BACKGROUND
in all eukaryotic cells, intra-cellular trafficking connects the cell with its environment by the orderly transport of membranes and proteins via the exocytic and endocytic pathways.
in the exocytic pathway, proteins destined to be secreted or presented on the plasma membrane  are transported from the endoplasmic reticulum , through the golgi apparatus, to the pm.
in the endocytic pathway, proteins from the environment or the pm are shuttled via a set of endosomes to lysosomes.
the machinery and the mechanisms of intra-cellular trafficking are highly conserved among all eukaryotes , and some protein components are also conserved between the various steps of the pathways   <cit> .
ypt/rab gtpases are key regulators of this protein trafficking.
they are conserved both within a single genome between transport steps and across large phylogenetic distances  <cit> .
saccharomycescerivisiae cells contain  <dig> ypts, whereas human cells have ~ <dig> rabs  <cit> .
in s. cerivisiae, ypt <dig> and the functional pair ypt31/ <dig> regulate entry into and exit from the golgi, respectively  <cit> .
the mammalian rab1a and rab1b share ~70% identity with s. cerivisiae ypt <dig>  and rab1a can replace ypt <dig> in yeast knockout cells  <cit> .
the mammalian rab11a, rab11b and rab <dig> share ~60% identity with s. cerivisiae ypt31/ <dig> and regulate the same transport steps: exit from the golgi and endosome-to-golgi transport  <cit> .
ypt/rabs are activated by specific nucleotide exchangers, called guanine nucleotide exchange factors .
gefs for different ypt/rab paralogs do not share sequence similarity and are therefore harder to identify.
in s. cerivisiae, the multi-subunit complex trapp was identified as the gef for both ypt <dig>  <cit> , and ypt31/ <dig>  <cit> .
trapp is a modular complex that exists in two forms: trappi and trappii  <cit> .
we recently showed that trappi acts as a ypt <dig> gef, whereas trappii functions as a ypt31/ <dig> gef  <cit> .
the s. cerivisiae trappi was shown to function in er-to-golgi transport.
it contains seven subunits that co-precipitate as a ~ <dig> kda complex from yeast cell lysates  <cit> .
there is considerable evidence for the structural and functional conservation of the trappi complex  from fungi to animals  <cit>  .
s. cerivisiae subunit
bet3
trs31
trs33
bet5
trs20
trs23
trs85/gsg1
trs120
trs130
trs65/kre11
<dig> information from incyte genomic and sgd databases, 7/3/06
<dig> bet <dig> and bet <dig> families was defined based on sequence and structure similarities  <cit> .
trappii is a ~ <dig> kda complex that contains three subunits in addition to those of trappi: two large, essential subunits , trs <dig> and trs <dig>  and one small, non-essential subunit, trs <dig>  <cit> .
in yeast, trs <dig> was implicated in late golgi transport, whereas trs <dig> was suggested to function in endosome-to-golgi transport  <cit> .
the conservation of the trappii complex is less clear than that of trappi  <cit> .
blast analyses show that the closest mammalian homologues of trs <dig> and trs <dig>  nibp and tmem <dig>  respectively, share a ~20% identity with the s. cerivisiae proteins over about a third of the protein  as compared to 23–34% identity for five of the seven trappi-specific proteins and 56% for bet <dig> .
only tmem <dig> was shown to co-precipitate with the human trapp complex  <cit> , and there is no functional evidence for a role for either of these proteins in protein trafficking.
instead, nibp and tmem <dig> were implicated in very different functions, nf-kappab signaling and as a membrane channel, respectively  <cit> .
there is no obvious mammalian ortholog for trs <dig>  however, current evidence supports a role for trs <dig> in the s. cerivisiae trappii complex.
first, deletion of trs <dig> is synthetic lethal with deletion of the non-essential trappi subunit, trs <dig>  this synthetic lethality can be rescued by over-expression of ypt <dig>  <cit> .
in addition, trs65/kre <dig> was shown to function in cell-wall biogenesis  <cit> , a process that is dependent on protein transport.
because of the low sequence conservation by blast analysis of the trappii-specific subunits , and the discrepancy between their suggested functions in fungal and mammalian cells, it is important to establish any evolutionary conservation more precisely.
here, we show a phylogenetic analysis of the trappii-specific subunits.
we found that nearly every fully sequenced eukaryotic proteome present in the ncbi non-redundant protein database  contains one trs <dig> and one trs <dig> homolog.
we found three exceptions, which are noted in results.
in contrast, trs <dig> is conserved among many, but not all, unicellular eukaryotes, and not in any fully sequenced multi-cellular proteome.
the conservation of trs <dig> and trs <dig> is supported by conserved predicted secondary structure of whole proteins, and conserved domains that include highly conserved amino acids, which should also help in future functional analysis.
in addition, our analysis suggests that neither trs <dig> nor tmem <dig> is a trans-membrane protein, arguing against the suggestion that tmem <dig> functions as a membrane channel.
understanding the function of the human homologues is important, because they are candidates for several human disorders .
RESULTS
phylogenetic trees of trappii-specific subunits
we searched the non-redundant protein ncbi database on 07/ <dig> for sequences related to the three s. cerivisiae trappii-specific subunits: trs <dig>  trs <dig>  and trs <dig> .
we identified  <dig> trs120-related protein sequences and  <dig> trs130-related protein sequences.
we identified no more than one ortholog for each of these proteins in all examined genomes from fungi to vertebrates.
in the  <dig> fully and  <dig> largely sequenced genomes, we always identified exactly one copy.
we found sixteen trs65-related sequences, also no more than one per genome, but only in fungi.
further, even some fully sequenced fungi  lack any recognizable ortholog of this protein .
in addition to the sequences found in the ncbi database at the time of our search, we also looked at a  <dig> other largely completed genomes  that were at varying stages of assembly and annotation as of december  <dig>  in spite of the incomplete nature of these projects, we still found trs <dig> and trs <dig> in  <dig> genomes, and trs <dig> in  <dig> genomes.
we show these results in table s1-b .
there are a few interesting exceptions to our observations of the universality of trs120- and trs130-related sequences in eukaryotes.
the three eukaryotic genomes that did not contain trs <dig> and trs <dig> are: the fungus encephalitozoon ecuniculi, the amoebazoan entamoeba histolytica and the archaeplastid cyanidioschyzon merolae.
it seems reasonable to suggest that the absence of trs <dig> and trs <dig> from the proteome of the first organism can be explained by the fact that it is an obligate intracellular parasite that might lack some essential cellular machinery.
in agreement, three essential trappi subunits are also missing in its genomes .
e, histolytica cmerolae, however, are well-sequenced genomes, the latter is a red alga with a very small genome.
interestingly, the two genomes are also missing one essential trappi subunit each .
if the absence of trapp subunits in these organisms is confirmed, it might shed light on the evolution of the intra-cellular trafficking machinery.
the full-length protein sequences from the ncbi non-redundant  database were used to construct un-rooted phylogenetic trees for each trappii-specific subunit employing programs described in methods.
we used two different methods to construct the trees: paup's distance method, and phyml's maximum likelihood  method.
both methods yielded essentially identical topologies, except for branches near the center of the tree representing far-flung subunit-family members.
only branch lengths differed significantly between the two methods, with the ml method giving consistently ~ <dig> fold longer branch lengths.
because we could estimate branch retention probabilities using paup, we show just the paup method results in figure  <dig>  the reliability of each branch position was assessed by distance-based bootstrap analysis and indicated by red  to green  colors.
in almost all cases branches were maintained in almost all bootstrap trials, excepting only those close to the center of the tree, which reflect the details of ancient divergence between phylogenetic groups.
the colored pie sections delineate groups of proteins in accord with tree branching and taxonomic relationships.
there are seven basic groups: animals , plants , slime molds , and four fungal  groups saccharomycetes , pezizimycotina , basidiomycota , and schizosaccharomycetes .
grouping in the tree corresponds in almost all cases to recognized taxonomic groupings  <cit> .
the sole exception was yarrowia lipolytica , which is a saccharomycete, but falls  with the pezizomycotinas on the trs <dig> tree.
multiple alignment analysis of trappii-specific subunits
multiple alignments and the domain structure of the trappii-specific subunits are shown in figures  <dig>   <dig>   <dig> and figures s1-s <dig> .
domains were defined by inspection of the raw alignment and figures s1-s <dig>  highly conserved  amino acids in the alignment  are shown at the bottom of the diagram as short vertical lines, and are detailed in the corresponding supplementary tables .
there is a number of particularly interesting hc amino acids, including those that are invariant and rare  or invariant and potentially catalytic .
these hc amino acids are indicated with asterisks in the multiple alignment and in the supplemental tables .
finally, conserved domains, as indicated by alignment strength and hc amino acids are boxed  and the alignment of these conserved domains from representative organisms is shown in figures  <dig>   <dig>   <dig>
multiple alignment analysis of trs120-related proteins is shown in figures  <dig> and s <dig>  this figure shows five well-conserved domains across the length of trs <dig>  each characterized by high-quality sequence similarities and by the presence of hc amino acids in the alignment.
these domains vary in size from  <dig> to  <dig> amino acids.
larger-scale sequence conservation and hc amino acids agree with one another: hc amino acids  are found only in the otherwise conserved regions
trs <dig> is essential for s. cerivisiae cell viability.
however, truncation of the c-terminal third of trs <dig> in s. cerivisiae , which includes part of conserved domain iv and all of domain v, results in a temperature-sensitive growth phenotype.
deletions further n-terminal of this point are lethal  <cit> .
therefore, we predict that domains i, ii, and iii and part of iv are important for the essential interactions of trs <dig>
multiple alignment analysis of trs130-related proteins is shown in figures  <dig> and s <dig>  in this figure, the worms caenorhabditiselegans and caenorhabditisbriggsiae are slightly different in that they share a c-terminal deletion.
alignment and hc amino acids show three trs <dig> domains ranging from 33– <dig> amino acids.
we found no significant hc amino acids  outside of these domains.
domains i and ii contain many highly unusual hc amino acids.
consequently, we predict that these domains are critical for essential interactions or functions of trs <dig>
domain iii is unlikely to be strongly important.
it is shorter and contains no significant hc amino acids.
truncation c-terminal to the arrow shown in this domain in s. cerivisiae results in a temperature-sensitive growth phenotype  <cit> .
while it is true that this domain might be important for the stability of the trs <dig> protein, based on the low level of the protein in mutant cells  <cit> , it might not be true for all organisms, since the two worm species share a c-terminal truncation of trs <dig>  that includes part of trs130-ii and all of trs130-iii.
multiple alignments of trs65-related proteins are shown in figures  <dig> and s <dig>  the protein is common for saccharomyceta and pezizomycotina subphyla , but not all fungi.
table s <dig>  shows that trs <dig> also occurs in at least two other non-fungal unicellular eukaryotes.
since trs <dig> is only found in some fungi, we used all three fungal groups shown in figure  <dig> that contain trs <dig> sequences.
hc amino acids  and alignment qualities show that these organisms share four conserved domains varying in size 40– <dig> amino acids.
although no mutagenesis data exists for these proteins, there are some highly unusual amino acids in domains ii, iii, and iv.
therefore we suggest that they are important for trs <dig> function.
the predicted secondary structure of trs <dig> and trs <dig> is conserved
we suspected that protein secondary structure was conserved in addition to its primary sequence.
since no crystal data is currently known for the three trappii-specific subunits, we predicted their secondary structures using prof  <cit> .
this program employs multiple sequence alignments to predict the helical, beta-sheet, or coiled-coil nature of each position in the primary sequence of a group of proteins.
we examined these predictions for all the trs <dig>  trs <dig>  and trs <dig> proteins, using the clustal alignment employed to construct the trees and alignments in figures  <dig>   <dig>   <dig>   <dig> for the input to prof. color-coded bars, representing the predicted secondary structures  are shown in figures  <dig>  s <dig> and s <dig>  along with the conserved domains and hc amino acids described in figures  <dig>   <dig>   <dig>  to be sure that differences among evolutionary groups were in agreement with these results, we also chose three groups of species that contain a large number of sequenced proteins  and that are evolutionary far from each other: animals , and two fungi groups, saccharomycetes , and pezizomycotina , and show these results in figures s <dig> and s <dig>
surprisingly, the predicted secondary structure of trs <dig> and trs <dig> is highly conserved not only in domains that are conserved for primary structure, but also along the whole sequence of these proteins, even where there is low sequence identity between the groups .
therefore, the predicted secondary structure of these two proteins is conserved better than their primary structure.
similar analysis of trs <dig> among the saccharomycetes and pezizomycotina fungal groups shows conservation of predicted secondary structure among members of the same group.
however, these predictions do not agree well between the two subphyla .
this suggests that unlike trs <dig> and trs <dig>  the interactions, and perhaps the specific functions, of trs <dig> are not well conserved even among all fungi.
for the three protein families, we searched all studied sequences  for potential structural motifs using smart  <cit> .
we found some isolated smart hits, but none were consistent across a whole group .
notably, none of the isolated hits overlapped the conserved domains shown in figs  <dig>   <dig>   <dig>  portions of the conserved domains of trappii-specific subunits seem good prospects for inclusion in future releases of such motif-finding engines.
trs <dig> is a membrane-associated protein
based on sequence analysis, the s. cerevisiae trs <dig> and its human homolog were suggested in  <dig> to contain trans-membrane domains .
in fact, the human homologue was named tmem <dig>  for trans-membrane epilepsy myoclonus  <cit> .
however, the yeast trapp complex is only membrane associated, because it can be removed from membranes by salt but not by detergent  <cit> , even though this was not shown specifically for trs <dig>
because the s. cereviciae trs <dig> contains two putative trans-membrane domains  <cit> , we wished to determine whether this protein is a trans-membrane or a membrane-associated protein.
trs <dig> isolated from yeast cell lysates is found in the p <dig>  particulate fraction of cell lysates , which floats with membranes on an optiprep gradient .
if trs <dig> were a trans-membrane protein, it could be extracted from the membrane by detergent, but not by salt.
however, treatment of the p <dig> fraction with salt, and not with detergent, yields soluble trs <dig> .
therefore, the yeast trs <dig> behaves as a membrane-associated protein, and not as a trans-membrane protein.
these results imply that the observed hydrophobic domains do not function as tmds, but rather are important for the proper folding or interactions of trs <dig>
this idea that tmem <dig> and trs <dig> are trans-membrane proteins was based on sequence analysis, so we revisited this sequence analysis for the human tmem <dig>  previously, sequence analysis of the human tmem <dig> suggested that the protein contained two  or multiple  <cit>  trans-membrane domains .
we tested the human tmem <dig> sequence for putative tmds using sosui  <cit>  and smart  <cit>  on 6/ <dig>  and no tmds were detected by these programs.
note that smart uses tmhmm <dig>  to predict tmds, a method which has been shown to be among the most accurate at such tasks  <cit> .
we suspect that the tmd-detecting algorithms have become more discriminating in the intervening years and suggest that, like the s. cerivisiae trs <dig>  the human protein is a membrane-associated protein, not a trans-membrane protein as suggested by its original name.
discussion
we show here that the trappii-specific subunits, trs <dig> and trs <dig>  are conserved from fungi to animals.
this conclusion is based on sequence and predicted secondary structure analyses.
multiple alignments analysis reveals conserved domains with highly conserved amino acids in each protein family.
predicted secondary structure analysis reveals conservation in the clustal/hc-derived domains as well as in the intervening sequences.
the conservation of the predicted secondary structure and the fact that the secondary structure of these proteins is conserved better than their primary structure supports the analysis presented here.
this suggests that trappii, like trappi, is a conserved complex, and that the function of both trapp complexes, in the regulation of trafficking through the golgi apparatus, is also conserved.
in contrast, the non-essential subunit, trs <dig>  is conserved only between some fungi.
the trs65-related proteins also share conserved domains with highly conserved amino acids.
however, their secondary structures as predicted by prof are not highly conserved between two evolutionary distant fungal groups, saccharomycetes  and pezizomycotina .
the fact that trs <dig> is not essential for viability in s. cerivisiae, and is only weakly conserved among some unicellular eukaryotes, suggests that trs <dig> has a fungi-specific function.
indeed, it was implicated in s. cerivisiae in cell-wall biogenesis  <cit> .
alternatively, other organisms may have a functionally similar subunit that does not share sequence similarity with trs <dig>
the s. cerivisiae trapp complexes act as activators, gefs, for the golgi ypts  <cit> .
in general, ypt/rab gefs are large protein complexes  <cit> , and there is no similarity between gefs for different ypt/rab paralogs.
this suggests the recruitment of ypt/rab gefs from divergent families.
the reason for this might stem from the divergence of factors that regulate these gefs, about which, very little is currently known.
thus, the diversity of the ypt/rab gefs suggests that gefs that function in different cellular compartments are regulated by diverse upstream factors.
alternatively, ypt/rab gefs might have diverged beyond recognition, which is hard to believe in light of the conservation of the whole trafficking machinery.
unlike the ypt/rabs, for which the number between fungi and mammalian cells increases by about seven fold, the number of trappi and trappii subunits per genome remains constant through evolution .
this ratio of rab/gef suggests that in mammalian cells trapp acts as a gef for more rabs than in s. cerivisiae, perhaps for whole groups of ypt1/rab1- and ypt31/rab11-related proteins  <cit> .
alternatively, more animal gefs evolved during evolution that do not share sequence similarity with the fungal gefs.
domains and functions
the conservation of the primary and the secondary structures of trs <dig> and trs <dig> suggests that they cooperatively execute an important cellular function.
we propose that conservation of secondary structure of the well-conserved domains  probably reflects conservation of catalytic functions and/or protein-protein interactions of these domains.
the latter are known to place constraints on the divergence of residues in contact surfaces.
in contrast, conservation of the secondary structure of the intervening sequences might be required for the three-dimensional orientation of the well-conserved domains.
we have previously suggested a role for the trapp complex in the coordination of entry into and exit from the golgi  <cit> , and for the trappii-specific subunits, in the specificity switch of the gef activity of trapp from ypt <dig> to ypt 31/ <dig> .
based on our functional analysis of the s. cerivisiae trs <dig> and trs <dig>  we propose a number of protein-protein interactions for the two essential trappii-specific subunits.
we predict at least two protein-protein interactions for trs <dig>  with trs <dig> and with trappi  <cit> .
the four trs <dig> conserved domains , which are essential for s. cerevisiae, are candidates for these interactions.
for trs <dig>  we predict at least two protein-protein interactions and a possible catalytic role in the ypt <dig> gef activity of trappii  <cit> .
the two conserved trs <dig> domains that are essential for s. cerevisiae viability, i and ii, are candidates for these functions.
the identification of trs120- and trs130-conserved domains and hc amino acids will allow future functional analysis of the separate domains of these large proteins.
proposed roles for the mammalian trs120- and trs130-related proteins
we suggest that the role of the two trapp complexes as ypt/rab gefs and their coordination function are conserved from fungi to animals.
however, the mammalian orthologs of trs <dig> and trs <dig> were implicated in other cellular processes.
the mammalian trs120-related protein, nibp was implicated in nf-kappab signaling  <cit> .
while the suggested functions of the fungal and mammalian orthologs of trs <dig> do not overlap, they do not contradict each other, and it is possible that trafficking through the golgi in mammalian cells is regulated by nf-kappab signaling.
in contrast, the mammalian trs130-related protein, tmem <dig> , was implicated in a function distinct from protein trafficking.
while the s. cerevisiae trapp, including trs <dig>  is a membrane associated complex, tmem <dig> was suggested to be a trans-membrane protein and to function as a membrane channel  <cit> .
our analyses suggest that both the s. cereviciae trs <dig>  and the human tmem <dig>  are not trans-membrane proteins.
based on our alignment and predicted secondary structure analyses, we propose that the function of trs <dig> as a specificity switch for trapp's gef activity is conserved.
these ideas can now be tested.
trs <dig> and trs <dig> and human disorders
regulation of critical steps of the exocytic pathway is bound to be crucial in understanding the basis of every disease that is connected with secretion of a substance or presentation of a receptor on the plasma membrane.
indeed, several trappi subunits were implicated in human diseases.
for example, a missense mutation in human bet5/trappc <dig> results in the expression of antigenic peptides in melanoma  <cit> ; and the sedl/trs <dig> gene is responsible for sedt, an xlinked skeletal disorder  <cit> .
the two mammalian orthologs of trs <dig> and trs <dig> were also implicated in human disorders: nibp, the trs <dig> ortholog, was implicated in neurogenerative disorders, based on its connection to nf-kappab signaling  <cit> .
the trs <dig> ortholog, tmem <dig>  is a candidate for several human disorders, including certain types of epilepsy, autoimmune, and holoprosencephaly disorders, based on genetic linkage studies mapping genes responsible for these disorders to a chromosome region that includes tmem <dig>  <cit> .
therefore, resolving the probably conserved function of the mammalian nibp and tmem <dig> proteins should help elucidate the basis for these disorders.
CONCLUSIONS
trapp is a modular protein complex that regulates entry into and exit from the golgi, which is a cellular compartment central to multiple trafficking pathways.
trappi, the complex that functions in the entry to the golgi, is highly conserved.
we have recently shown that the two yeast essential trappii-specific subunits are required for changing the activity of trappi to that of trappii  <cit> .
here, we explored the conservation of the trappii-specific subunits.
we show that the primary and secondary structures of the two essential subunits, trs <dig> and trs <dig>  are conserved from yeast to man, whereas the non-essential trs <dig> is conserved only among some unicellular eukaryotes.
we suggest that the role of the human orthologs of trs <dig> and trs <dig>  nibp and tmem <dig>  respectively, in the regulation of intra-cellular trafficking is also conserved.
conserved domains and highly conserved amino acids in these domains should help future functional studies of these proteins in higher eukaryotes.

 in all eukaryotic cells, intra-cellular trafficking connects the cell with its environment by the orderly transport of membranes and proteins via the exocytic and endocytic pathways. in the exocytic pathway, proteins destined to be secreted or presented on the plasma membrane are transported from the endoplasmic reticulum, through the golgi apparatus, to the pm. in the endocytic pathway, proteins from the environment or the pm are shuttled via a set of endosomes to lysosomes. the machinery and the mechanisms of intra-cellular trafficking are highly conserved among all eukaryotes, and some protein components are also conserved between the various steps of the pathways. ypt/rab gtpases are key regulators of this protein trafficking. they are conserved both within a single genome between transport steps and across large phylogenetic distances. saccharomycescerivisiae cells contain ypts, whereas human cells have ~ rabs. in s. cerivisiae, ypt and the functional pair ypt31/ regulate entry into and exit from the golgi, respectively. the mammalian rab1a and rab1b share ~70% identity with s. cerivisiae ypt and rab1a can replace ypt in yeast knockout cells. the mammalian rab11a, rab11b and rab share ~60% identity with s. cerivisiae ypt31/ and regulate the same transport steps: exit from the golgi and endosome-to-golgi transport. ypt/rabs are activated by specific nucleotide exchangers, called guanine nucleotide exchange factors. gefs for different ypt/rab paralogs do not share sequence similarity and are therefore harder to identify. in s. cerivisiae, the multi-subunit complex trapp was identified as the gef for both ypt, and ypt31/. trapp is a modular complex that exists in two forms: trappi and trappii. we recently showed that trappi acts as a ypt gef, whereas trappii functions as a ypt31/ gef. the s. cerivisiae trappi was shown to function in er-to-golgi transport. it contains seven subunits that co-precipitate as a ~ kda complex from yeast cell lysates. there is considerable evidence for the structural and functional conservation of the trappi complex from fungi to animals. s. cerivisiae subunit bet3 trs31 trs33 bet5 trs20 trs23 trs85/gsg1 trs120 trs130 trs65/kre11 information from incyte genomic and sgd databases, 7/3/06 bet and bet families was defined based on sequence and structure similarities. trappii is a ~ kda complex that contains three subunits in addition to those of trappi: two large, essential subunits, trs and trs and one small, non-essential subunit, trs. in yeast, trs was implicated in late golgi transport, whereas trs was suggested to function in endosome-to-golgi transport. the conservation of the trappii complex is less clear than that of trappi. blast analyses show that the closest mammalian homologues of trs and trs nibp and tmem respectively, share a ~20% identity with the s. cerivisiae proteins over about a third of the protein as compared to 23–34% identity for five of the seven trappi-specific proteins and 56% for bet. only tmem was shown to co-precipitate with the human trapp complex, and there is no functional evidence for a role for either of these proteins in protein trafficking. instead, nibp and tmem were implicated in very different functions, nf-kappab signaling and as a membrane channel, respectively. there is no obvious mammalian ortholog for trs however, current evidence supports a role for trs in the s. cerivisiae trappii complex. first, deletion of trs is synthetic lethal with deletion of the non-essential trappi subunit, trs this synthetic lethality can be rescued by over-expression of ypt. in addition, trs65/kre was shown to function in cell-wall biogenesis, a process that is dependent on protein transport. because of the low sequence conservation by blast analysis of the trappii-specific subunits, and the discrepancy between their suggested functions in fungal and mammalian cells, it is important to establish any evolutionary conservation more precisely. here, we show a phylogenetic analysis of the trappii-specific subunits. we found that nearly every fully sequenced eukaryotic proteome present in the ncbi non-redundant protein database contains one trs and one trs homolog. we found three exceptions, which are noted in results. in contrast, trs is conserved among many, but not all, unicellular eukaryotes, and not in any fully sequenced multi-cellular proteome. the conservation of trs and trs is supported by conserved predicted secondary structure of whole proteins, and conserved domains that include highly conserved amino acids, which should also help in future functional analysis. in addition, our analysis suggests that neither trs nor tmem is a trans-membrane protein, arguing against the suggestion that tmem functions as a membrane channel. understanding the function of the human homologues is important, because they are candidates for several human disorders.  phylogenetic trees of trappii-specific subunits we searched the non-redundant protein ncbi database on 07/ for sequences related to the three s. cerivisiae trappii-specific subunits: trs trs and trs. we identified trs120-related protein sequences and trs130-related protein sequences. we identified no more than one ortholog for each of these proteins in all examined genomes from fungi to vertebrates. in the fully and largely sequenced genomes, we always identified exactly one copy. we found sixteen trs65-related sequences, also no more than one per genome, but only in fungi. further, even some fully sequenced fungi lack any recognizable ortholog of this protein. in addition to the sequences found in the ncbi database at the time of our search, we also looked at a other largely completed genomes that were at varying stages of assembly and annotation as of december in spite of the incomplete nature of these projects, we still found trs and trs in genomes, and trs in genomes. we show these results in table s1-b. there are a few interesting exceptions to our observations of the universality of trs120- and trs130-related sequences in eukaryotes. the three eukaryotic genomes that did not contain trs and trs are: the fungus encephalitozoon ecuniculi, the amoebazoan entamoeba histolytica and the archaeplastid cyanidioschyzon merolae. it seems reasonable to suggest that the absence of trs and trs from the proteome of the first organism can be explained by the fact that it is an obligate intracellular parasite that might lack some essential cellular machinery. in agreement, three essential trappi subunits are also missing in its genomes. e, histolytica cmerolae, however, are well-sequenced genomes, the latter is a red alga with a very small genome. interestingly, the two genomes are also missing one essential trappi subunit each. if the absence of trapp subunits in these organisms is confirmed, it might shed light on the evolution of the intra-cellular trafficking machinery. the full-length protein sequences from the ncbi non-redundant database were used to construct un-rooted phylogenetic trees for each trappii-specific subunit employing programs described in methods. we used two different methods to construct the trees: paup's distance method, and phyml's maximum likelihood method. both methods yielded essentially identical topologies, except for branches near the center of the tree representing far-flung subunit-family members. only branch lengths differed significantly between the two methods, with the ml method giving consistently ~ fold longer branch lengths. because we could estimate branch retention probabilities using paup, we show just the paup method results in figure the reliability of each branch position was assessed by distance-based bootstrap analysis and indicated by red to green colors. in almost all cases branches were maintained in almost all bootstrap trials, excepting only those close to the center of the tree, which reflect the details of ancient divergence between phylogenetic groups. the colored pie sections delineate groups of proteins in accord with tree branching and taxonomic relationships. there are seven basic groups: animals, plants, slime molds, and four fungal groups saccharomycetes, pezizimycotina, basidiomycota, and schizosaccharomycetes. grouping in the tree corresponds in almost all cases to recognized taxonomic groupings. the sole exception was yarrowia lipolytica, which is a saccharomycete, but falls with the pezizomycotinas on the trs tree. multiple alignment analysis of trappii-specific subunits multiple alignments and the domain structure of the trappii-specific subunits are shown in figures and figures s1-s. domains were defined by inspection of the raw alignment and figures s1-s highly conserved amino acids in the alignment are shown at the bottom of the diagram as short vertical lines, and are detailed in the corresponding supplementary tables. there is a number of particularly interesting hc amino acids, including those that are invariant and rare or invariant and potentially catalytic. these hc amino acids are indicated with asterisks in the multiple alignment and in the supplemental tables. finally, conserved domains, as indicated by alignment strength and hc amino acids are boxed and the alignment of these conserved domains from representative organisms is shown in figures multiple alignment analysis of trs120-related proteins is shown in figures and s this figure shows five well-conserved domains across the length of trs each characterized by high-quality sequence similarities and by the presence of hc amino acids in the alignment. these domains vary in size from to amino acids. larger-scale sequence conservation and hc amino acids agree with one another: hc amino acids are found only in the otherwise conserved regions trs is essential for s. cerivisiae cell viability. however, truncation of the c-terminal third of trs in s. cerivisiae, which includes part of conserved domain iv and all of domain v, results in a temperature-sensitive growth phenotype. deletions further n-terminal of this point are lethal. therefore, we predict that domains i, ii, and iii and part of iv are important for the essential interactions of trs multiple alignment analysis of trs130-related proteins is shown in figures and s in this figure, the worms caenorhabditiselegans and caenorhabditisbriggsiae are slightly different in that they share a c-terminal deletion. alignment and hc amino acids show three trs domains ranging from 33– amino acids. we found no significant hc amino acids outside of these domains. domains i and ii contain many highly unusual hc amino acids. consequently, we predict that these domains are critical for essential interactions or functions of trs domain iii is unlikely to be strongly important. it is shorter and contains no significant hc amino acids. truncation c-terminal to the arrow shown in this domain in s. cerivisiae results in a temperature-sensitive growth phenotype. while it is true that this domain might be important for the stability of the trs protein, based on the low level of the protein in mutant cells, it might not be true for all organisms, since the two worm species share a c-terminal truncation of trs that includes part of trs130-ii and all of trs130-iii. multiple alignments of trs65-related proteins are shown in figures and s the protein is common for saccharomyceta and pezizomycotina subphyla, but not all fungi. table s shows that trs also occurs in at least two other non-fungal unicellular eukaryotes. since trs is only found in some fungi, we used all three fungal groups shown in figure that contain trs sequences. hc amino acids and alignment qualities show that these organisms share four conserved domains varying in size 40– amino acids. although no mutagenesis data exists for these proteins, there are some highly unusual amino acids in domains ii, iii, and iv. therefore we suggest that they are important for trs function. the predicted secondary structure of trs and trs is conserved we suspected that protein secondary structure was conserved in addition to its primary sequence. since no crystal data is currently known for the three trappii-specific subunits, we predicted their secondary structures using prof. this program employs multiple sequence alignments to predict the helical, beta-sheet, or coiled-coil nature of each position in the primary sequence of a group of proteins. we examined these predictions for all the trs trs and trs proteins, using the clustal alignment employed to construct the trees and alignments in figures for the input to prof. color-coded bars, representing the predicted secondary structures are shown in figures s and s along with the conserved domains and hc amino acids described in figures to be sure that differences among evolutionary groups were in agreement with these results, we also chose three groups of species that contain a large number of sequenced proteins and that are evolutionary far from each other: animals, and two fungi groups, saccharomycetes, and pezizomycotina, and show these results in figures s and s surprisingly, the predicted secondary structure of trs and trs is highly conserved not only in domains that are conserved for primary structure, but also along the whole sequence of these proteins, even where there is low sequence identity between the groups. therefore, the predicted secondary structure of these two proteins is conserved better than their primary structure. similar analysis of trs among the saccharomycetes and pezizomycotina fungal groups shows conservation of predicted secondary structure among members of the same group. however, these predictions do not agree well between the two subphyla. this suggests that unlike trs and trs the interactions, and perhaps the specific functions, of trs are not well conserved even among all fungi. for the three protein families, we searched all studied sequences for potential structural motifs using smart. we found some isolated smart hits, but none were consistent across a whole group. notably, none of the isolated hits overlapped the conserved domains shown in figs portions of the conserved domains of trappii-specific subunits seem good prospects for inclusion in future releases of such motif-finding engines. trs is a membrane-associated protein based on sequence analysis, the s. cerevisiae trs and its human homolog were suggested in to contain trans-membrane domains. in fact, the human homologue was named tmem for trans-membrane epilepsy myoclonus. however, the yeast trapp complex is only membrane associated, because it can be removed from membranes by salt but not by detergent, even though this was not shown specifically for trs because the s. cereviciae trs contains two putative trans-membrane domains, we wished to determine whether this protein is a trans-membrane or a membrane-associated protein. trs isolated from yeast cell lysates is found in the p particulate fraction of cell lysates, which floats with membranes on an optiprep gradient. if trs were a trans-membrane protein, it could be extracted from the membrane by detergent, but not by salt. however, treatment of the p fraction with salt, and not with detergent, yields soluble trs. therefore, the yeast trs behaves as a membrane-associated protein, and not as a trans-membrane protein. these results imply that the observed hydrophobic domains do not function as tmds, but rather are important for the proper folding or interactions of trs this idea that tmem and trs are trans-membrane proteins was based on sequence analysis, so we revisited this sequence analysis for the human tmem previously, sequence analysis of the human tmem suggested that the protein contained two or multiple trans-membrane domains. we tested the human tmem sequence for putative tmds using sosui and smart on 6/ and no tmds were detected by these programs. note that smart uses tmhmm to predict tmds, a method which has been shown to be among the most accurate at such tasks. we suspect that the tmd-detecting algorithms have become more discriminating in the intervening years and suggest that, like the s. cerivisiae trs the human protein is a membrane-associated protein, not a trans-membrane protein as suggested by its original name. discussion we show here that the trappii-specific subunits, trs and trs are conserved from fungi to animals. this conclusion is based on sequence and predicted secondary structure analyses. multiple alignments analysis reveals conserved domains with highly conserved amino acids in each protein family. predicted secondary structure analysis reveals conservation in the clustal/hc-derived domains as well as in the intervening sequences. the conservation of the predicted secondary structure and the fact that the secondary structure of these proteins is conserved better than their primary structure supports the analysis presented here. this suggests that trappii, like trappi, is a conserved complex, and that the function of both trapp complexes, in the regulation of trafficking through the golgi apparatus, is also conserved. in contrast, the non-essential subunit, trs is conserved only between some fungi. the trs65-related proteins also share conserved domains with highly conserved amino acids. however, their secondary structures as predicted by prof are not highly conserved between two evolutionary distant fungal groups, saccharomycetes and pezizomycotina. the fact that trs is not essential for viability in s. cerivisiae, and is only weakly conserved among some unicellular eukaryotes, suggests that trs has a fungi-specific function. indeed, it was implicated in s. cerivisiae in cell-wall biogenesis. alternatively, other organisms may have a functionally similar subunit that does not share sequence similarity with trs the s. cerivisiae trapp complexes act as activators, gefs, for the golgi ypts. in general, ypt/rab gefs are large protein complexes, and there is no similarity between gefs for different ypt/rab paralogs. this suggests the recruitment of ypt/rab gefs from divergent families. the reason for this might stem from the divergence of factors that regulate these gefs, about which, very little is currently known. thus, the diversity of the ypt/rab gefs suggests that gefs that function in different cellular compartments are regulated by diverse upstream factors. alternatively, ypt/rab gefs might have diverged beyond recognition, which is hard to believe in light of the conservation of the whole trafficking machinery. unlike the ypt/rabs, for which the number between fungi and mammalian cells increases by about seven fold, the number of trappi and trappii subunits per genome remains constant through evolution. this ratio of rab/gef suggests that in mammalian cells trapp acts as a gef for more rabs than in s. cerivisiae, perhaps for whole groups of ypt1/rab1- and ypt31/rab11-related proteins. alternatively, more animal gefs evolved during evolution that do not share sequence similarity with the fungal gefs. domains and functions the conservation of the primary and the secondary structures of trs and trs suggests that they cooperatively execute an important cellular function. we propose that conservation of secondary structure of the well-conserved domains probably reflects conservation of catalytic functions and/or protein-protein interactions of these domains. the latter are known to place constraints on the divergence of residues in contact surfaces. in contrast, conservation of the secondary structure of the intervening sequences might be required for the three-dimensional orientation of the well-conserved domains. we have previously suggested a role for the trapp complex in the coordination of entry into and exit from the golgi, and for the trappii-specific subunits, in the specificity switch of the gef activity of trapp from ypt to ypt 31/. based on our functional analysis of the s. cerivisiae trs and trs we propose a number of protein-protein interactions for the two essential trappii-specific subunits. we predict at least two protein-protein interactions for trs with trs and with trappi. the four trs conserved domains, which are essential for s. cerevisiae, are candidates for these interactions. for trs we predict at least two protein-protein interactions and a possible catalytic role in the ypt gef activity of trappii. the two conserved trs domains that are essential for s. cerevisiae viability, i and ii, are candidates for these functions. the identification of trs120- and trs130-conserved domains and hc amino acids will allow future functional analysis of the separate domains of these large proteins. proposed roles for the mammalian trs120- and trs130-related proteins we suggest that the role of the two trapp complexes as ypt/rab gefs and their coordination function are conserved from fungi to animals. however, the mammalian orthologs of trs and trs were implicated in other cellular processes. the mammalian trs120-related protein, nibp was implicated in nf-kappab signaling. while the suggested functions of the fungal and mammalian orthologs of trs do not overlap, they do not contradict each other, and it is possible that trafficking through the golgi in mammalian cells is regulated by nf-kappab signaling. in contrast, the mammalian trs130-related protein, tmem, was implicated in a function distinct from protein trafficking. while the s. cerevisiae trapp, including trs is a membrane associated complex, tmem was suggested to be a trans-membrane protein and to function as a membrane channel. our analyses suggest that both the s. cereviciae trs and the human tmem are not trans-membrane proteins. based on our alignment and predicted secondary structure analyses, we propose that the function of trs as a specificity switch for trapp's gef activity is conserved. these ideas can now be tested. trs and trs and human disorders regulation of critical steps of the exocytic pathway is bound to be crucial in understanding the basis of every disease that is connected with secretion of a substance or presentation of a receptor on the plasma membrane. indeed, several trappi subunits were implicated in human diseases. for example, a missense mutation in human bet5/trappc results in the expression of antigenic peptides in melanoma; and the sedl/trs gene is responsible for sedt, an xlinked skeletal disorder. the two mammalian orthologs of trs and trs were also implicated in human disorders: nibp, the trs ortholog, was implicated in neurogenerative disorders, based on its connection to nf-kappab signaling. the trs ortholog, tmem is a candidate for several human disorders, including certain types of epilepsy, autoimmune, and holoprosencephaly disorders, based on genetic linkage studies mapping genes responsible for these disorders to a chromosome region that includes tmem. therefore, resolving the probably conserved function of the mammalian nibp and tmem proteins should help elucidate the basis for these disorders.  trapp is a modular protein complex that regulates entry into and exit from the golgi, which is a cellular compartment central to multiple trafficking pathways. trappi, the complex that functions in the entry to the golgi, is highly conserved. we have recently shown that the two yeast essential trappii-specific subunits are required for changing the activity of trappi to that of trappii. here, we explored the conservation of the trappii-specific subunits. we show that the primary and secondary structures of the two essential subunits, trs and trs are conserved from yeast to man, whereas the non-essential trs is conserved only among some unicellular eukaryotes. we suggest that the role of the human orthologs of trs and trs nibp and tmem respectively, in the regulation of intra-cellular trafficking is also conserved. conserved domains and highly conserved amino acids in these domains should help future functional studies of these proteins in higher eukaryotes. 
the non-essential subunit, trs is conserved only among many fungi and some unicellular eukaryotes. ypt/rab gtpases and their gef activators regulate intra-cellular trafficking in all eukaryotic cells. here, we show a phylogenetic analysis of the three trappii-specific subunits. however, we show here that the yeast trs does not function as a trans-membrane protein, and the human tmem does not contain putative tmds. multiple alignment analysis of each trappii-specific subunit revealed conserved domains that include highly conserved amino acids. the conserved domains and amino acids discovered here can be used for functional analysis that should help to resolve the differences in the assigned functions of these proteins in fungi and animals. in s. cerivisiae, the modular trapp complex acts as a gef for the golgi gatekeepers: ypt and the functional pair ypt31/ while trappi, which acts in early golgi, is conserved from fungi to animals, not much is known about trappii, which acts in late golgi and consists of trappi plus three additional subunits. currently, nibp is implicated in signaling, and tmem is suggested to have trans-membrane domains and to function as a membrane channel. the mammalian orthologs of trs and trs nibp and tmem respectively, are candidates for human disorders. one copy of each of the two essential subunits, trs and trs is present in almost every fully sequenced eukaryotic genome.
 ypt/rab gtpases and their gef activators regulate intra-cellular trafficking in all eukaryotic cells. in s. cerivisiae, the modular trapp complex acts as a gef for the golgi gatekeepers: ypt and the functional pair ypt31/ while trappi, which acts in early golgi, is conserved from fungi to animals, not much is known about trappii, which acts in late golgi and consists of trappi plus three additional subunits.  here, we show a phylogenetic analysis of the three trappii-specific subunits. one copy of each of the two essential subunits, trs and trs is present in almost every fully sequenced eukaryotic genome. moreover, the primary, as well as the predicted secondary, structure of the trs120- and trs130-related sequences are conserved from fungi to animals. the mammalian orthologs of trs and trs nibp and tmem respectively, are candidates for human disorders. currently, nibp is implicated in signaling, and tmem is suggested to have trans-membrane domains and to function as a membrane channel. however, we show here that the yeast trs does not function as a trans-membrane protein, and the human tmem does not contain putative tmds. the non-essential subunit, trs is conserved only among many fungi and some unicellular eukaryotes. multiple alignment analysis of each trappii-specific subunit revealed conserved domains that include highly conserved amino acids.  we suggest that the function of both nibp and tmem in the regulation of intra-cellular trafficking is conserved from yeast to man. the conserved domains and amino acids discovered here can be used for functional analysis that should help to resolve the differences in the assigned functions of these proteins in fungi and animals. 