with a specific subfamily mipe from green algae. according to our analysis, none of the analysed mips from heterokonts are relatives to nips, which transports silicic acid, on the phylogeny, and have dissimilar ar/r filter.  heterokonts, like other organisms, contain a variety of mips, which could allow for the transport of substances, such as water, glycerol, urea, carbon dioxide, etc. we found that heterokonts contain mips that belong to different subfamilies, such as pip, gip, and mipe. the most surprising finding is that during their evolution, heterokonts acquired unique genes, such as those that encode the mips of the lip subfamily. these unusual proteins encoded by these genes are only distantly related to typical aqua- or glyceroporins, and are characterised by a specific motif and the composition of the ar/r filter. notably, none of mips from heterokonts have any similarities with nips that are responsible for transporting silicic acid. 
we compared the predicted amino acid sequences of mips from several algae species of the phylum heterokontophyta with the sequences of mips from other organisms. these proteins are important in cells and organisms because they allow for passive transmembrane transport of water and other small, uncharged polar molecules. the mip sequences of the studied chromists fell into several clusters that belonged to different groups of mips from a wide variety of organisms from different kingdoms. two of these proteins belong to plasma membrane intrinsic proteins, four of them belong to glpf-like intrinsic proteins, and one of them belongs to a specific mipe subfamily from green algae.
 proteins of the major intrinsic protein family, or aquaporins, have been detected in almost all organisms. these proteins are important in cells and organisms because they allow for passive transmembrane transport of water and other small, uncharged polar molecules.  we compared the predicted amino acid sequences of mips from several algae species of the phylum heterokontophyta with the sequences of mips from other organisms. multiple sequence alignments revealed motifs that were homologous to functionally important npa motifs and the so-called ar/r-selective filter of glyceroporins and aquaporins. the mip sequences of the studied chromists fell into several clusters that belonged to different groups of mips from a wide variety of organisms from different kingdoms. two of these proteins belong to plasma membrane intrinsic proteins, four of them belong to glpf-like intrinsic proteins, and one of them belongs to a specific mipe subfamily from green algae. three proteins belong to the unclassified mips, two of which are of bacterial origin. eight of the studied mips contain an npm-motif in place of the second conserved npa-motif typical of the majority of mips. the mips of heterokonts within all detected clusters can differ from other mips in the same cluster regarding the structure of the ar/r-selective filter and other generally conserved motifs.  we proposed placing nine mips from heterokonts into a new group, which we have named the lips. the possible substrate specificities of the studied mips are discussed. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aquaporinschromistaheterokontophytamajor intrinsic proteinsissue-copyright-statement© the author 2014 