BACKGROUND
guanine nucleotide exchange factors  are obligatory components of signaling cascades regulated by small gtp-binding proteins .
their biochemical activity is to stimulate the dissociation of the tightly bound gdp nucleotide from the small g protein in response to cellular signals.
thereby, they favor the binding of the more abundant cellular gtp, organizing the active conformation of the small g protein which can recruit its effectors .
each small g protein family features its own ensemble of gefs characterized by a conserved catalytic domain responsible for nucleotide exchange, which is generally combined with non-catalytic domains that define the spatio-temporal conditions of activation.
in the case of small g proteins of the arf family, which are major regulators in membrane trafficking , the exchange domain is a conserved module of ~ <dig> amino acids called the sec <dig> domain  <cit> .
its biochemical  and structural  <cit>  mechanisms have been investigated in detail.
remarkably, the sec <dig> domain is the only domain that is conserved in all arfgefs  and it is to some extent interchangeable between species  <cit> .
in contrast, little is known about the functions of the other domains, which are likely to determine intracellular localization of arfgefs and their responsiveness to specific signals.
as for most small g proteins, arf family members are outnumbered by arfgefs in many species.
in humans for instance,  <dig> arf proteins have been identified, and there are at least  <dig> proteins carrying a sec <dig> domain, of which most have been characterized as bona fide arfgefs .
thus an individual arf protein may be activated by more than one gef, emphasizing that essential aspects in building up the arf responses may be encoded by the modular architecture of their gefs.
sequence similarity in the non-catalytic regions forms the basis for the classification of arfgefs into subfamilies.
<dig> subfamilies are currently identified in eukaryotes with sizes ranging from small , to medium  and large  arfgefs .
large arfgefs comprise two subfamilies which we will refer to as the big and gbf subfamilies after the name of their human representatives.
the gbf subfamily includes human gbf <dig>  <cit> , arabidopsis gnom  <cit>  and saccharomyces gea <dig> and gea <dig>  <cit> , the big subfamily human big <dig> and big <dig>  <cit>  and yeast sec7p  <cit> .
an additional subfamily called ralf is found in rickettsie and legionella bacteria, likely acting on an host arf pathway  <cit> .
analysis of the cyh and efa <dig> subfamilies, present only in multicellular animals, and that of the large arfgefs, found in all eukaryotes, have yielded most of the functional data currently available.
cyh and efa <dig> are active on arf <dig> at the plasma membrane where they may function in the crosstalk of membrane traffic, cytoskeleton dynamics and signalling in endosomal pathways .
most members of the big and gbf subfamilies characterized so far function in vesicular trafficking at the golgi  <cit> , except for big <dig>  which also localizes on recycling endosomes  <cit> , and gnom which acts in the endosomal recycling pathway  <cit> .
the domain architecture of non-catalytic regions of arfgefs, hence their contribution to specific aspects of the build-up of the arf response, is essentially not established except for those arfgefs with domains found in other classes of cellular regulators.
the known domains include membrane-interacting ph domains in the cyh , efa <dig>  <cit>  and possibly brag/loner <cit>  subfamilies, and a putative f-box in the fbs subfamily  <cit> , a protein-protein interaction domain that has been involved in the recruitment of substrates to the scf ubiquitination machinery.
coiled-coil structures have also been predicted in the n-terminus of the cyh subfamily and in the c-terminus of the efa <dig> subfamily.
in cyh, they are involved in dimerization  <cit> , recruitment of partners  <cit>  and golgi targeting  <cit> , and in actin remodeling functions in the case of efa <dig>  <cit> .
on the other hand, although the functions of big and gbf subfamilies have been the subject of many investigations, their architecture is barely described, making it difficult to associate biochemical activities with their molecular structure.
here we investigate the domain architecture in the big and gbf subfamilies, including all sequences from fully annotated eukaryotic genomes and our novel annotation of sec7-containing proteins from the paramecium tetraurelia alveolate.
sequence comparisons combined with secondary structures and splicing patterns analysis identifies five novel domains that are conserved between big and gbf subfamilies, thus unifying them as a higher order subfamily with a probable common ancestor.
our analysis of sec7-domain containing sequences from paramecium also introduces a novel subfamily of arfgefs unique to alveolates, which we call tbs .
RESULTS
a conserved domain architecture in big and gbf subfamilies
the big and gbf subfamilies are the only arfgefs subfamilies common to all eukaryotes  <cit>  and the sole arfgefs present in plants  <cit>  .
they are therefore possible representatives of ancestral arfgef functions and may provide a model to understand the nature and implementation of activities associated with the exchange function carried by the conserved sec <dig> domain.
however, domain 'hunting' in big and gbf subfamilies was complicated by the facts that the sec <dig> domain is their only domain that could be identified from known domain repertoires, and that their poorly characterized non-catalytic regions were not found outside these arfgef subfamilies.
alternatively, we based our search of candidate structural domains in bigs and gbfs on the bioinformatics analysis of their own sequences, taking advantage of the growing number of sequences from fully annotated genomes from mammals, insects, plants, nematode, and fungi, to which we included our annotation of sec7-containing proteins from the newly sequenced genome of paramecium.
multiple alignments of  <dig> sequences  revealed that the big and gbf subfamilies share an unexpected conserved architecture .
two homology domains are located in n-terminus of the sec <dig> domain – the dcb  and hus  domains – and three in its c-terminus -the hds <dig> , hds <dig>  and hds <dig>  domains .
in arabidopsis gnom, the dcb domain is included in an n-terminal region of ~ <dig> residues involved in dimerization and possibly binding to cyclophilin <dig> and called the dimerization/cyclophilin binding region  <cit> , after which the new domain was named .
all domains are predicted to have a high content of α-helices that co-align in the multiple sequence alignments, reinforcing the prediction of sequence similarities and suggesting that these domains form folded structural units that may share common functional features.
except for the n-terminal dcb domain which is also found in the yeast protein ysl2p  <cit> , all of them are unique to these two arfgefs subfamilies within the detection limits of the blast search.
the hus domain features a remarkably conserved ndc motif, which we call the hus box, which is predicted to locate in a loop where it may be available for functional interactions .
the n- and c-terminal ends of bigs and gbfs are more variable, including an unusual enrichment in asp/glu or pro residues in some members.
a specific feature of big members is that their c-terminus is in general less variable than that of gbfs, and is predicted with a significant amount of secondary structures.
in contrast to the predicted structural domains, the intervening regions are highly variable in length and do not yield aligned sequences.
analysis of their amino-acid composition reveals a paucity of hydrophobic residues which is predicted to associate with an essentially unfolded conformation, suggesting that they act as linkers to tether the functional domains together.
a unnamed sequences are designated by their ncbi accession number, agi  locus numbers for at and tigr model temporary ids for os.
big and gbf subfamily members are in normal and bold characters respectively, except for pt members which have not been assigned to either subfamily .
species abbreviations are: ag, anopheles gambiae; ce, caenorhabditis.
elegans; dm, drosophila melanogaster; hs, homo sapiens; rn, rattus norvegicus; ca, candida albicans; nc, neurospora crassa; sc, saccharomyces cerevisiae; sp, schizosaccharomyces pombe; at, arabidopsis thaliana; os, oryza sativa; pt, parameciumtetraurelia).
to further investigate the predicted organization of bigs and gbfs in  <dig> conserved helical domains connected by variable linkers, splicing patterns of human bigs and gbfs were analyzed in the large number of cdnas and ests in the databases that correspond to gbf/big transcripts.
this revealed the use of alternate splice donor and acceptor sites predicted to yield proteins with insertions and deletions ranging from  <dig> to  <dig> residues, and a number of splice variants arising from exon skipping .
strikingly, all observed sequence variations occur in regions identified as linkers between conserved domains .
together with our domain analysis, this suggests that splicing at non-canonical exon/intron boundaries is only tolerated in regions of the protein where the impact upon folding of domains with essential function would be minimal.
a all changes were expressed relative to the reference sequence stored under accession number nm_ <dig> , nm_ <dig>  and nm_ <dig>  .
b all variants are supported by one or more cdna/ests as detailed in the aceview for each gene that can be obtained at  <cit> .
evolution of bigs and gfbs from a common ancestor
combined, our analysis reveals that the big and gbf subfamilies share the same overall domain organization, and are likely to descend from a common ancestor gene that duplicated first to form the big and gbf groups, and again within these groups to yield species-specific big and gbf members.
these two subfamilies can therefore be unified as a higher order arfgef subfamily , from which unrooted phylogenetic trees can be built .
unlike previous phylogenetic analysis which compared arfgefs based on their sec <dig> domains after diverging non-catalytic regions have been trimmed  <cit> , our trees were established from the simultaneous alignment of all  <dig> conserved domains , excluding variable linkers.
the same tree topology was obtained with both neighbor-joining and maximum likelihood methods, and was retained using any one of the new conserved domains alone .
bootstrap analysis strongly supported this topology for most branches.
only a few small branches located at the base of the groups were found in less than 60% of the trials in one of the two methods, but this never occurred with both methods simultaneously.
the tree topology strongly suggests that in most organisms, gbg members sort in separate branches, corresponding to their classification in bigs and gbfs.
remarkably, our annotation of sec7-containing proteins in the genome of paramecium reveals the first departure from this distribution, as all gbgs in this species are located in a single branch, which is closer to the bigs.
this unexpected tree topology may indicate that alveolates diverged from animals/fungi and plants before the duplication of an ancestral gbg into the big and gbf, and that gbgs in that organism are representative of this ancestral gene.
alternatively, duplication may have been followed by loss of gbf genes.
current knowledge of the phylogenetic branching of alveolates relative to the plants and animal/fungi branches does not permit resolution between those two possibilities.
gbgs in plants: refining the functional evolution of bigs and gbfs
in fungi and mammals, bigs and gbfs are represented by only one or two members, whose functions in vesicular trafficking at the golgi within each group appear largely overlapping  <cit> .
in contrast, plants encode a large number of gbgs in both the big and gbf branches but lack other arfgefs .
in arabidopsis, none of the gbgs map to duplicated chromosomes where identical functions may be encoded  <cit> .
in addition, comparative analysis with the rice genome nearing completion identifies at least five branches each represented by one rice and one or two arabidopsis homologs .
this correspondence between two highly divergent plant species indicates that gbgs diversified early during plant evolution, probably reflecting functional specialization along with the establishment of plant multicellularity.
while gnom has a plant-specific function in recycling plasma-membrane proteins needed for cell-cell communication and cell polarity establishment  <cit> , possibly closer to the function of efa <dig> or cyh subfamilies in metazoans, other plant gbgs are expected to fulfill the presumed ancient function of regulating golgi trafficking exemplified by mammalian and yeast gbgs.
comparison of orthologous pairs in plants further reveals that they have different sensitivities to brefeldin a  as predicted from the sequences of the binding site of the drug carried by the sec <dig> domain  <cit> .
this observation clearly illustrates that differences in outcome following brefeldina treatment may not reflect differences in underlying molecular mechanisms, but instead simply reflect neutral sequence differences at the sec <dig> domains between species.
in particular, not all bigs may be bfa-sensitive or gbfs bfa-resistant, unlike suggested by their original nomenclature.
a novel arfgef subfamily in alveolates
a remarkable evolutionary feature of arfgefs is that while gbgs seem to be ubiquitous to all eukaryotes, fungi and animals kingdoms evolved their own arfgefs subfamilies unrelated to those of the other kingdoms.
we thus addressed the question of whether paramecium, which has a large number of gbgs  but appears to lack the specialization into the big and gbf subgroups, has the same arfgef distribution as plants or features a second arfgef subfamily.
we thus searched the newly sequenced genome from paramecium tetraurelia and the available alveolate genomes from cryptosporidium parvum and tetrahymena thermophila for additional sec7-containing proteins.
this identified a novel putative arfgef subfamily characterized by the association of the sec <dig> domain with a tbc  domain , which was found only in the protists kingdom.
the tbc domain is predicted to carry a gap  activity towards small g proteins of the rab family  <cit> , suggesting a potential crosstalk between rab and arf pathways.
such a relationship between these two small g proteins families, which are major regulators of membrane traffic, would not be unprecedented, as for example the syt <dig> arfgef gene was identified in yeast by its genetic interactions with rab proteins in the exocytic pathway  <cit> .
interestingly, alveolates have specialized exocytic pathways based on a membrane organelle lying beneath the plasma membrane, the trichocyst, where this unique arfgef family may potentially function.
CONCLUSIONS
a conserved scenario for the activation of arf proteins by their gefs?
the identification of a conserved modular architecture in all gbg subfamily members suggests that the mechanistic basis for their activation of arf is likely to follow a similar scenario.
candidate functions for the conserved domains include oligomerization, the collection of input signals, membrane localization, regulation of the exchange activity, scaffolding of arf proteins to their downstream effectors, not excluding signaling to partners outside the arf pathways.
dimerization has been reported in the big subgroup for big <dig>  which forms heterodimers with the highly homologous big <dig> arfgef  <cit> , and in the gbf subgroup for gnom, which forms homodimers  <cit> .
the conservation of the dcb domain in gbgs, which is responsible for the dimerization of gnom, suggests that such a dimerization function may be general to this domain in gbgs.
another unresolved issue is the conservation of the cellular partners effecting the functions associated with the conserved domains.
our identification of an almost invariant motif in the hus domain argues in favor of this domain interacting with a conserved partner.
however, the ancient divergence into the big and gbf groups and their subsequent divergence into species-specific members suggest that specialized requirements are likely to have evolved in most organisms, possibly yielding less conserved partners outside the sec <dig> and hus domains.
finally, whereas in plants all arfgefs are predicted to function according to the scheme defined by the conserved domains, other species have additional arfgef subfamilies with a modular architecture unrelated to that of the gbg subfamily.
it is not known to what extent the gbg's scenario for arf activation will also apply to non-gbgs arfgefs, acting alone or in association with protein partners.
in the case of the gbgs, our definition of the structural homology domains as reported here should now provide a robust background for future investigations of their interactions and functions.

 guanine nucleotide exchange factors are obligatory components of signaling cascades regulated by small gtp-binding proteins. their biochemical activity is to stimulate the dissociation of the tightly bound gdp nucleotide from the small g protein in response to cellular signals. thereby, they favor the binding of the more abundant cellular gtp, organizing the active conformation of the small g protein which can recruit its effectors. each small g protein family features its own ensemble of gefs characterized by a conserved catalytic domain responsible for nucleotide exchange, which is generally combined with non-catalytic domains that define the spatio-temporal conditions of activation. in the case of small g proteins of the arf family, which are major regulators in membrane trafficking, the exchange domain is a conserved module of ~ amino acids called the sec domain. its biochemical and structural mechanisms have been investigated in detail. remarkably, the sec domain is the only domain that is conserved in all arfgefs and it is to some extent interchangeable between species. in contrast, little is known about the functions of the other domains, which are likely to determine intracellular localization of arfgefs and their responsiveness to specific signals. as for most small g proteins, arf family members are outnumbered by arfgefs in many species. in humans for instance, arf proteins have been identified, and there are at least proteins carrying a sec domain, of which most have been characterized as bona fide arfgefs. thus an individual arf protein may be activated by more than one gef, emphasizing that essential aspects in building up the arf responses may be encoded by the modular architecture of their gefs. sequence similarity in the non-catalytic regions forms the basis for the classification of arfgefs into subfamilies. subfamilies are currently identified in eukaryotes with sizes ranging from small, to medium and large arfgefs. large arfgefs comprise two subfamilies which we will refer to as the big and gbf subfamilies after the name of their human representatives. the gbf subfamily includes human gbf, arabidopsis gnom and saccharomyces gea and gea, the big subfamily human big and big and yeast sec7p. an additional subfamily called ralf is found in rickettsie and legionella bacteria, likely acting on an host arf pathway. analysis of the cyh and efa subfamilies, present only in multicellular animals, and that of the large arfgefs, found in all eukaryotes, have yielded most of the functional data currently available. cyh and efa are active on arf at the plasma membrane where they may function in the crosstalk of membrane traffic, cytoskeleton dynamics and signalling in endosomal pathways. most members of the big and gbf subfamilies characterized so far function in vesicular trafficking at the golgi, except for big which also localizes on recycling endosomes, and gnom which acts in the endosomal recycling pathway. the domain architecture of non-catalytic regions of arfgefs, hence their contribution to specific aspects of the build-up of the arf response, is essentially not established except for those arfgefs with domains found in other classes of cellular regulators. the known domains include membrane-interacting ph domains in the cyh, efa and possibly brag/loner subfamilies, and a putative f-box in the fbs subfamily, a protein-protein interaction domain that has been involved in the recruitment of substrates to the scf ubiquitination machinery. coiled-coil structures have also been predicted in the n-terminus of the cyh subfamily and in the c-terminus of the efa subfamily. in cyh, they are involved in dimerization, recruitment of partners and golgi targeting, and in actin remodeling functions in the case of efa. on the other hand, although the functions of big and gbf subfamilies have been the subject of many investigations, their architecture is barely described, making it difficult to associate biochemical activities with their molecular structure. here we investigate the domain architecture in the big and gbf subfamilies, including all sequences from fully annotated eukaryotic genomes and our novel annotation of sec7-containing proteins from the paramecium tetraurelia alveolate. sequence comparisons combined with secondary structures and splicing patterns analysis identifies five novel domains that are conserved between big and gbf subfamilies, thus unifying them as a higher order subfamily with a probable common ancestor. our analysis of sec7-domain containing sequences from paramecium also introduces a novel subfamily of arfgefs unique to alveolates, which we call tbs.  a conserved domain architecture in big and gbf subfamilies the big and gbf subfamilies are the only arfgefs subfamilies common to all eukaryotes and the sole arfgefs present in plants. they are therefore possible representatives of ancestral arfgef functions and may provide a model to understand the nature and implementation of activities associated with the exchange function carried by the conserved sec domain. however, domain'hunting' in big and gbf subfamilies was complicated by the facts that the sec domain is their only domain that could be identified from known domain repertoires, and that their poorly characterized non-catalytic regions were not found outside these arfgef subfamilies. alternatively, we based our search of candidate structural domains in bigs and gbfs on the bioinformatics analysis of their own sequences, taking advantage of the growing number of sequences from fully annotated genomes from mammals, insects, plants, nematode, and fungi, to which we included our annotation of sec7-containing proteins from the newly sequenced genome of paramecium. multiple alignments of sequences revealed that the big and gbf subfamilies share an unexpected conserved architecture. two homology domains are located in n-terminus of the sec domain – the dcb and hus domains – and three in its c-terminus -the hds, hds and hds domains. in arabidopsis gnom, the dcb domain is included in an n-terminal region of ~ residues involved in dimerization and possibly binding to cyclophilin and called the dimerization/cyclophilin binding region, after which the new domain was named. all domains are predicted to have a high content of α-helices that co-align in the multiple sequence alignments, reinforcing the prediction of sequence similarities and suggesting that these domains form folded structural units that may share common functional features. except for the n-terminal dcb domain which is also found in the yeast protein ysl2p, all of them are unique to these two arfgefs subfamilies within the detection limits of the blast search. the hus domain features a remarkably conserved ndc motif, which we call the hus box, which is predicted to locate in a loop where it may be available for functional interactions. the n- and c-terminal ends of bigs and gbfs are more variable, including an unusual enrichment in asp/glu or pro residues in some members. a specific feature of big members is that their c-terminus is in general less variable than that of gbfs, and is predicted with a significant amount of secondary structures. in contrast to the predicted structural domains, the intervening regions are highly variable in length and do not yield aligned sequences. analysis of their amino-acid composition reveals a paucity of hydrophobic residues which is predicted to associate with an essentially unfolded conformation, suggesting that they act as linkers to tether the functional domains together. a unnamed sequences are designated by their ncbi accession number, agi locus numbers for at and tigr model temporary ids for os. big and gbf subfamily members are in normal and bold characters respectively, except for pt members which have not been assigned to either subfamily. species abbreviations are: ag, anopheles gambiae; ce, caenorhabditis. elegans; dm, drosophila melanogaster; hs, homo sapiens; rn, rattus norvegicus; ca, candida albicans; nc, neurospora crassa; sc, saccharomyces cerevisiae; sp, schizosaccharomyces pombe; at, arabidopsis thaliana; os, oryza sativa; pt, parameciumtetraurelia). to further investigate the predicted organization of bigs and gbfs in conserved helical domains connected by variable linkers, splicing patterns of human bigs and gbfs were analyzed in the large number of cdnas and ests in the databases that correspond to gbf/big transcripts. this revealed the use of alternate splice donor and acceptor sites predicted to yield proteins with insertions and deletions ranging from to residues, and a number of splice variants arising from exon skipping. strikingly, all observed sequence variations occur in regions identified as linkers between conserved domains. together with our domain analysis, this suggests that splicing at non-canonical exon/intron boundaries is only tolerated in regions of the protein where the impact upon folding of domains with essential function would be minimal. a all changes were expressed relative to the reference sequence stored under accession number nm_, nm_ and nm_. b all variants are supported by one or more cdna/ests as detailed in the aceview for each gene that can be obtained at. evolution of bigs and gfbs from a common ancestor combined, our analysis reveals that the big and gbf subfamilies share the same overall domain organization, and are likely to descend from a common ancestor gene that duplicated first to form the big and gbf groups, and again within these groups to yield species-specific big and gbf members. these two subfamilies can therefore be unified as a higher order arfgef subfamily, from which unrooted phylogenetic trees can be built. unlike previous phylogenetic analysis which compared arfgefs based on their sec domains after diverging non-catalytic regions have been trimmed, our trees were established from the simultaneous alignment of all conserved domains, excluding variable linkers. the same tree topology was obtained with both neighbor-joining and maximum likelihood methods, and was retained using any one of the new conserved domains alone. bootstrap analysis strongly supported this topology for most branches. only a few small branches located at the base of the groups were found in less than 60% of the trials in one of the two methods, but this never occurred with both methods simultaneously. the tree topology strongly suggests that in most organisms, gbg members sort in separate branches, corresponding to their classification in bigs and gbfs. remarkably, our annotation of sec7-containing proteins in the genome of paramecium reveals the first departure from this distribution, as all gbgs in this species are located in a single branch, which is closer to the bigs. this unexpected tree topology may indicate that alveolates diverged from animals/fungi and plants before the duplication of an ancestral gbg into the big and gbf, and that gbgs in that organism are representative of this ancestral gene. alternatively, duplication may have been followed by loss of gbf genes. current knowledge of the phylogenetic branching of alveolates relative to the plants and animal/fungi branches does not permit resolution between those two possibilities. gbgs in plants: refining the functional evolution of bigs and gbfs in fungi and mammals, bigs and gbfs are represented by only one or two members, whose functions in vesicular trafficking at the golgi within each group appear largely overlapping. in contrast, plants encode a large number of gbgs in both the big and gbf branches but lack other arfgefs. in arabidopsis, none of the gbgs map to duplicated chromosomes where identical functions may be encoded. in addition, comparative analysis with the rice genome nearing completion identifies at least five branches each represented by one rice and one or two arabidopsis homologs. this correspondence between two highly divergent plant species indicates that gbgs diversified early during plant evolution, probably reflecting functional specialization along with the establishment of plant multicellularity. while gnom has a plant-specific function in recycling plasma-membrane proteins needed for cell-cell communication and cell polarity establishment, possibly closer to the function of efa or cyh subfamilies in metazoans, other plant gbgs are expected to fulfill the presumed ancient function of regulating golgi trafficking exemplified by mammalian and yeast gbgs. comparison of orthologous pairs in plants further reveals that they have different sensitivities to brefeldin a as predicted from the sequences of the binding site of the drug carried by the sec domain. this observation clearly illustrates that differences in outcome following brefeldina treatment may not reflect differences in underlying molecular mechanisms, but instead simply reflect neutral sequence differences at the sec domains between species. in particular, not all bigs may be bfa-sensitive or gbfs bfa-resistant, unlike suggested by their original nomenclature. a novel arfgef subfamily in alveolates a remarkable evolutionary feature of arfgefs is that while gbgs seem to be ubiquitous to all eukaryotes, fungi and animals kingdoms evolved their own arfgefs subfamilies unrelated to those of the other kingdoms. we thus addressed the question of whether paramecium, which has a large number of gbgs but appears to lack the specialization into the big and gbf subgroups, has the same arfgef distribution as plants or features a second arfgef subfamily. we thus searched the newly sequenced genome from paramecium tetraurelia and the available alveolate genomes from cryptosporidium parvum and tetrahymena thermophila for additional sec7-containing proteins. this identified a novel putative arfgef subfamily characterized by the association of the sec domain with a tbc domain, which was found only in the protists kingdom. the tbc domain is predicted to carry a gap activity towards small g proteins of the rab family, suggesting a potential crosstalk between rab and arf pathways. such a relationship between these two small g proteins families, which are major regulators of membrane traffic, would not be unprecedented, as for example the syt arfgef gene was identified in yeast by its genetic interactions with rab proteins in the exocytic pathway. interestingly, alveolates have specialized exocytic pathways based on a membrane organelle lying beneath the plasma membrane, the trichocyst, where this unique arfgef family may potentially function.  a conserved scenario for the activation of arf proteins by their gefs? the identification of a conserved modular architecture in all gbg subfamily members suggests that the mechanistic basis for their activation of arf is likely to follow a similar scenario. candidate functions for the conserved domains include oligomerization, the collection of input signals, membrane localization, regulation of the exchange activity, scaffolding of arf proteins to their downstream effectors, not excluding signaling to partners outside the arf pathways. dimerization has been reported in the big subgroup for big which forms heterodimers with the highly homologous big arfgef, and in the gbf subgroup for gnom, which forms homodimers. the conservation of the dcb domain in gbgs, which is responsible for the dimerization of gnom, suggests that such a dimerization function may be general to this domain in gbgs. another unresolved issue is the conservation of the cellular partners effecting the functions associated with the conserved domains. our identification of an almost invariant motif in the hus domain argues in favor of this domain interacting with a conserved partner. however, the ancient divergence into the big and gbf groups and their subsequent divergence into species-specific members suggest that specialized requirements are likely to have evolved in most organisms, possibly yielding less conserved partners outside the sec and hus domains. finally, whereas in plants all arfgefs are predicted to function according to the scheme defined by the conserved domains, other species have additional arfgef subfamilies with a modular architecture unrelated to that of the gbg subfamily. it is not known to what extent the gbg's scenario for arf activation will also apply to non-gbgs arfgefs, acting alone or in association with protein partners. in the case of the gbgs, our definition of the structural homology domains as reported here should now provide a robust background for future investigations of their interactions and functions. 
based on sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from paramecium, we have investigated the domain architecture of large arfgefs of the big and gbf subfamilies, which are involved in golgi traffic. small g proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors that stimulate the exchange of the tightly bound gdp nucleotide by gtp. in the case of small g proteins of the arf subfamily, which are major regulators of membrane trafficking, gefs form a heterogeneous family whose only common characteristic is the well-characterized sec catalytic domain. we also report a novel arfgef subfamily with a domain organization so far unique to alveolates, which we name tbs. the catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. in contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains. multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. our analysis unifies the big and gbf subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific arfgefs have subsequently evolved.
 small g proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors that stimulate the exchange of the tightly bound gdp nucleotide by gtp. the catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. in the case of small g proteins of the arf subfamily, which are major regulators of membrane trafficking, gefs form a heterogeneous family whose only common characteristic is the well-characterized sec catalytic domain. in contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown.  based on sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from paramecium, we have investigated the domain architecture of large arfgefs of the big and gbf subfamilies, which are involved in golgi traffic. multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. we also report a novel arfgef subfamily with a domain organization so far unique to alveolates, which we name tbs.  our analysis unifies the big and gbf subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific arfgefs have subsequently evolved. our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains. 