The Complexity of Vesicle Transport Factors in Plants Examined by Orthology Search
et al. (2014) The Complexity of Vesicle Transport Factors in Plants Examined by Orthology
Search. PLoS ONE 9(5): e97745. doi:10.1371/journal.pone.0097745
The Complexity of Vesicle Transport Factors in Plants Examined by Orthology Search
Puneet Paul 0
Stefan Simm 0
Oliver Mirus 0
Klaus-Dieter Scharf 0
Sotirios Fragkostefanakis 0
Enrico Schleiff 0
Gordon Langsley, Institut national de la sante et de la recherche medicale - Institut Cochin, France
0 1 Department of Biosciences Molecular Cell Biology of Plants, 2 Cluster of Excellence Frankfurt, 3 Center of Membrane Proteomics; Goethe University Frankfurt , Frankfurt/ Main , Germany
Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae and Arabidopsis thaliana, which revealed 240 different proteinaceous factors either experimentally proven or predicted to be involved in vesicle transport. In here, we performed an orthologue search using two different algorithms to identify the components of the secretory pathway in yeast and 14 plant genomes by using the 'core-set' of 240 factors as bait. We identified 4021 orthologues and (co-)orthologues in the discussed plant species accounting for components of COP-II, COPI, Clathrin Coated Vesicles, Retromers and ESCRTs, Rab GTPases, Tethering factors and SNAREs. In plants, we observed a significantly higher number of (co-)orthologues than yeast, while only 8 tethering factors from yeast seem to be absent in the analyzed plant genomes. To link the identified (co-)orthologues to vesicle transport, the domain architecture of the proteins from yeast, genetic model plant A. thaliana and agriculturally relevant crop Solanum lycopersicum has been inspected. For the orthologous groups containing (co-)orthologues from yeast, A. thaliana and S. lycopersicum, we observed the same domain architecture for 79% (416/527) of the (co-)orthologues, which documents a very high conservation of this process. Further, publically available tissue-specific expression profiles for a subset of (co-)orthologues found in A. thaliana and S. lycopersicum suggest that some (co-)orthologues are involved in tissue-specific functions. Inspection of localization of the (co-)orthologues based on available proteome data or localization predictions lead to the assignment of plastid- as well as mitochondrial localized (co-)orthologues of vesicle transport factors and the relevance of this is discussed.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Vesicle transport ensures the exchange of macromolecules and
proteins between different compartments and the endomembrane
system. Membrane-bound vesicles mediate the transport of cargo
from a donor to a target compartment . Different routes have
been identified (Fig. 1). The forward flow (anterograde) starts with
vesicle transport from endoplasmic reticulum (ER) to Golgi, from
which vesicles flow to various organelles and the plasma
membrane (PM; secretory pathway). In addition, vesicles are
transported from PM to vacuoles via endosomes (annotated as
endocytic pathway) and a retrieval mechanism known as
retrograde pathway, which delivers escaped proteins or lipids
back to their residential compartments [4,5]. Moreover, reports
also suggest vesicle transport from ER to chloroplasts , ER to
peroxisomes [7,8] and mitochondria to peroxisomes . However,
ER - chloroplast (PLAM; Plastid Associated Membranes) and ER
mitochondria (MAMs; Mitochondrial Associated Membranes)
contact sites are also discussed to function in lipid/protein and
lipid exchange, respectively .
Each of the pathways involves a specific set of molecular
processes acting in a series of events [13,14]. The budding of the
vesicle entails (i) selection of cargo followed by (ii) recruitment of
the vesicle coat proteins and (iii) scission of the vesicle. The fusion
of vesicle commences with (iv) its trafficking to target membrane
along the cytoskeleton, (v) recognition of the vesicle at the target
compartment by tethering factors and (vi) the fusion of vesicle
and the target membrane mediated by SNAREs soluble NSF
(Nethylmaleimide sensitive factor) attachment protein receptors.
Besides the underlying commonality, distinctions exist in coat
proteins and their recruitment processes, as well as in the involved
regulatory GTPases, tethering factors, and the SNARE proteins.
Three major types of vesicles defined by their coat proteins are
discussed: COP-II (coat protein complex-II), COP-I and Clathrin
Coated Vesicles (CCVs; Fig. 1). COP-II vesicles mediate the flow
from ER to cis-Golgi while COP-I vesicles account for the counter
flow from Golgi to ER and intra-Golgi traffic . CCVs are
involved in the subsequent endocytic traffic flow . In addition,
retromer and ESCRT (endosomal sorting required for transport)
coat complexes are also known to play a crucial role in endosomal
trafficking pathways .
Similar to the animal and fungal system, plants have all the
major components involved in vesicle-mediated transport
[13,18,19]. It was noted that plants possess a high number of
(co-)orthologues for the respective factors; coat proteins, Rab
GTPases, SNAREs, etc. . It is also discussed that the plant
secretory system possesses certain distinctive features in
comparison to the yeast, namely the absence of the ER-Golgi intermediate
compartment (ERGIC), a drastically reduced mobility of Golgi
stacks , and an activity of the trans-Golgi network (TGN) as an
early endosome  to name a few.
At present, majority of the knowledge concerning vesicle
transport in plants has been conducted for the model plant A.
thaliana [13,14,18,19,21]. Thus, we used the available information
on vesicle transport factors from the model systems A. thaliana and
yeast to define orthologous groups from the proteomes of 14
different plant species. We discuss the results with a special focus
on agriculturally relevant crop plant Solanum lycopersicum (tomato),
which represents the model plant system for studying fleshy fruit
development, ripening and wound response . However, we
did not inspect the time point of duplication in relation to
speciation, because definition of paralogues  was not in focus
of our analysis. Thus, we used the term orthologue for
representing genes of two different species derived from a single
common ancestor, while the term (co-)orthologue has been used to
designate the orthologous relationships due to lineage-specific
duplication . The detection of (co-)orthologues was achieved
by the bi-directional BLAST-dependent orthologue search
algorithms OrthoMCL and PGAP. The experimentally proven and
bioinformatically predicted vesicular transport proteins of A.
thaliana and yeast corresponding to core-set of 240 factors were
used as bait to detect putative proteins and group of
(co-)orthologues. The (co-)orthologues were discussed in some
detail for the model systems yeast, A. thaliana and S. lycopersicum
concerning domain architecture and intracellular localization,
while the tissue-specific expression analysis was performed for the
two plant species. Based on our results, we provide an overview
concerning conservation and diversification of orthologues to
factors involved in the vesicle transport systems in Viridiplantae.
Materials and Methods
Database composition and orthologue search
Literature search for proteins involved in vesicle transport was
performed for the two model systems S. cerevisiae and A. thaliana as
described . Manual confirmation of the yeast and A. thaliana
proteins described to be involved in the vesicular transport was
performed by screening existing literature for each single protein
based on the SGD (http://www.yeastgenome.org/; Table S1 [28
109]) and TAIR (http://www.arabidopsis.org/; Table S2
[21,110206]) databases. The protein sequences were categorized
as bioinformatically predicted or experimentally proven. For all
identified factors in S. cerevisiae and A. thaliana the corresponding
protein sequences were extracted from http://www.yeastgenome.
org (S. cerevisiae - April 2012) and http://www.arabidopsis.org (A.
thaliana - TAIR10).
Orthologue identification is based on the strategy defined by
Paul et al. , which used two different orthologue search
algorithms for 14 plant genomes and yeast. These different
algorithms are based on different approaches. The combination of
OrthoMCL and PGAP were used in order to improve the
accuracy of detecting false positives and false negatives. In brief,
the PGAP (pan genome analysis pipeline) in which InParanoid
and MultiParanoid (method MP) are implemented was used to
cluster sequences of S. cerevisiae, A. thaliana and S. lycopersicum
(ITAG2.3 http://solgenomics.net) in their respective orthologous
Orthologue identification in S. cerevisiae, A. thaliana, S. lycopersicum
and 12 other plant species was performed using OrthoMCL 
to identify orthologous groups for more than three species in a less
time-consuming clustering and also to compare the different
predictions. The plant genomes were extracted from (i) B. distachyon
(bradi1.2 with GAEVAL http://www.plantgdb.org), (ii) C.
reinhardtii (JGI v4 with GAEVAL http://www.plantgdb.org), (iii) G.
max (Glyma1 http://www.plantgdb.org), (iv) L. japonicus (Lj1.0
http://www.plantgdb.org), (v) M. truncatula (Mt3.5v5 http://jcvi.
org), (vi) O. sativa (MSU Version 7.0 with GAEVAL http://www.
plantgdb.org), (vii) P. patens (Phypa1.6 http://phytozome.net), (viii)
P. trichocarpa (Ptr v2.0 with GAEVAL http://www.plantgdb.org),
(ix) S. tuberosum (PGSC v3.4 http://potatogenome.net), (x) S. bicolor
(JGI Sbi1 http://www.plantgdb.org), (xi) V. vinifera (Genescope
12X http://genoscope.cns.fr), and (xii) Z. mays (B73 RefGen v2
http://www.plantgdb.org). All genomes downloaded from
PlantGDB  have verified annotations of genes in relation to
alternative splicing and gene fusions/fissions by gene annotation
evaluation algorithm (GAEVAL) . OrthoMCL filtered away
nine poor-quality sequences by our evaluation process based on
the protein sequence length (,10 amino acids) and percent of stop
codons (marked by asterisks; .20%). The results derived from
both orthologue prediction algorithms (OrthoMCL, PGAP) were
used to check for consistency and automatically combined to
generate the list of the vesicle transport components in yeast, A.
thaliana and S. lycopersicum. For all other plant species we only rely
on the results of OrthoMCL.
Protein family scan from Pfam (Version 26.0)  was
performed to predict functional domains of the protein sequences
comprising different vesicle components. Moreover, order and
similarity of domains of the (co-)orthologues in a respective
orthologous group was analyzed automatically by customized
Python scripts (www.python.org). The name of the Pfam domain is
indicated when discussed and the description of the individual
domains is available in the Pfam database (http://pfam.sanger.ac.
uk/). The comparison of domains of (co-)orthologues within one
orthologous group was done in relation to the detected domains
and their order of occurrence. Based on this, we distinguished
three classes; the first class (Class I) means the similar domains and
their identical order of occurrence. Class II means that additional
parts or at least some of the domains occur in both orthologues
referring to their partial similarity in domain architecture, whereas
class III means that both orthologues share no similarity in their
domain architecture. For comparison of domains in the respective
orthologous groups, we used bait as starting point for our analysis,
which is classified concerning their reliability to be involved in
vesicular transport based on experimentally proven or
bioinformatically predicted proteins of yeast and A. thaliana. The major
bait of each orthologous group is marked with an asterisk (*) and
the minor baits are marked with plus (+).
Localization analysis for (co-)orthologues of the identified factors
was performed with a high certainty approach for A. thaliana, while
a low certainty procedure was undertaken for other plant species
and yeast, because for the latter only predictors that allowed
massive sequence analysis were used.
High certainty approach. The prediction was based on
publically available experimental data; Green Fluorescent Protein
(GFP) based localization studies and mass spectrometry (MS) data.
Further, experimental information for chloroplast and
mitochondria localized (co-)orthologues (Table S3, Table S4) as well as for
the other compartments was extracted from SUBA3 , FTFLP
 and PPDB . This information was used to build a
consensus on the majority basis. All (co-)orthologues without
experimentally confirmed localization were assigned to a
particular compartment using 20 different localization predictors
provided by SUBA3 , which represents the consensual
localization via bare majority. Additionally, we utilized the
annotation provided by TAIR as well as in the literature based
on experimental studies for individual protein with respect to their
localization to verify the localization data of the high throughput
analyses via mass spectrometry or GFP fluorescence.
Low certainty approach. For other plant species,
experimental evidences for intracellular localization are largely absent.
Thus, we selected YLoc, WoLF PSORT, TargetP, Predotar,
MitoPred and ChloroP from SUBA3 localization predictor
bundle, which enable the automation of the localization approach
by submitting $2 sequences at once. The predictor YLoc 
and WoLF PSORT  distinguish between 11 different
compartments (extracellular, nucleus, Golgi, ER, mitochondrion,
plastid, plasma membrane, peroxisome, vacuole, cytosol and
cytoskeleton), while TargetP  and Predotar  are highly
accepted to distinguish between chloroplast, mitochondria and
secretory pathway localization. In addition to the
multi-compartment localization predictor, we use MitoPred  as
mitochondrial specific and ChloroP  as chloroplast specific localization
predictor to strengthen the results, because both predictors are
specifically trained to detect proteins with the respective signals.
The localization results of YLoc and WoLF PSORT for vacuole,
ER, Golgi, plasma membrane are merged and represented as
Cluster analysis of expression data
We downloaded microarray expression data from nine different
tissues for A. thaliana (Table S5); (i) flower (4 samples, GSE32193);
(ii) fruit (3 samples, GSE28446); (iii) ovules (2 samples, GSE27281);
(iv) mature pollen (4 samples, GSE17343); (v) root (2 samples,
GSE21504); (vi) anther (3 samples, GSE18225); (vii) seedlings and
whole plant (23 samples, GSE5629); (viii) shoot and stem (41
samples, GSE5633); and (ix) leaf (59 samples, GSE5630) while for
S. lycopersicum seven different tissues (Table S5) were considered
(GSE19326, (i) cotyledons: 2 samples; (ii) hypocotyledons: 2
samples; (iii) 3-weeks old leaves: 3 samples; (iv) 5-weeks old leaves:
3 samples; (v) roots: 3 samples and GSE22300, (vi) fruit: 3 samples;
(vii) flower: 1 sample). The raw CEL data of the samples of both
organisms were normalized using the APT (Affymetrix Power
Tools) software package  with RMA (Robust Multichip
Average) . Further, to avoid overweighting of certain tissues
with multiple samples we considered mean expression level from a
maximal number of four samples for each tissue by performing
hierarchical clustering. The RMA normalized expression data
from a maximum of four samples per tissue of both organisms
were used to build the average, which was used to cluster
independently by using a k-means clustering algorithm (Pycluster
1.50). The number of clusters (k) for the k-means clustering was
limited to 10, which was determined by performing the clustering
for 1 to 50 clusters and then plotting the distance to the optimal
solution (Fig. S1) (iii) the available Affymetrix IDs for the vesicle
transport proteins from the GeneChips of A. thaliana (GPL198 alias
ATH1-121501) and S. lycopersicum (GPL4741) were identified and
used for clustering the genes encoding vesicle transport proteins
(iv) for detecting the expression for different tissues more easily the
median of the samples concerning the tissues and clusters was
Bioinformatic detection of orthologues to factors
involved in vesicle transport
We performed literature search for factors involved in vesicle
transport pathways and extracted 212 factors corresponding to
different pathways in A. thaliana [14,21,147,177] and 45 factors in
S. cerevisiae [223,224]. From the initial set of 257 factors, we
realized an overlap of 17 factors identified for both species thus
yielding 240 different factors used as core-set. The core-set
contains 8 factors for the COP-II, 16 for COP-I, 18 for Clathrin
Coated Vesicles (CCV), 20 for Retromers and ESCRTs, 68 for
Rab GTPases, 45 for Tethering factors and 65 for SNAREs (Fig. 1,
Table 17, Table S6S19). The core-set was further analyzed to
discriminate between experimentally proven or bioinformatically
predicted protein sequences (Table S1 , Table S2
[21,110206]). The same holds true for the (co-)orthologues
identified for yeast and A. thaliana described below, for which
existing literature was screened using SGD (http://www.
yeastgenome.org/; Table S1 ) and TAIR (http://www.
arabidopsis.org/; Table S2 [21,110206]).
The core-set of factors was used to detect the likely orthologous
groups in the 14 analyzed plant genomes and S. cerevisiae via
OrthoMCL (Fig. 2, Table S6S12). The proteome sequences of
the species are subjected to an all-against-all BLASTP to find
reciprocal best similarity pairs between species (putative
orthologues) and reciprocal best similarity pairs within species (putative
co-orthologues). Both pairs are used to define species normalized
similarity matrices, which are then used to classify orthologous
groups via Markov clustering. Consequently, we identified 4021
different (co-)orthologues corresponding to 14 plant genomes via
OrthoMCL search. For most of the plant genomes the number of
(co-)orthologues ranges between 200 and 300, whereas yeast and
Chlamydomonas reinhardtii contains nearly 120 (co-)orthologues and
Glycine max nearly 500 (Fig. 2).
In general, 150 of the initial set of 240 vesicle transport factors
are conserved in algae (C. reinhardtii), moss (P. patens), monocots and
dicots, whereas eight tethering factors (RUD3, IMH1, VPS3,
DSL1, SEC39, VPS51, TRS85, TRS65) could only be identified
in yeast. For the majority of the analyzed factors at least one
(co)orthologue is observed in most of the analyzed plants. Moreover,
multiple (co-)orthologues have been found in the analyzed plant
species for most of the vesicle transport factors (Table S6S12). In
turn, orthologues to 29 factors are only absent in C. reinhardtii,
while orthologues to 15 factors seem to be only present in
monocots and dicots. Interestingly, orthologues to 31 factors seem
to be specific for A. thaliana or dicots in general (Table S19).
In addition, PGAP with implemented InParanoid and
MultiParanoid-like algorithms (see Materials and Methods) was
employed to complement the OrthoMCL analysis in case of S.
cerevisiae, A. thaliana and S. lycopersicum (Table S13S19). We
combine the results of both algorithms to reduce the number of
false negatives. The algorithm uses the pairwise similarity scores
between two species based on an all-against-all BLASTP. The
constructed orthologous groups consist of two seed orthologues
identified by a reciprocal best-hit search between two organisms.
Further, more sequences are added to the orthologous group on
basis of their similarity to the corresponding seed orthologue. The
pairwise orthologous groups of more than two species are merged
concerning their overlap.
Both BLAST-dependent orthologue search algorithms perform
an all-versus-all BLAST of the protein sequences to detect pairs,
which is more sensitive and reliable than a unidirectional BLAST
search. Further, the orthologue search was used to detect groups of
orthologous genes from different plant species, which allowed the
detection of so called (co-)orthologues due to lineage-specific
duplications . Consequently, we identified 129 different (co-)
orthologues for S. cerevisiae corresponding to 171 factors of the
core-set of 240 factors, because some of the (co-)orthologues of
different vesicular transport factors fall in the same orthologous
groups. The 340 and 307 different (co-)orthologues for A. thaliana
and S. lycopersicum could be assigned to 231 and 223 factors,
respectively. The genes not related to the vesicle transport are
discussed in the following sections.
Domain analyses of identified orthologues to vesicle
Orthologues typically perform equivalent functions (Koonin,
2005), but they are not necessarily involved in the same cellular
process. However, if in addition to the inferred orthology the same
domain architecture and same protein localization is observed, the
likelihood that the identified protein performs the function in a
similar cellular process as the bait is very high (e.g. ). Thus,
we inspected the domain architecture of the proteins from S.
cerevisiae, A. thaliana and S. lycopersicum as an additional hint for an
involvement of the identified (co-)orthologues in vesicle transport
For the analysis of the domain architecture, we used bait on the
basis of its reliability for being involved in vesicular transport as
per existing literature (Table S1 , Table S2 [21,110
206]). Thus, (co-)orthologues with an experimental proven
evidence is preferentially used as major bait, while
bioinformatically predicted protein sequences are only used as major bait (*) in
the case where no experimental evidence is available for the
orthologue in the respective group (Tables 17). In case, when .1
bait have been identified, we used the sequence of the yeast
proteins as major bait (*) and the (co-)orthologues of A. thaliana as
minor bait (+).
Further, for analyzes of the domain architecture of the
orthologues in different orthologous groups, three classes have
been defined (see Materials and Methods). Starting from the major
bait (*) the domain architecture of all other (co-)orthologues within
one group were compared to the major bait and classified
accordingly. For the orthologous groups containing
(co-)orthologues from yeast, A. thaliana and tomato, we observed the same
domain architecture (class I) for ,79% (416/527) of the (co-)
orthologues, which indicates a very high conservation of this
process (Tables 17). Overall, analyzes of domain architecture of
detected 776 (co-)orthologues in the three species (A. thaliana, S.
lycopersicum, S. cerevisiae) lead to the assignment of 629 (co-)
orthologues to class I and 127 (co-)orthologues to class II using
the respective major bait for the domain annotation (Tables 17).
Different domain architectures within the same orthologous
groups can be interpreted as gain, loss or swap of functionality of
some genes . Further, there are different orthologous groups
detected for the same vesicular transport factor, which might be
the result of whole genome duplication (WGD) in plants. For some
proteins we even find orthologues with entirely different domain
structure (class III), like for Sec17, VPS54, SYP61, TYN11 and
TYN12 orthologues (Figs. 3a, S2). The (co-)orthologues of the
Sec17 protein in yeast, A. thaliana and S. lycopersicum display
different domain architecture from each other.
In some cases, we observed the presence of additional domains
in identified orthologues when compared to the bait (class II), e.g.
for COG4 or Sec26p (Fig. 3b). COG4 orthologues in all three
species contain the COG4 domain (PF08318), while additional
domains exist in the N-terminal region of the A. thaliana orthologue
(At4g01400) and in the C-terminal region of tomato orthologue
II 3 1 1 1 2 1 2
+ * + * + *
B y * * * * *
3 M rp m
n ;t e
V V S 6
(Solyc07g056010; Fig. 3b; Table S11, Table S18, Table S26).
These additional domains might provide additional regulatory
features but do not argue against an involvement of these
orthologues in vesicle transport. The same situation is found for
the orthologues of Sec26p (Fig. 3b; Table S7, Table S14, Table
S22). While the yeast protein contains only an Adaptin N domain
(PF01602), the two proteins found to be orthologue in A. thaliana
and tomato contain an additional Coatamer beta C domain
(PF07718) at the C-terminus. Again, this additional domain
supports a function in vesicle transport rather than contradicting
an involvement in this process.
Finally, in some cases a domain is absent in the identified
orthologue (class II) as seen for the Sec16 proteins (Fig. 3c, Table
S6, Table S13, Table S21). The yeast Sec16 (YPL085W) contains
three domains annotated as Sec16_N (PF12935), Sec16
(PF12932), and Sec16_C (PF12931), while Sec16_N is not present
in the (co-)orthologues found in A. thaliana and in S. lycopersicum.
However, Sec16_N appears not to be essential for the function
 and thus, the one (co-)orthologue in A. thaliana (At5g47490)
and the found two in S. lycopersicum (Solyc08g007340,
Solyc08g007360) which possess Sec16 and Sec16_C domains
(Fig. 3c) might indeed be involved in vesicle transport. The second
A. thaliana (co-)orthologue (At5g47480) contains only the Sec16
domain and thus might be involved in a process distinct from
COP-II vesicle transport because the Sec16_C domain is essential
for the association of yeast Sec16 to Sec23 . Thus, in case of
the absence of domains a manual inspection was needed to judge
the involvement of each of the orthologues in vesicle transport.
However, in some cases, we find at least two of the above
described cases, e.g. SFT11 (Fig. S2, Table S21S27).
In light of the predicted vesicle transport system in chloroplasts
[141,177], we analyzed the localization of the identified
(co)orthologues in plants by using publically available experimental
data (GFP and mass spectrometry data; see Materials and
Methods) from SUBA3 , FTFLP  and PPDB 
for A. thaliana. Moreover, we also looked for the annotation
provided by TAIR as well as the evidence in literature concerning
localization of specific proteins (Table S3). For (co-)orthologues
without experimental confirmed localization, we used a consensus
of 20 different localization predictors provided by SUBA3 to
assign the presumable localization (see Material and Methods). In
parallel, we predicted the localization for the detected
(co)orthologues found in other plant species. However, we limited
the number of tools used to 6 programs, which allowed fully
automated prediction (Table S4). Consequently, the previous
localization studies concerning vesicle transport factors are
compared with our approach for chloroplasts (Table 8) and
mitochondria (Table 9). Specific factors and characteristics are
presented in respective sections below.
COP-II vesicles deliver cargo from the site of synthesis at the
ER to cis-Golgi . Primarily, Sec16 defines the site of assembly
of COP-II units [84,228]. With the exception of C.reinhardtii (0), we
found 25 (co-)orthologues for Sec16 in all the plants analyzed as
discussed above (Table S6, Table S13; A. thaliana: 2; S. lycopersicum:
After assembly site definition, the small G-protein of the Ras
superfamily Sar1 is activated by the ER-localized guanine
exchange factors (GEF) Sec12 and Sed4 [229,230]. We observed
18 (co-)orthologues for Sar1 (6/5 in A. thaliana/S. lycopersicum),
with one (co-)orthologue (At5g18570) localized in chloroplast as
experimentally confirmed (Table 8) . For the GEF factors we
observed 24 (co-)orthologues in plants (3/1 in A. thaliana/S.
A + + + + + + *
B y * * * * * * * * * * * * * * * * * * * * * * * * * * * *
A I 1 1 1 1 2 2
2 3 4 1 0
5 5 5 5 0 3 1 3 5 2
GO SP SP SP SP te3 te5 rs2 rs2 rs3 rs3 rs8 rs1
C V V V V B B T T T T T T
1 1 1 1 1 1 1 1 2
V V V V T S S 6 t i n
lycopersicum; Table S6, Table S13), but it needs to be mentioned
that the orthologues found have a Sec12-like domain architecture
The activated Sar1 exposes an N-terminal amphipathic a-helix
facilitating its insertion into the membrane and leading to
deformation of the ER membrane [231,232]. Subsequently,
Sar1 interacts with the GTPase-activating protein Sec23 to recruit
the Sec23Sec24 heterodimer to form the pre-budding complex
 in which Sec24 recruits the cargo [234,235]. In the analyzed
plants, we identified up to eight (co-)orthologues for Sec23 (6/4 in
A. thaliana/S. lycopersicum) and for Sec24 (4/4 in A. thaliana/S.
lycopersicum; Table S6, Table S13). Interestingly, At4g01810
(Sec23), At3g44340 and At4g32640 (Sec24) are described as
putatively chloroplast-localized (Table 8) [141,177]. By our
approach we confirmed the assignment of one Sec23 (co-)
orthologue (At4g01810) as plastid-localized, but both Sec24 (co-)
orthologues (At3g44340, At4g32640) were assigned to the plasma
membrane and cytosol based on experimental evidence (Suba-MS;
Table 8, Table S3) [236,237]. However, one (co-)orthologue of
Sec24 in both, A. thaliana and tomato (At2g27460 and
Solyc11g068500) does not carry the Gelsolin domain
(PF00626), which is reflected by their smaller protein lengths
(Table S13). Further, based on the structural context it is not
entirely clear whether this domain is indeed essential for Sec24
After formation of the pre-budding complex, an outer coat is
formed by Sec13 and Sec31 [43,238] to shape the membrane for
bud formation . We identified 310 and 29 (co-)orthologues
for Sec13 and Sec31 in plants, respectively. Previously, two of the
Sec13 (At2g43770, At3g49660) have been assigned as chloroplast
proteins [141,177], while we predict an additional
chloroplastlocalized protein (At1g68690; Table 8). However, contradicting to
the previously described chloroplast localization for At2g43770,
we predict cytosolic localization (Table 8). Furthermore,
At3g49660 as well as At4g02730 have been described as
components of the H3K4 methyltransferase complexes localized
in the nucleus . Similarly, one of the (co-)orthologue of Sec13
in yeast (YBR175W) is assigned to perform function in histone
methylation . Thus, the orthologue cluster of Sec13 contains
proteins involved in two distinct cellular processes.
The Sec31 (co-)orthologues in A. thaliana At5g38560 and
At2g45000 have been assigned as chloroplast proteins, and we
predict a chloroplast localization for the Sec31 (co-)orthologue
At1g68690 as well (Table 8) [141,177]. However, At5g38560 and
At2g45000 have been experimental via literature localized to
plasma membrane (Suba-MS, TAIR) and nucleus (Suba-GFP,
TAIR), respectively (Table 8, Table S3). In line, At5g38560 has
been assigned as putative proline-rich extensin-like receptor kinase
8 , while At2g45000 was assigned as nuclear pore protein 62
(AtNUP62) . In addition, only At1g18830, At3g63460 and
Solyc01g088020 show a similar domain architectures as the yeast
bait (Fig. S2, Table 1, Table S21).
In case of S. lycopersicum, 3 out of 5 Sec13 and all Sec31 (co-)
orthologues are predicted as plastid-localized proteins (Table 8,
Table S3), however, in the light of the discrepancy between
prediction and experimental evidence for A. thaliana Sec31
proteins, the prediction for S. lycopersicum Sec31 has to be taken
Finally, the newly configured COP-II vesicle is detached from
the ER uncoated by the activity of Sec23  and moves towards
the target membrane. For this factor we identified 6 (co-)
orthologues in A. thaliana and 5 in tomato, all with identical
domain structure suggesting that this process involves a multitude
of factors in plants (Table 1, Table S21).
COP-I vesicles mediate the bidirectional transport within the
Golgi network (percolating model) [240,241] and from Golgi
apparatus back to the ER . The formation of COP-I vesicles
is initiated by the small GTPase of the Ras superfamily Arf1 which
in GDP-bound state is adhered to p24 receptors, a group of type-I
transmembrane proteins . With the exception of ARF1D,
which is only found in A. thaliana, we detected orthologues for all
Arf1 or Arf-like proteins in all plant species analyzed (Table S7,
Table S14) . Further, two ARF1A proteins in A. thaliana
(At5g14670, At3g62290) are predicted to be
mitochondriallocalized (Table 9, Table S3, Table S4), but this prediction is
not yet supported by experimental evidence. Similarly, the yeast
(YDL137W, YDL192W) and S. lycopersicum (Solyc05g005190,
Solyc01g008000) (co-)orthologues were also predicted to be
mitochondrial-localized as per our analysis (Table S3).
The GEF factors involved in COP-I vesicle transport contains a
Sec7 domain and mediate the exchange of Arf1-GDP to
Arf1GTP leading to the exposure of its myristoylated N-terminal
amphipathic helix for membrane-anchoring [244,245].
Subsequently to its activation, en bloc recruitment of coatomer unit takes
place . The coatomer unit is composed of two multi-subunit
complexes F-COP (cargo selective; b, c, d and f subunits) and
BCOP (cage forming; a, b9 and e subunits) . All of these
coatomer proteins have been identified in plants (Table S7, Table
S14). After assembly, COP-I vesicle traverse to the recipient
compartment and the Arf1 GTPase-activating protein (ArfGAP)
catalyses the Arf1 hydrolysis facilitating the uncoating of the
In general, (co-)orthologues for all factors of COP-I vesicle have
been found in all analyzed plant genomes (Table S7, Table S14)
and for ,60% COP-I (co-)orthologues in A. thaliana, experimental
evidence (either GFP or mass spectrometry data) for localization
exists (Table S3). However, Z. mays, G. max, P. patens and S.
tuberosum encode higher number of (co-)orthologues for most of the
components than other analyzed plants (Table S7). Furthermore,
the plant F-COP b (Sec26) and F-COP d (RET2p) have distinct
domain architecture in comparison to the yeast proteins (Table 2).
For F-COP b (Sec26), the A. thaliana and S. lycopersicum proteins
possess an additional domain coatamer_beta_c (PF07718) domain
that is probably used to regulate the function of N-terminal
domain (Adaptin_N: PF01602; Table S22) . In contrast,
FCOP d (RET2p) in A. thaliana and S. lycopersicum do not contain the
clat_adaptor_s (PF01217) domain found in the yeast protein
(Table 2, Table S22). One of the identified plant F-COP f (RET3;
At1g08520) was found in chloroplast  and has been described
as Mg-chelatase subunit D (CHLD)  and thus, is involved in a
different cellular process.
Interestingly, except Solyc03g121800, the (co-)orthologues of
FCOP f in S. lycopersicum have also been predicted as
plastidlocalized (Table S3, Table S4). All identified plant (co-)orthologues
for GNOM-type GEF have a Sec7_N domain (PF12783), which
is absent in the corresponding yeast proteins (YEL022W,
YJR031C; Table S22), however, this domain does not argue
against their involvement in vesicle transport.
Clathrin-coated vesicles (CCVs) deliver cargo from PM and
TGN to endosomes . The coatomer of CCVs consists of three
light chains bound to three heavy chains, which form a polyhedral
lattice [2,249]. Further, adapter protein (AP) complexes form
the cargo-selective subunit of CCVs . In general, four
AP-complexes are known: AP-1 to -4. The AP-1 complex (c, b1,
m1 and s1) functions in vesicle formation at TGN and endosomal
compartments, while the AP-2 complex (a, b2, m2 and s2) is
involved in recruiting cargo proteins from the PM
[58,122,175,250]. The AP-3 (d, b3, m3, s3) and AP-4 complex
(e, b4, m4, s4) are presumed to play a functional role in
TGNendosomal route and may be associated with clathrin .
The components of CCVs have been identified in plants and by
manual inspection (Table 3). We did not detect any assigned
function distinct from vesicle transport for the proteins in A.
thaliana and yeast. The factors are by large comparable in their
protein length and by the number of (co-)orthologues between A.
thaliana and S. lycopersicum (Table 3, Table S8, Table S15), but we
observed certain distinctions in the domain architecture of the
plant (co-)orthologues to the yeast factors. For example, one (co-)
orthologue of the AP1-m1 subunit (Solyc04g026830) lacks the
Clathrin adaptor complex small chain domain (PF01217), while
AP2-a subunit (Solyc11g066760) lacks the Adaptin C-terminal
domain (PF02883) and the alpha adaptin AP29 domain (PF02296;
Table S23), both known to regulate clathrin-bud formation .
This poses a high uncertainty for the assignment of the three
detected (co-)orthologues as vesicle component.
In contrast, the b1/29 subunit of AP1 and 2 in plants possess an
additional B2-adapt-app C (PF09066) and Alpha_adaptin C2
(PF02883) domain when compared to the yeast protein (Table
S23). However, the existence of the latter domain in other yeast
proteins YPR029C (U9-AP1) and YBL037W (a-AP2) might
compensate for the loss of this domain. Moreover, from the
localization analysis, we predicted mitochondrial-localized (co-)
orthologues for AP1, AP2 and AP3 factors in yeast, A. thaliana and
S. lycopersicum (Table S3, Table S4). In addition, one (co-)
orthologue of heavy chain in A. thaliana (At3g08530) was
experimentally (FTFLP, PPDB) and via literature (TAIR) localized
to plastid and plasma membrane (Table 8, Table S3).
Retromer and ESCRT complexes
The retromer coat complex possessing a cargo-recognition unit
(Vps26, Vps29a and Vps35) and sorting nexins (SNX) are known
to recycle the receptor proteins back from endosomes . On
the contrary, ESCRTs are involved in concentrating and sorting
ubiquitinated membrane proteins into invaginations of endosomal
membrane (intra-lumenal vesicles ILVs) thereby forming
multivesicle body (MVB/s) [252,253]. Later ILVs release the destined
proteins into the vacuole/lysosomes.
We identified orthologues to the described factors in all
analyzed plants (Table S9). Analyzing the (co)-orthologues to
SNX proteins we realized that all three yeast SNX proteins
contain the typical PX domain (PF00787), which is a structural
domain involved in phosphoinositide binding and thus in
membrane targeting , but one of the yeast (YJL036W) as
well as a tomato protein (Solyc09g010130) does not carry VPS5
domain (PF09325; Table 4, Table S24). The comparison between
(co-)orthologues identified in A. thaliana and S. lycopersicum
manifested that most of the factors have a similar architecture
with the exception of SNF7a (Table 4). The latter is exclusively
found in A. thaliana; as well as VPS2 and VPS31/Bro1, for which
the tomato sequences are significantly shorter than the sequences
in A. thaliana (Table S16, Table S24).
In general, we detected experimental evidence by SUBA3,
PPDB or FTFLP or the annotations provided by TAIR for the
localization of only 32% Retromer and ESCRT proteins in A.
thaliana (Table S3). We observed that majority of the identified
PIP3P-binding proteins in A. thaliana are localized to the cytosol
(FTFLP, Suba-MS, PPDB, TAIR) or endosomes (TAIR), wherein
the cargo recognition components were detected as either
cytosolic (FTFLP, PPDB, TAIR) or Golgi localized proteins
(TAIR; Table S3). In turn, the majority of the S. lycopersicum (co-)
orthologues for retromer units were predicted to be cytosolically
localized (Table S3). Further, most of the ESCRT components in
Cyto & Nucleusk
Cyto & Nucleusk
A. thaliana were predicted to be localized to the nucleus, while a
few were confirmed via literature or experiments to be localized to
the cytosol (At3g12400; TAIR) or plasma membrane (Suba-MS,
TAIR) wherein the S. lycopersicum (co-)orthologues were predicted
as cytosolic, nuclear, mitochondrial and plastidial proteins (Table
S3). Again, in the light of the experimental evidence for the A.
thaliana proteins, the prediction for the S. lycopersicum proteins has
to be confirmed in future.
Rab GTPases emerge as universal regulators for multiple events
ranging from vesicle formation, uncoating and transport to
tethering process, and to the final vesicle fusion . GTP
bound Rab proteins recruit effector-molecules (e.g. adaptors
tethering factors kinases phosphatases and motors) to facilitate
vesicle traffic [256,257]. These proteins have been used as a
markers for different compartments; RabB and RabD are known
to be localized at Golgi, RabE at ER and Golgi, RabG at vacuoles
and RabA to recycling endosomes while other Rabs (RabC and
RabF) are expected to be localized at endocytic compartments
In line with their importance for the cargo recognition, we
identified Rab GTPases belonging to all groups (A to H) in all
plant species (Table S10, Table S17). In contrast to the above
described factors, we did not identify significant differences in the
domain architecture in any of the identified (co-)orthologues. In
addition, we did not detect any distinct function proposed for the
mentioned A. thaliana and yeast (co-)orthologues. While yeast
possesses one representative member for majority of the groups,
we detected multiple (co-)orthologues in plants (Table S10, Table
S17). Most of the classes (A to H) contain Ras domain; PF00071
typical for small GTPases with the exception of RabE1b, which
A. thaliana Id
instead possess domains typical for GTP-binding elongation factor
family proteins and the small GTPases containing a ADP
Ribosylation Factor type GTPase domain (Table S25).
Interestingly, for 66% of the Arabidopsis Rab GTPases, experimental
evidence (GFP and mass spectrometry) for their localization is
available (Table S3). Moreover, most of the Rab proteins from
class A are experimentally known to be localized to trans-Golgi
network or endosomes [258,259], while 3 and 5 proteins from Rab
B and E, respectively, are experimentally known to be localized to
Golgi or pre-vacuolar compartment (Table S3) .
Remarkably, all the Rab proteins are highly conserved in their domain
architectures and belong to class I (Table 5).
The Sec1-Munc (SM)-family of proteins which are known to
form an association with Qa family of SNAREs  are present
in all plants as well (Table S10, Table S17) and in almost all cases
they contain a so-called Sec domain (Sec1 family PF00995; Table
S25). This domain generally characterizes proteins involved in
vesicle transport processes like exocytosis .
Tethering factors act upstream of SNAREs to facilitate
membrane recognition before fusion . Two types of tethering
factors are discussed: homodimeric-tethering factors with
elongated coiled-coil regions  and multi-subunit tethering complexes
(MTCs) . Coiled-coil tethers are long rod-like structures
possessing heptad repeats . In yeast, four coiled-coil tethers
have been described: Uso1 (p115), COY1 (CASP), RUD3/GRP1
(GMAP210) and Imh1 (Golgin-245) [34,42,52,67,223]. In plants,
we only found orthologues to the homodimeric-tethering factors
Uso1 and COY1 (Table S11, Table S18). The two plant (co-)
orthologues of Uso1 (At3g27530, Solyc08g081410) are only half
the size of their yeast counterpart (YDL058W) but contain the
Heazlewood et al.
A. thaliana protein
13 of 14
13 of 14
11 of 14
11 of 14
13 of 14
10 of 14
10 of 14
10 of 14
11 of 14
* Predictions according to our analysis, Cyto- cytoplasm, Mito- mitochondria, PM- plasma membrane; bold indicates genes for which a mitochondrial localization is most
likely based on Heazlewood et al.  and the prediction of mitochondrial localized orthologues in many plants but without further literature evidence; a Johansen et
al. 2009 ; b Heazlewood et al. 2004 .
characteristic domains Uso1_p115_head and Uso1_p115_C
(PF04869 and PF04871; Table 6, Table S26), and thus, might
indeed be involved in vesicle transport. Interestingly, the COY1
orthologue in A. thaliana (At3g18480) is putatively described to be
chloroplast-localized , but experimental evidence for its
localization to Golgi exists (Suba-MS, FFTLP, PPDB; Table 8,
Table S3) .
Further, four major MTC complexes are discussed. (i) HOPS
(homotypic fusion & vacuole protein sorting/class-C vacuole
protein sorting (Vps), (ii) an extension of HOPS annotated as
CORVET (class C core vacuole/endosome tethering), (iii) the
complex associated with tethering containing helical rods
(CATCHR) constituting Exocyst, COG, DSL1, and GARP
complexes, and (iv) the transport protein particle complex,
With the exception of Vps3, all HOPS and CORVET complex
components (Vps3, Vps8, Vps11, Vps16, Vps18, Vps33, Vps39
and Vps 41) are found in plants (Table 6). However, Vps39
(HOPS) and Vps8 (CORVET) are class II proteins, which differs
slightly in the domain architecture comparing plants and yeast (co-)
orthologues (Tabsle 6, Table S11), but are predicted to possess
similar localization to that of yeast protein.
The four CATCHR complexes are conserved to a different
extent. With the exception of Tip20 (At1g08400) , we could
not identify orthologues to components of the DSL1 complex
(Dsl1 and Sec39; Table S18, Table S26). However, we could not
detect (co-)orthologue of Tip20 in tomato, which is detected in
other plant species (Table S18). For COGs, (co-)orthologues for all
have been identified in both S. lycopersicum and A. thaliana (Table
S18), . Interestingly, 6 COG orthologues in A. thaliana have
been putatively described to be chloroplast-localized , while
from our analysis we predicted 3 of 6 (co-)orthologues as
plastidlocalized (COG1, 3 and 5; Table 8, Table S4), COG4 as
cytosolically localized, COG6 as mitochondrial and COG8 as
Golgi localized. However, one (COG2, At4g24840) is
experimentally confirmed (Suba-MS, TAIR) to have a vacuolar localization
(Table 8, Table S3) .
Additionally, we identified orthologues to the three GARP
components: Vps52 Vps53 and Vps54 but not to Vps51
(YKR020W; Table 6, Table S11). In contrast to the other
CATCHR families, we identified orthologues to all eight Exocyst
components (Sec3, 5, 6, 8, 10, 15, Exo70 and 84), which also show
the same domain architecture as the corresponding yeast protein,
except the Sec10 (Table 6, Table S18). For Exo70 we observed a
large number of (co-)orthologues in all plant genomes (Table S11,
Table S18), however, previous studies showed even larger set of
genes representing the Exo70s in A. thaliana (23 putative
homologues) , while we detected only 14 of 23 in the
orthologus group corresponding to yeast Exo70. Furthermore, 5 of
the 14 Exo70 (co-)orthologues in A. thaliana have been predicted as
chloroplast-localized , while we detected contradictory
localization based on experimental evidences (FTFLP, PPDB) for
two (co-)orthologues (At3g55150, At5g59730; Table 8) . In
addition, we identified three more Exo70 (co-)orthologues
(At5g03540, At1g07000, At5g61010) with evidence via
experiments and literature to be localized to cytosol, nucleus or plasma
membrane (PPDB, FTFLP; Table S3) . Remarkably, with
the exception of Trs85 (YDR108W) and Trs65 (YGR166W), other
components of TRAPP-I & TRAPP-II complex have been
detected in plants (Table 6, Table S18). From the manual
inspection, we did not detect any A. thaliana or yeast (co-)
orthologue to be involved in a process other than vesicle transport.
SNAREs act as a universal adapter facilitating the fusion of
vesicle and recipient compartment. SNARE proteins possess a
signature SNARE motif (6070 amino acids) arranged in heptad
repeats which play a role in establishing hetero-oligomeric
interactions . Based on the presence of conserved glutamine
(Q) or arginine (R) in the center of the SNARE domain, SNAREs
are classified into two groups: Q- and R- SNARES . In
general, Q-SNAREs (Qa, Qb, Qc, Qb+Qc- SNAREs) are
localized on the target compartment whereas R-SNAREs reside
on the vesicle . A SNARE complex is composed of four
intertwined a-helices; three distinct Q-SNAREs and one
RSNARE . The complex formation enforces a tight association
between the opposing membranes thereby initiating the fusion
In accordance with their reported importance , we
identified orthologues for almost all SNARE types in both A.
thaliana and S. lycopersicum (Table S19), which are comparable on
the basis of protein length and their domain architecture (Table 7,
Table S27). Moreover, with few exceptions (Syp112, Pen1, VTI13,
SYP61, SYP72, SYP73, and Snap29), we could detect SNARE
orthologues in all other plant species as well (Table S12). Further,
the plant-specific SNAREs in A. thaliana, NPSN (novel
plantspecific SNARE) [19,119] At2g35190, At3g17440 and At1g48240
are experimentally confirmed to be plasma membrane localized
(Table S3) . The two (co-)orthologues of NPSN in S.
lycopersicum, Solyc08g077550 and Solyc12g098950 were predicted
to be localized to cytosol, nuclei or Golgi. Furthermore, majority
of the Qa SNAREs in A. thaliana were experimentally identified in
the PM (Table S3) . From the previous studies, SYP21
(At5g16830), SNAP33 (At5g61210), VAMP726 (At1g04760) are
putatively described as chloroplast-localized , while we
detected contradictory localizations compared to the existing
experimental evidences (SUBA3, FTFLP, PPDB; Table 8, Table
The detected (co-)orthologues of VTI12, with the exception of
At2g24645, At2g24681 and At2g24696, have been described as
members of B3 superfamily of proteins and are referred as REM
proteins [133,181]. Only At4g31610 is experimentally
characterized as AtREM1, while other (co-)orthologues are putatively
classified as REMs [133,181]. Moreover, At4g00260 has been
discussed as MEE45 as well, which plays role in embryo sac
development . Thus, considering the existing literature and
analyzing the domain architecture (Table 7), at stage it is not clear
whether the detected (co-)orthologues of VTI12 have a role in
vesicle transport or not.
Co-regulated clusters of vesicle transport encoding
Having identified (co-)orthologues for most of the components
involved in vesicle transport we aimed at identification of
coregulated clusters of genes. To this end, we used publically
available expression data (Table S3, Materials and Methods). The
tissue expression data were extracted from the Gene Expression
Omnibus (GEO)  for 9 and 7 different tissues and
developmental stages of A. thaliana and S. lycopersicum, respectively
(Table S5). We performed k-means clustering for both organisms
independently. To avoid overweighting of a certain tissue due to a
higher number of samples we performed hierarchical clustering of
the data for respective tissue to select four representative samples
showing a median expression profile. The mean of the tissue
samples were further considered as basis for the k-means
clustering. The number of clusters (k) was limited to 10 because
of the decreased gradient in analyzing the distance to the optimal
cluster solution (Fig. S1; Table S28, Table S29). The available
data allowed the analysis of 282 of the 340 genes (82%) from A.
thaliana (Fig. 4a; Table S28) and 149 of 307 S. lycopersicum genes
(48%, ,25% of which belongs to Rab GTPases) identified in here
as (co-)orthologues of factors involved in the vesicle transport
(Fig. 4b; Table S29).
In A. thaliana, we detected two clusters (annotated as Atha_9 and
Atha_10) of significantly higher expression in mature pollen with
respect to other tissues (Fig. 4a). Atha_9 mostly contains
orthologues to Rab GTPases and SNARE proteins (32/48) (Table
S28), while cluster Atha_10 consists of orthologues to SNARE
proteins (18/46), Sec31 (3) and ESCRT components (5; Table
The genes of the clusters Atha_2, Atha_3 and Atha_7 exhibited
lower expression in mature pollen, and genes of cluster Atha_3
show reduced expression in siliques (Fig. 4a). The Atha_2 cluster is
composed of (co-)orthologues from COP-II (Sec23, Sec24 and
Sec13), clathrin units (b4-AP4, b3-AP3, b1/29-AP1), SNAREs
(Qa, R and Qc), Rab GTPases (C, D and E), tethering factors
(COG3, COY1, BET3 and BET5). Similarly, Atha_7 represents
(co-)orthologues of all COP-II units; Sec13 Sec31, Sec23, Sec24,
GEF and GTPase, and Atha_3 exhibits (co-)orthologues of cage
and cargo selective units of COP-I vesicle (Table S28).
Genes of the clusters Atha_5 and Atha_8 have enhanced
expression in roots or seedlings (in case of Atha_8). In turn, the
genes of cluster Atha_1 show the highest expression in siliques, the
genes of cluster Atha_4 in ovules and pollen, while the genes of
cluster Atha_6 do not show a preferential tissue expression
(Fig. 4a). Atha_1 represents (co-)orthologues for ESCRT I (VPS23
VPS37), ESCRT II (VPS36), and ESCRT III (VPS2 DID2)
factors, while cluster Atha_5 majorly consists of Rab GTPases and
SNARES (11/17; Table S28) in addition to Retromers, ESCRT
and clathrin units.
The A. thaliana (co-)orthologues to 25 factors are present in
different clusters according to their expression pattern. In addition,
for 15 factors at least one (co-)orthologue is classified in a distinct
cluster, while for four factors all (co-)orthologues were classified in
the same cluster (Table S28). Consistently, for S. lycopersicum we
found 15 factors with all (co-)orthologues in different clusters, 7
factors with at least one (co-)orthologues in a different cluster and
only for two factors a classification of all (co-)orthologues in the
same cluster (Table S29). Thus, the presence of (co-)orthologues in
different clusters strongly suggests possible distinct and overlapping
Comparing the clusters for A. thaliana (Fig. 4a, Table S28) with
S. lycopersicum (Fig. 4b, Table S29), we observed that genes in
cluster Slyc_2 and Slyc_3 show an enhanced expression in roots
similar as observed for A. thaliana co-expression clusters Atha_5
and Atha_8, but not as drastic. Similarly, the Slyc_9 cluster shows
an enhanced expression in cotyledon and hypocotyls comparable
to the enhanced expression in seedlings for Atha_8. For the
clusters Slyc_1, Slyc_6, Slyc_8 and Slyc_10 we do not find a
significant alteration of expression, which is comparable to
Atha_6. Furthermore, genes of Slyc_7 are higher expressed in
fruits, which is comparable to the expression behavior of Atha_1
i.e. expressed more in siliques. In contrast to the A. thaliana genes,
we found a specific set of genes, which is highly expressed in
flowers (Slyc_4) and another set, which has low expression in
cotyledon and hypocotyl (Slyc_5). Slyc_4 consists of
(co-)rthologues of SNAREs (Sec18, NPSN, SYP21/22/23, GOS12, VAMP,
SNAP33) and Rab GTPases (D, E and F), while the Slyc_5 cluster
possess (co-)rthologues of SNAREs (Sec22, VAMP, Use11,
SFT11/12, MEMB11/12), Rab GTPases (A, B, C, D and G),
ESCRTs (Vps37, 2, 25, 22). Unfortunately, for S. lycopersicum a
large dataset for expression in pollen or ovules is not available.
While inspecting the overlap between the genes found in S.
lycopersicum and A. thaliana clusters with similar regulation we did
not find a large overlaps with respect to factors assigned with
specific pathways, which in part might be explained by the
different datasets analyzed. More likely, this might also suggest
that evolution has led to the co-regulation of distinct components
corresponding to different pathways.
Further, we analyzed the confirmed or predicted localization of
the proteins encoded by the genes of different clusters (Fig. 5).
Correlating the localization and expression for specific tissues, no
obvious pattern could be observed. Interestingly, A. thaliana and S.
lycopersicum differ in their amount of vesicle proteins localized to
specific compartments. For A. thaliana, a high amount of plasma
membrane localized proteins (represented as endomembrane;
Fig. 5) were observed, while in S. lycopersicum the localization to
plastids and the cytosol was dominating. The latter result might be
biased by the large amount of experimentally confirmed
localization of A. thaliana proteins and only the localization predictions for
the S. lycopersicum proteins.
The complexity of the vesicle transport system in plants
We identified (co-)orthologues of components involved in vesicle
transport in 14 plant species and yeast (Fig. 2; Table S6S12).
These (co-)orthologues were based on the core-set of 240 factors
extracted from literature in yeast or A. thaliana (Fig. 1). In yeast,
(co-)orthologues for 171 factors were identified (Tables 17).
However, this is reflected by 129 (co-)orthologues only, as some of
the factors belong to the same orthologous group. In addition, with
the exception of one (co-)orthologue for Sec13, all (co-)orthologues
have been assigned to class I suggesting an involvement in vesicle
transport. For the 69 remaining factors described to be involved in
vesicle transport in A. thaliana, orthologues do not exist in yeast,
namely for Rab GTPases (29; Table 5, Table S17), SNAREs (26;
Table 7, Table S19), COP-I vesicles, CCVs (5 each; Table 2,
Table 3, Table S14, Table S15) and ESCRTs (4; Table 4, Table
In the 14 plant genomes a total of 4021 (co-)orthologous
sequences corresponding to the core-set of factors are identified.
Only 8 tethering factors found in yeast are not observed in plants
(Table 6, Table S18); namely Rud3/Grp1 and Imh1 (coiled coils),
Vps3 (CORVET), Dsl1 and Sec39 (DSL1 complex), Vps51
(GARP complex), as well as Trs85 and Trs65 (TRAPP-I and II).
The highest number of genes per factor is present in G. max (481;
,2 genes/factor) and P. trichocarpa (341; .1 genes/factor). This
may be credited to recent whole genome duplications (WGD)
[270,271]. It might be speculated that the time that has passed
after WGD was not sufficient to deselect redundant factors of
duplicated regions. The lowest number of (co-)orthologues is
obtained for C. reinhardtii (118; ,1 genes/factor), which is
consistent with its small genome size. Moreover, the number of
(co-)orthologues is relatively constant in all monocots with the
exception of Z. mays. The latter reflects that Z. mays is the only
investigated monocot with recent whole genome duplication
. In contrast, the analyzed dicots show a higher variation
in the number of identified (co-)orthologues of the vesicle transport
factors as several dicots had a recent whole genome dupli-/
triplications (Table S20).
Species specificities in the proteome of vesicle transport factor
exist as well. For example, no (co-)orthologue for Sec16 (COP-II,
Table S6) or the light chain of the triskelion and AP3 complex is
found in C. reinhardtii (CCVs; Table S8). Similarly, S. tuberosum and
M. truncatula do not possess light chains of the triskelion (Table S8).
However, it cannot be excluded that the absence of factors might
result from incomplete gene annotation of the respective genomes
and should be taken with care. Generally, plants appear to show a
higher complexity with respect to the vesicle transport when
compared to yeast (Table S612, Table S20). This on the one
hand results from the existence of chloroplast as additional
organelle and on the other hand most likely reflects certain tissue
specificity in the expression pattern of individual genes.
With respect to the core-set of 240 factors, 340
(co)orthologues in A. thaliana and 307 in S. lycopersicum are assigned
to orthologous groups (Tables 17, Table S13S19), 275 A. thaliana
and 232 S. lycopersicum protein sequences are of class I, and thus
most likely involved in vesicle transport. The difference in the
number of (co-)orthologues is mainly accounted by Rab GTPase
(,4.5 times more in A. thaliana and tomato, Table S17) and
SNARES (34 times more in A. thaliana and tomato, Table S19).
Furthermore, 149 of the 212 factors described for A. thaliana have
been identified in all plants. Interestingly, 7 factors are specifically
found only in A. thaliana, namely ARF1D, RabA4b, Raba4e,
Rabc2b, RabG1, KEULLE and SYP24 (Table 17).
Tissue-specificity of vesicle factors in A. thaliana and S.
Clustering of the available tissue specific expression studies
based on S. lycopersicum GeneChip (9,200 transcripts) containing
149 of the 307 different (co-)orthologues for vesicle transport
revealed a comparable behavior for most genes. In general, the
clusters showed a higher expression in roots, flowers and fruits
when compared to hypocotyl and cotyledon tissues and an
intermediate expression in leafs (Fig. 4b). Only two clusters show a
significantly different expression, namely low expression in fruits
(cluster Slyc_3), which is only represented by one RABH1
orthologue (Table S29), or high expression in hypocotyl and
cotyledon and low expression in roots (cluster Slyc_9). Again, this
cluster represents only 3 orthologues, one to RABE1a, one to
RABE1b and one to Arl8-like. Thus, our analysis only presents a
first indication, but for a final conclusion more experimental data
In contrast, the publically available expression data for A.
thaliana genes is sufficient to justify conclusions (Fig. 4a).
Remarkably, (co-)orthologues for all components of COP-II vesicle are
represented in cluster Atha_7, which display a high expression in
all tissues except of pollen (Fig. 4a, Table S28). This might suggest
that a pollen-specific COP-II composition exists, which is
supported by the clustering of one Sec16, one Sec13, one
Sar1like and one Sec31 (class II) orthologue in Atha_9/Atha_10, which
represent cluster with high expression exclusively in pollen. In
turn, cluster Atha_9/Atha_10 contains genes coding for
orthologues of all inspected complexes except of CCV-transport factors
(Fig. 4a, Table S28). This suggests that the pollen specificity of
vesicle transport is rather defined by specific expression of RAB
and SNARE genes, of which about 35% of all found (co-)
orthologous genes are present in cluster Atha_9/Atha_10.
Atha_5 is the only other cluster which unifies a set of genes with
a tissue specific expression, namely with highest expression in roots
(Fig. 4a, Table S28). The analysis documents that no orthologues
to COP-I, COP-II and CCV factors (with the exception of
m1AP1) is specifically expressed in roots, while most of the genes
found in this cluster are RAB orthologues. Thus, while pollen
specific-expressed orthologues exist for many components, roots
do not represent a tissue with a large specific set of vesicle
Vesicle transport systems in both chloroplasts and
Based on bioinformatics approaches, a vesicle transport system
has been discussed to be present inside the chloroplasts [141,177].
This analysis was extended here by utilizing experimental
evidences, a multitude of prediction server and localization
prediction for orthologues from all plants analyzed (Fig. 6, Table
S4). Thus, while 26 factors in A. thaliana were previously proposed
to be involved in chloroplast-localized vesicle transport system
[141,177], we predict chloroplast localization for 15 proteins in
plants, namely four COP-II (Sec13, Sec31, Sec23 and Sar1-like),
three for COP-I (F-COP and Sec7-type), two CCVs (Heavy chain
and AP4-b4), one RAB GTPase (RABE1) and vive tethering factor
components (Vps5, Exo70, COG1, COG3 and COG5; Table 8).
From this result it is tempting to speculate that the chloroplast
vesicle transport system is similar the COP-II system for transport
from ER to Golgi. Nevertheless, the presented large-scale analysis
supports the previous proposal of a chloroplast intrinsic vesicle
transport system [141,177]. However, for most of the factors the
chloroplast localization has to be experimentally confirmed,
particularly for the central components of the vesicles; the cage
and cargo-selective units.
Unexpectedly, we also realized (co-)orthologues for which a
mitochondrial localization is predicted or even experimentally
confirmed (Table 9). However, in contrast to the chloroplast
inventory which is dominated by COP-II components and
tethering factors, most of the proteins predicted to be
mitochondrial localized are (co-)orthologues for CCVs components (Fig. 6,
Table S3, Table S4). Approaching the MitoMiner database ,
we observed experimental evidence based on GFP tagging or mass
spectrometry for mitochondrial localization in yeast for some
(co)orthologues of Rab GTPases, tethering factors and CCV
component as well. Nevertheless, the mitochondrial localization
of these factors has not been discussed till date. If one considers
that (i) a third of yeast mitochondrial proteome shows dual
localization and (ii) that proteins with dual localization have a
weaker mitochondrial targeting signal , it is possible that at
least some of these proteins are indeed mitochondrial-localized.
At stage, a mitochondrial vesicle transport system has not been
described. However, at a theoretical level, vesicle-like structures
have been proposed to be involved in cristae formation . The
Cristae fissionfusion model suggests that transiently formed
vesicles are implicated in the propagation of the cristae
membranes, through budding off from pre-existing cristae and
fusion with the inner membrane at different site . Consistent
with this idea, Mulkidjanian et al.  suggested that the
intracellular vesicles of purple bacteria like Rhodobacter capsulatus (e.
g. Borghese et al. ) discussed as close relative to the ancestral
endosymbiont leading to mitochondria  are the evolutionary
precursor of cristae. In line, mitochondrion internal vesicle-like
structures have been reported in mitochondria of patients with
defective gene functions which cause pathological conditions, or
during reconstruction of the matrix compartment after extensive
osmotic swelling [279,280] as well as in degenerating
mitochondria in vascular bundle in petals of open Dendrobium cv. Lucky
Duan flowers . Therefore, one can speculate that some of the
components identified in this study as mitochondrial-localized
factors are involved in the formation of cristae as the induction of
membrane curvature is comparable to vesicle formation .
Nevertheless, the exact need for mitochondrial-localized vesicle
transport factor remains elusive and is subject to verification.
Figure S1 Determination of number of clusters (k) for k-means
clustering. Shown are the distances of the clustering from the
optimal solution (dividing each factor to a single cluster) using
enumerated amount of clusters. The k-means clustering is
performed for A. thaliana (black dot) and S. lycopersicum (white dot)
using 1 to 50 clusters. The red dashed line marks the number of
clusters used for the clustering in this study where the logarithmic
distance to the optimal solution has a decreased slope.
Figure S2 Domain architecture of different classes. Shown are
the domain architecture of (co-)orthologues within one
orthologous group for the factors (a) Ret3p, (b) Sec26p, (c) Sec31, (D)
SFT11 in yeast, A. thaliana and S. lycopersicum.
Table S1 Literature reference for all the yeast (co-)orthologues
for the vesicular transport factors from the SGD.
Table S2 Literature reference for all the Arabidopsis
(co-)orthologues for the vesicular transport factors from the TAIR.
Table S3 Localization analysis for yeast, Arabidopsis, and
tomato (co-orthologues) for COP-II components. For yeast and
tomato, Yloc, WoLF PSORT, MitoPred, ChloroP, Target P,
Predotar predictors were used and the consensus was built. The
score given (for e.g. 1 of 2 or 2 of 3) refers to the prediction given
by X of the Y predictors. In contrast, for Arabidopsis publically
available experimental data; GFP (green fluorescent protein)
localization/mass spectrometry (SUBA3, FTFLP, PPDB) were
utilised. We also looked into the annotation given by TAIR
database with a provided reference (PMID). Further, if no
experimental evidence existed, we used the consensus of 20
different predictors to assign the probable localizations (as in
SUBA3) and the score is presented respectively (for e.g. 11/19 or
5/14 etc.). Highlighted cells signify experimental evidence for the
particular (co-)orthologue.PM: plasma membrane, VACU:
vacuole, PLAS: plastid, MITO: mitochondria, NUCL: nucleus;
CYTO: cyoplasm, GOLG: golgi, ER: endoplasmic reticulum,
PERO: peroxisome, EX-CE: extra-cellular, CY-SK: cytoskeleton.
The experimentally proven localisations are highlighted.
Table S4 Localization analysis of chloroplast or mitochondrial
localized (co-)orthologues in other analysed plant species in
context of chloroplast or mitochondrial-localized A. thaliana (co-)
orthologues. The highlighted cells shows that the respective factor
has 7 or .7 of the 14 plant species possessing the similar
localization. Except A. thaliana, Yloc, WoLF PSORT, MitoPred,
ChloroP, Target P, Predotar predictors were used to build a
consensus for all other plant species.
Table S5 GEO IDs considered for downloading microarray
data for both A. thaliana and S. lycopersicum for clustering analysis.
Table S6 The orthologues of COP-II components identified via
OrthoMCL in all the species discussed.
Table S7 The orthologues of COP-I components identified via
OrthoMCL in all the species discussed.
Table S8 The orthologues of Clathrin coated vesicles
components identified via OrthoMCL in all the species discussed.
Table S9 The orthologues of retromer and ESCRT components
identified via OrthoMCL in all the species discussed.
Table S10 The orthologues of Rab GTPases components
identified via OrthoMCL in all the species discussed.
Table S11 The orthologues of tethering factors components
identified via OrthoMCL in all the species discussed.
Table S12 The orthologues of SNARE components identified
via OrthoMCL in all the species discussed.
Table S13 The COP-II-coated vesicle components of yeast, A.
thaliana and tomato identified via OrthoMCL and PGAP.
Table S14 The COP-I-coated vesicle components of yeast, A.
thaliana and tomato identified via OrthoMCL and PGAP.
Table S15 The Clathrin-Coated Vesicle (CCVs) transport
factors of yeast, A. thaliana and tomato identified via OrthoMCL
Table S16 The Retromer and ESCRT transport factors of
yeast, A. thaliana and tomato identified via OrthoMCL and PGAP.
Table S17 The Rab GTPase components of yeast, A. thaliana
and tomato identified via OrthoMCL and PGAP.
Table S18 The Tethering factors of yeast, A. thaliana and tomato
identified via OrthoMCL and PGAP.
Table S19 The Q and R-SNARE components of yeast, A.
thaliana and tomato identified via OrthoMCL and PGAP.
Core-set of plant orthologues for vesicle transport
Table S21 Domain architecture of components of COP-II
coated vesicles in Yeast, A. thaliana and S. lycopersicum.
Table S22 Domain architecture of COP-I components in yeast,
A. thaliana and S. lycopersicum.
Table S23 Domain architecture of clathrin coated vesicle
components in yeast, A. thaliana and S. lycopersicum.
Table S24 Domain architecture of retromer and
components in yeast, A. thaliana and S. lycopersicum.
Table S25 Domain architecture of RabGTPase components in
yeast, A. thaliana and S. lycopersicum.
Table S26 Domain architecture of tethering factors in yeast, A.
thaliana and S. lycopersicum.
Table S27 Domain architecture of SNARE components in
yeast, A. thaliana and S. lycopersicum.
Table S28 A. thaliana genes with their description sorted
according to the clusters.
Table S29 S. lycopersicum genes with their description sorted
according to the clusters.
Disclaimer: This manuscript is a contribution of the SPOT-ITN. We thank
Maik S. Sommer for critical discussion of the manuscript.
Conceived and designed the experiments: ES. Analyzed the data: SS PP.
Wrote the paper: ES SS PP. Performed the computational analysis: SS.
Performed the literature search: PP. Helped in critically analyzing the
subject: KDS OM SF. Helped in the final version of the manuscript: KDS
OM SF. Read and approved the final manuscript: SS PP ES KDS OM SF.
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