Program in Evolutionary Biology, Canadian Institute for Advanced Research, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, N.S., B3H 4H7, Canada.
* Author for correspondence (e-mail: jdacks{at}is2.dal.ca )
Accepted 25 January 2002
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Summary |
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Key words: SNARE, Protist, Golgi
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Introduction |
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SNAREs have been implicated in vesicle tethering
(Ungermann et al., 2000),
docking (Ungermann et al.,
1998
), and fusion (Nickel et
al., 1999
). Some authors have suggested that SNAREs from the
minimal fusion machinery (Sollner et al.,
1993
) and are responsible for the specificity of vesicle transport
in the eukaryotic cell (McNew et al.,
2000
). Other workers downplay the importance of SNARES,
particularly in fusion (Peters et al.,
2001
) and as the minimal machinery
(Wickner and Haas, 2000
).
One class of T-snares, the syntaxins, form a clearly delineated protein
family based on primary and secondary structure
(Bennett et al., 1993).
Syntaxins have three N-terminal regulatory helices
(Parlati et al., 1999
),
denoted A, B and C, interspaced with linker regions of variable size. These
are linked to the SNARE motif (Fasshauer
et al., 1998
), a coiled-coil forming helix that generally ends in
a transmembrane spanning domain, although some syntaxins lack this anchor
(Low et al., 2000
).
Syntaxin proteins can themselves be classified into various paralog
families (Bennett et al.,
1993), each associated with either a step in the transport
pathway, or an intracellular location. There are a number of plasma membrane
(PM) localized syntaxins (syntaxins 1-4, 11, knolle, syr 1, sso 1 and -2)
collectively referred to herein as syntaxin PM homologs. Other syntaxin
paralogs may be associated with endoplasmic reticulum (ER), Golgi or
endosomes. Syntaxins have also been implicated in reassembly of Golgi
(Rabouille et al., 1998
) and
transitional ER (Roy et al.,
2000
), perhaps suggesting a role for syntaxins in maintaining
organellar stability and the identity of an intracellular compartment.
The majority of functional work on SNAREs has been performed using animal
neuronal (Bennett et al., 1992)
and fungal secretion models (Banfield et
al., 1995
; Sollner et al.,
1993
). However, with the exception of a few studies from plants
(Lauber et al., 1997
;
Leyman et al., 1999
) and a
single syntaxin from the slime mold Dictyostelium discoideum
(Bogdanovic et al., 2000
),
there is little understanding of the diversity of syntaxins among eukaryotes.
In a recent bioinformatic study we suggested that an early eukaryotic ancestor
had a relatively complex endomembrane machinery including a primitive syntaxin
(Dacks and Doolittle, 2001
).
This BLAST-based analysis used only partial and publicly available genome
sequences and so was not suited to examine syntaxin evolution in-depth. A more
phylogenetically diverse sampling of full syntaxin sequences would allow us
to: (1) determine details of events in the evolution of the syntaxin
superfamily; and (2) more effectively deduce functional constraints on
syntaxin primary structure from patterns of evolutionary conservation. In
addition, while there have been several phylogenetic classifications of
syntaxins, some failed to test the robustness of their result by resampling
(Bogdanovic et al., 2000
;
Sanderfoot et al., 2000
) and
none accounted for important variables in phylogenetic reconstruction such as
among site rate variation or invariant sites
(Bogdanovic et al., 2000
;
Sanderfoot et al., 2000
;
Wang et al., 1997
). These are
well known to have major effects on phylogenetic analyses
(Efron et al., 1996
;
Felsenstein, 1978
;
Lockhart et al., 1996
;
Yang, 1994
) and so a thorough
phylogenetic analysis of the syntaxin gene family is warranted.
We have undertaken to expand the available taxonomic diversity of syntaxin sequences using a combined bioinformatic and molecular biology approach. We have identified and sequenced seven different syntaxins from various protists and present the first rigorous molecular phylogenetic analysis of the syntaxin gene family.
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Materials and Methods |
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The syntaxins identified from the Giardia genome project were based on single pass genomic reads. It was therefore necessary to amplify the genes and confirm their double strand sequences. This was done using exact match primers GsynAXF1- TCATCGCTCCTA- GCTACG and GsynXXR2-GTACAGTGCAGCATTTGGCG for Giardia syntaxin PM and primers Gsyn7X1F-GCTCAAACTTGTC- GAAGG and Gsyn7X2R-TAAGCACAGCTCATTGCC for Giardia syntaxin 16.
5' and 3' fragments of a syntaxin 5 homolog were detected in the Trypanosoma brucei GSS database. The complete Open Reading Frame (ORF) was obtained (including the missing internal portion) by amplifying the gene from T. brucei genomic DNA using exact match primers TBS5X1F-CTCCAACTATGGTTGTAGAGC and TBS5X2R-ATTTCATTGCCTTGAGACGGC designed to the respective GSS fragments. All amplified fragments were cloned into Topo 2.1 vector (Invitrogen Carlsbad VA). Plasmids resulting from these amplifications and all ESTs with the exception of the MY-F08 clones were transformed into TOP10 cells.
The syntaxins identified from ESTs were either based on single pass or partial sequencing reads (not covering the entire ORF). The complete sequencing of each ORF was performed on an ABI 377 sequencer with two clones of each syntaxin ORF sequenced fully in both directions.
Alignments
To aid in alignment of the syntaxins, each syntaxin sequence was analyzed
using the secondary structure prediction software at the EMBL site
(www.embl-heidelberg.de/Services/index.html
). This was followed by alignment of the predicted helix regions of all
syntaxins using Clustal X (Thompson et
al., 1997). The raw Clustal output was then pared down by eye and
only regions of unambiguously alignable sequence were used. This resulted in a
final, global alignment of 68 taxa and 87 sites corresponding to the conserved
SNARE motif of representatives from all syntaxin families. Several
sub-datasets consisting of pairwise combinations of the five syntaxin families
were also constructed. Paralog specific alignments were made, yielding a
plasma membrane syntaxin alignment of 24 taxa and 112 sites and a non-plasma
membrane alignment of 18 and 123 sites. All alignments are available upon
request.
Phylogeny
Protein maximum likelihood (ML) analysis was done either using Puzzle
(Strimmer and von Haeseler, 1997) with a gamma correction for among-site-rate
variation plus a correction for invariant sites (8 plus 1 rate categories)
estimated from the dataset or by using ProtML 2.2
(Adachi and Hasegawa, 1996)
with a q1000 search for each dataset. Relative estimated log likelihood values
(RELLs) were calculated using Mo12con (A. Stoltzfus, personal communication).
The topology shown for each dataset is the best ProtML tree, but with branch
lengths estimated in Puzzle to incorporate gamma and invariant sites. Maximum
likelihood distance analyses were performed using Puzzle (Strimmer and von
Haeseler, 1997) to calculate ML distance matrices in coordination with
Puzzleboot (A. Roger and M. Holder;
http://members.tripod.de/korbi/puzzle/
). These matrices were then analyzed using Neighbor from the Phylip package
(Felsenstein, 1995) with jumbling. All bootstrap support values are based on
100 replicates.
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Results |
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Physical attributes of the new syntaxin ORFs
The predicted proteins of all syntaxin sequences obtained were well within
the normal size range for syntaxin proteins
(Table 1) and probably share
the characteristic conserved secondary structure. The Trypanosoma
brucei syntaxin 5 sequence had a slightly longer linker region between
helices 1 and 2 (as estimated by secondary structure prediction software) than
other syntaxin 5 orthologues. The Porphyra yezoensis syntaxin PM ORF
encodes a C-terminal extension of 54 amino acids after the end of its
transmembrane domain. Given the previous data on syntaxin membrane insertion
(Bennett et al., 1993), this
region presumably forms a luminal/extracellular extension.
Global phylogeny
In order to determine the evolutionary affinities of the syntaxin genes
obtained, a global dataset was assembled of syntaxins from as broad a
taxonomic range as possible. Because of the limited sequence conservation of
the syntaxin genes, predicted secondary structure of the deduced protein
sequences was used as a guide for sequence alignment. So as to be sure of the
homology of the regions analyzed, only the unambiguously alignable coiled-coil
forming region and the transmembrane domain were used in the global alignment.
This yielded a dataset of 68 taxa and 87 aligned sites, which was analyzed
using maximum-likelihood (ML) and ML distance methods. As seen in
Fig. 1, the syntaxin 5 and
syntaxin 6 families were robustly delineated and separated from the other
syntaxin genes (bootstrap support values E and F). The syntaxin 16/TLG2,
endosomal and plasma-membrane-associated syntaxin families were reconstructed,
but not strongly supported.
|
Because the syntaxin 16/TLG2, endosomal localized and syntaxin PM clades
were poorly supported in the global phylogeny, we sought to test their
monophyly by explicitly asking whether one syntaxin family was robustly
separated from another. This was done by performing phylogenetic analysis on
all pairwise combinations of syntaxin families, the results of which are
summarized in Table 2. In
accordance with the global phylogeny, the support for the monophyly of
syntaxin 5 and 6 families is very strong. Moreover, the syntaxin 7/Vam 3 and
syntaxin PM clades were now reconstructed with significantly improved
bootstrap support. The low Puzzle support values for the syntaxin PM family
may be due to a recently documented problem of quartet puzzling methods in
dealing with sequences with high rates of evolutionary change
(Ranwez and Gascuel, 2001).
However, Bootstrap support for the separation between the syntaxin 16/TLG2
clade and the syntaxin 7 and syntaxin PM clades was only moderate. In no case
was there significant conflict between the placement of the outgroup roots
within a syntaxin family, thus discounting the possibility of strong
paraphyly. Overall, this suggests that the syntaxin paralogs are monophyletic
and allows us to assign, with moderate confidence, the Giardia
syntaxin 16 sequence, as well as the Giardia and Porphyra PM
syntaxins. Thus all of the syntaxins that we obtained were assignable to
specific families, despite their long branch nature in several cases.
|
Plasma membrane syntaxin phylogeny.
To better assess internal relationships within the plasma membrane
associated syntaxins, a sub-dataset was assembled. This enabled the
unambiguous alignment of helix A in addition to the regions previously
aligned, bringing the total number of sites in this dataset to 112. Unrooted
phylogenies clearly delineated the syntaxin PM families of animals, plants and
fungi (Fig. 2). There appears
to be at least two separate cases of expansion in the syntaxin PM family, one
in the metazoan line (Fig. 2,
pink box) and one in the streptophytes
(Fig. 2, orange box).
|
Within the animal sequences, the syntaxin 1 genes from both vertebrates and
invertebrates (Fig. 2, bracket
A) were robustly separated from the human and mouse syntaxin PM subfamilies.
In addition, the syntaxin 4 proteins were separated from the syntaxins 1, 2
and 3 with moderate support (Fig.
2, bracket B). Sanderfoot et al. noted that the SNARE complement
of Arabidopsis thaliana had been expanded
(Sanderfoot et al., 2000);
this is seen particularly in the syntaxin PM family. The plant syntaxins are
robustly separated from the red algal syntaxin and form a number of internally
resolved clades. From our analysis it appears likely that the expanded
complement will be common to higher plants since the Capsicum annum
knolle protein is robustly placed with the Arabidopsis thaliana
knolle. This indicates that the duplication of some plant SNAREs occurred
before the separation of these two plant lineages.
Non-plasma membrane syntaxin phylogeny
A dataset of representative syntaxin 5, 16/TLG2 and endosomal homologs was
also constructed. This allowed for the unambiguous alignment of helix A from
these paralogs, although reliable alignment was not possible between these and
the syntaxin PM sequences. The new dataset included 18 taxa and 123 sites. The
syntaxin 5 clade was robustly reconstructed under all methods of phylogenetic
analysis. The syntaxin 16/TLG2 clade was consistently recovered, despite the
long branch nature of the G. intestinalis sequence
(Fig. 3).
|
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Discussion |
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Evolution of syntaxin functional residues
Syntaxins are classified as `Q snares'
(Fasshauer et al., 1998) based
on the presence of a functionally critical glutamine (Q) residue; this
glutamine was reliably alignable and conserved in all syntaxins that we
obtained (Fig. 4Aa).
|
Calcium ions are known to play an important role in membrane fusion events
(reviewed by Wickner and Haas,
2000). Bezprozvanny et al. identified Ala240 and Val244 in
syntaxin 1A as critical to the protein's interaction with
1B, the pore-forming unit of the N-type Ca2+
channels (Bezprozvanny et al.,
2000
). When compared across syntaxins, there appears to be little
pattern to the conservation at these positions
(Fig. 4Ac,d). There is
variability even within the animal syntaxin 1A versus -B, but the
Giardia syntaxin PM, and some Arabidopsis syntaxin PM
homologs do have the conserved residues. As well the animal syntaxin 7 and
syntaxin 5 proteins have the appropriate alanine and valines. On the other
extreme the Dictyostelium syntaxin 5 has a large portion of this
region deleted entirely. The functional implication of this variability is a
matter for experimental investigation.
Neuronal sec1 binds to syntaxin 1A in animal nerve cells and appears to act
as a regulatory inhibitor (Schulze et al.,
1994). Studies with neuronal sec1 showed that mutating several
residues including isoleucine 236, leucine 165 and glutamate 166, in syntaxin
1A significantly decreased sec1 binding
(Misura et al., 2000
). We
found that the isoleucine residue is conserved in all paralogs that we
identified with the exception of syntaxin 6, where it was replaced with
leucine or phenylalanine (Fig.
4Ab). This underlines the general importance of this position for
sec1 binding, and its potential as a target for site-directed mutagenesis
studies aimed at examining differential syntaxin 6 function or mechanism with
respect to the other syntaxins. Unfortunately, it was not possible to align
the region containing leucine 165 and glutamate 166 between the various
syntaxin paralogs. However, it was possible to align it within the syntaxin PM
proteins (Fig. 4B). The
homologous position to Leu165 is always hydrophobic, including a leucine in
the G. intestinalis and a valine in the P. yezoensis
syntaxin PM sequences, respectively. The next position is slightly less well
conserved with an aspartate in the Giardia sequence and a glutamine
in the Porphyra sequence. These two positions are also well conserved
in the S. cerevisiae sso1 gene and in the A. thaliana syr1
and knolle sequences, indicating the general functional importance of this
region. As this region is one of the binding sites for botulism toxin, it
should be of considerable interest for studies aimed at the understanding of
syntaxin function.
The minimal syntaxin complement of the `adictyosomal' taxon
Giardia intestinalis might include Golgi-associated syntaxins
The protozoan Giardia intestinalis causes the disease Giardiasis
and is the most commonly found intestinal parasite throughout the world
(Adams and Perkin, 1985). Based
on molecular evidence and its lack of mitochondria and several other membrane
bounded organelles, it has also been proposed as one of the earliest evolving
eukaryotes (Cavalier-Smith,
1998
; Leipe et al.,
1993
; see also Embley and
Hirt, 1998
). Giardia, in its feeding form, possesses no
clearly identifiable Golgi apparatus. However, it is possible to induce the
appearance of Golgi structure and function in Giardia during
encystation (Lujan et al.,
1995
). Therefore, the adictyosomal status of Giardia is a
matter of contention.
We amplified, sequenced and analyzed two clear syntaxin paralogs from
Giardia intestinalis. Interestingly, both of the paralog families to
which the Giardia syntaxins were assigned are associated with
transport steps involving the Golgi apparatus
(Abeliovich et al., 1998;
Sollner et al., 1993
). This
seems to provide further evidence for the cryptic presence of a Golgi
apparatus in Giardia.
Syntaxin complements of animals, fungi and Arabidopsis range from
7 to 24 known genes (Bock et al.,
2001; Sanderfoot et al.,
2000
). In our search of the Giardia genome only two
clearly identifiable syntaxin sequences were found. We have amplified, but not
analyzed two other potential syntaxin homologs from the Giardia
genome database (J.B.D. and W.F.D., unpublished). Although other syntaxins may
be present, it seems unlikely that sufficient syntaxin paralogs will be
identified in Giardia to equal the standard eukaryotic diversity.
Syntaxin diversity in Giardia may be reduced to a minimal complement
as Giardia is known to have undergone reduction in response to its
parasitic lifestyle (Roger,
1999
). Since our two analyzed sequences have each been assigned to
specific paralogs, this seems to indicate loss rather than divergence of
Giardia prior to the duplication of the syntaxin families. It will be
interesting to see, when the entire Giardia genome is complete, what
is the final syntaxin complement and to compare it with other, non-reduced,
single-celled eukaryotes.
Duplications in animal and plant plasma membrane syntaxins
The syntaxin PM family contains two sets of nested duplications, one in the
animal and one in the plant lineage (Fig.
2). This expansion of the syntaxins involved in Golgi to plasma
membrane transport seems to have occurred twice independently and represents
an interesting case of parallel evolution. Since the red algal
(Porphyra) syntaxin PM homolog emerges prior to the monophyletic
green plant clade, it appears that the expansion of the syntaxin PM sub-family
occurred after the red and green algal divergence
(Moreira et al., 2000). A
syntaxin PM from a green alga might allow us to further narrow the time frame
for the beginning of the expansion process in plants. Having a comparable
sampling of syntaxins from a plant in addition to Arabidopsis as well
as further information regarding the biological roles of the predicted
syntaxins from the A. thaliana genome should shed light on the
evolution of this syntaxin sub-family within green algae and land plants.
In the animal lineages, syntaxins 1-4 are well characterized. All are
involved in exocytosis but, while syntaxin 4 is constitutively sent to the
basolateral region of epithelial cells, syntaxins 2 and 3 are apically
associated (Low et al., 2000).
Since syntaxin 4 emerges prior to the clade formed by the syntaxin 1-3
paralogs (Fig. 2, bracket B),
it is possible that the first duplication of animal PM syntaxins was
associated with the evolution of cell polarity in metazoa. Syntaxin 1 is found
in the nerve synapse and is involved in neurotransmitter release
(Bennett et al., 1992
). Since
syntaxin 1 is clearly separated from syntaxins 2 and 3
(Fig. 2, bracket A) and since
syntaxin 1 is present in both vertebrate and invertebrates, we propose that
the syntaxin 1 duplication is associated with the evolution of the nervous
system and occurred prior to the vertebrate/invertebrate split.
Paralog duplications and the ancient nature of the syntaxin
system
Recently we noted that syntaxin protein genes were present in diverse
eukaryotic genomes, prompting us to speculate the presence of a primitive
syntaxin in an early eukaryotic ancestor
(Dacks and Doolittle, 2001).
The phylogeny of the syntaxin families based on full length protozoan gene
sequences allows us to deduce the timing of divergence versus duplication
events in the evolution of the syntaxin superfamily. If a taxon contains at
least two syntaxins from different known paralog families, then the
duplication events giving rise to those families must have occurred prior to
the divergence of that taxon. Since Phytophthora species have both
syntaxins 5 and 6, the duplication giving rise to these two syntaxins occurred
prior to the origin of Phytophthora and by extension the rest of the
heterokonts. The same argument can be made, although less strongly in
accordance with the lower bootstrap support, for Dictyostelium
discoideum (syntaxins 5 and 7), Plasmodium falciparum (syntaxins
5 and PM) and Giardia intestinalis (syntaxins 16/TLG2 and PM).
Finally the robust assignment of the Trypanosoma brucei syntaxin 5
indicates that the duplication that gave rise to the syntaxin 5 family
occurred prior to the divergence of Trypanosoma. Similar logic can be
applied for Chlamydomonas, but with respect to the syntaxin 6 family
duplication.
More generally, we have expanded evidence for the presence of syntaxin
paralogs from a broad range of eukaryotes, speaking to the universal role of
the different syntaxin families in the vesicular transport machinery. We can
now more strongly infer the ancient nature of the syntaxin system and conclude
that the diversification into paralog families began early in eukaryotic
evolution. This raises the possibility that syntaxins could have been involved
in the origin or early evolution of the endomembrane system. One major
obstacle in first evolving a permanent internal membrane system would be
establishing a system that is both stable in the identity of its compartments
and its maintenance within the cell, and yet dynamic enough to accommodate
incoming and outgoing vesicles. In vitro reconstitution assays have shown that
syntaxins not only play a role in vesicular transport but also in organellar
reconstruction (Rabouille et al.,
1998; Roy et al.,
2000
). This implies that they could be partially responsible for
the `identity' of an organellar compartment and might have been able to
fulfill the early role necessary for internal membrane stability and
flexibility.
Syntaxins are critical components of the vesicular transport machinery from human to yeast. Here we have assembled and extended the evidence that they are likely to be present in all eukaryotes, even those deemed primitive. By analyzing syntaxins from across eukaryotic diversity, we begin to get a glimpse of some events in the evolution of the syntaxin system, from recent animal or plant-specific paralog expansions all the way back to the earliest evolutionary stages of the eukaryotic internal membrane system.
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Acknowledgments |
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