BACKGROUND
the model organism saccharomyces cerevisiae has contributed greatly to our current understanding on eukaryotic genes, their products, and their functions.
decades of study have resulted in an extensive knowledge on yeast physiology, genetics, and the molecular functions and interactions of its proteins.
furthermore, this unicellular eukaryotic system is well suited for the study of basic cellular processes which are often conserved in higher eukaryotes.
because of its ease for genetic modification and fast growth, yeast became the system of choice for in vivo protein analyses from other eukaryotes.
s. cerevisiae was used, for example, to perform proteome-wide analysis of the human protein-protein interaction networks  <cit> , to systematically analyze protein-dna interaction networks of the nematode c. elegans <cit> , and to produce high-value bioactive plant secondary metabolites through metabolic engineering approaches  <cit> .
large scale genomics approaches to uncover protein function are adopted more and more in the current era of systems biology research.
to cope with the large amounts of constructs needed, scientists make use of high-throughput cloning technologies, such as the gatewaytm technology , which is based on the site-specific recombination system from the bacteriophage lambda that facilitates the integration of the phage’s dna into the escherichia coli chromosome  <cit> .
dna segments that are flanked by the appropriate recombination sites in a standard vector  can easily be transferred to a compatible vector  for functional analysis .
multisite gatewaytm uses modified recombination sites to allow the combination of multiple dna segments in one single in vitro recombination reaction.
the segments are joined in a pdest in a predefined order and orientation, maintaining the reading frame and with low risk for mutations  <cit>  .
three-segment multisite gatewaytm makes it possible to easily make any combination of a promoter, gene and tag without the need of redesigning new destination vectors for each new experimental approach.
the ability to choose a promoter allows varying temporal, spatial, and quantitative control of gene expression, while different possible tags enable the inclusion of fluorescent protein tags for localisation or epitope tags for detection or purification, or the creation of protein chimeras .
the flexibility introduced by the multisite gatewaytm technology is illustrated by the large amount of “building blocks” already made available as pentr clones by several research groups  <cit> .
existing pentr collections include orfeomes from multiple prokaryotic and eukaryotic organisms  <cit> .
single-segment pdest vectors are available for virtually all commonly used systems, such as drosophila  <cit> .
moreover, for plants an extensive repertoire of pdest for two- and three-segment multisite gatewaytm has been established  <cit> .
three-segment multisite gatewaytm destination vectors are also available for gram-positive bacteria  <cit> .
in s. cerevisiae, single-segment pdest vectors are available for yeast two-hybrid screens , and two-segment  multisite gatewaytm vectors have been described  <cit> .
an extensive set of single-fragment gatewaytm vectors was constructed, allowing n-terminal fusions with four fluorescent tags, and c-terminal fusions with five different fluorescent tags, an affinity tag and an epitope tag under the control of the inducible gal <dig> or constitutive gpd promoter  <cit> .
alternatively, tags that are not present in this vector set can be fused to the gene of interest through 2-step pcr fusion  <cit>  before performing the bp reaction.
however, to our knowledge no three-segment multisite gatewaytm pdest vectors exist for s. cerevisiae to date.
as a consequence, a large number of commonly used protein tags already available as pentr clones are not readily applicable in the organism that - together with e. coli - is the workhorse of molecular biology.
in this paper we present a set of eleven functionally validated three-segment multisite gatewaytm pdest vectors for use in s. cerevisiae.
the vector set features the four most commonly used auxotrophic markers for selection of yeast transformants combined with three different replication mechanisms.
the presence of the cyc <dig> terminator in these vectors allows construction of any promoter::orf:tag combination.
in addition, a number of useful entry clones harbouring commonly used yeast promoters and entry clones with protein tags to be used with these pdests are presented and validated.
these vectors have been appended to our collection of ‘gateway™ vectors for functional studies’ and can be ordered through the website http://gateway.psb.ugent.be/.
finally, we illustrate the applicability of this vector set by confirming the formation of a ternary complex between the jasmonate zim-domain , the novel interactor of jaz  and the topless  proteins, with ninja acting as a bridging protein.
this complex was previously shown to be involved in jasmonate signalling in the model plant arabidopsis   <cit> .
RESULTS
construction of multisite gatewaytm vectors
the multisite gatewaytm cassette of pkm43gw  flanked by attb <dig> and attb <dig> sites, was pcr amplified adding xhoi and saci restriction sites .
the amplification product was subsequently cloned into the xhoi and saci sites present in the backbones of the vectors of the pag-series  <cit> .
this step replaced the original gatewaytm cassette and the eukaryotic promoter from the pag vectors, while maintaining the cyc <dig> terminator.
ligation products were transformed into ccdb resistant e. coli cells .
the pag vectors contain an ampicillin resistance gene for selection in e. coli, one of four different auxotrophic selection markers  and one of three different replication determinants  thus giving rise to a multisite gatewaytm vector set  for expression in s. cerevisiae .
we did not succeed in creating the integrating vector with the his <dig> autotrophic marker .
*pmgxyz where mg indicates the presence of the multisite gatewaytm cassette; x stands for the presence  or absence  of replication determinants on the vector; y stands for the replication determinant: integrating , cen  or 2 μ ; z indicates the auxotrophic selection marker: his <dig> , trp <dig> , leu <dig>  or ura <dig> .
testing the versatility of the system
to illustrate the flexibility of this vector collection, we took advantage of a few “building blocks” for multisite gatewaytm cloning that were available in-house as entry clones .
one of the assets of multisite gatewaytm destination vectors is that any promoter of interest can be used, provided it is available as an entry clone flanked by attl <dig> and attr <dig> sites.
to achieve this, we cloned two constitutively active promoters  and a galactose-inducible promoter  into pdonr p4-p1r .
the inducibility of the gal <dig> promoter was verified by expression in yeast of a heterologous arabidopsis gene fused to a nls-flag-his tag under the control of the gal <dig> promoter using pmg <dig> .
another advantage of the multisite gatewaytm system is that several different tag sequences are readily available  and can easily be introduced to acquire translational fusions, since the att sites do not disturb the reading frame.
for proof-of-concept, we cloned different plant genes  in a c-terminal translational fusion with either a v <dig>  c-myc, or flag-his tag.
to make these constructs compatible with protein interaction studies in yeast, we additionally fused each epitope tag with a nuclear localization signal  derived from sv <dig>  thereby creating the nls-3xv <dig>  nls-3xc-myc, and nls-3xflag-6xhis tags, respectively.
such constructs allow avoiding false negative experimental outcomes that result from protein localisation in different cellular compartments, for instance.
tagged plant proteins were expressed in yeast under the control of the constitutive gpd or adh <dig> promoters in different vectors from our set.
total protein extracts were obtained and the expressed proteins were visualized through immunoblot analysis .
the flag-his tag is particularly suitable when protein purification under denaturing conditions is needed, while, at the same time, low background detection on immunoblots is desired.
to express c-terminal translational fusions by means of our promoters, tags and destination vectors, entry vectors containing the goi should be cloned with a start codon and without a stop codon, and the manufacturer’s guidelines regarding the reading frame should be adopted .
organisms for which orfeome collections contain vectors that match these criteria include several bacterial collections , human and mouse  <cit> , plants , viruses  <cit>  and yeasts .
caution is advised since some orfeome collections contain entry clones both with and without stop codon  <cit> .
orfeome collections that comprise only goi’s provided with a stop codon, as is the case for some bacterial and viral orfeomes  are not compatible with our vectors.
other orfeomes in which the goi is cloned without a start and without a stop codon , are not compatible with our current promoters but can be used for expression of c-terminal translational fusions with our vectors provided the promoter is cloned followed by a start codon.
since this orfeome list is non-exhaustive and, as described above, different approaches are used when cloning the goi, the compatibility of available pentr collections should always be corroborated either in silico before assembling the different sequences into expression vectors or through epitope tag detection in immunoblots.
finally, the destination vector set presented here also allows expression of n-terminal fusions, provided the att sites flanking the different fusion components are adapted accordingly.
value of the vector set
the value of this vector set was exemplified by a yeast three-hybrid  experiment, in which we investigated the formation of the ternary protein complex by the arabidopsis jaz <dig>  ninja, and tpl proteins.
we have proposed recently that the adaptor protein ninja bridges the jaz proteins to tpl proteins and thereby forms a repressor complex that blocks the cellular programs regulated by the jasmonates, ubiquitous plant hormones that regulate various aspects of plant growth, development, and survival  <cit> .
tpl interacts with the ethylene responsive factor–associated amphiphilic repression  motif that is present in ninja  <cit>  but absent in most jaz proteins, including jaz <dig>  some jaz proteins however, i.e.
jaz <dig> to jaz <dig>  contain ear motifs themselves and are capable of direct interaction with tpl  <cit> .
the n-terminal domain of tpl contains the lise and cthl domains and was previously shown to be essential for binding the ear motif in aux/iaa proteins  <cit> .
therefore we cloned this part  as a bait protein for y2h.
in agreement with the proposed models, the interaction of tpl-n with ninja was confirmed but tpl-n could not interact with jaz <dig> .
commonly used y2h vectors such as pgadt <dig> and pgbkt <dig>  are designed such that the bait and prey fusion proteins are targeted to the same subcellular compartment  and are equipped with an epitope tag, ha and c-myc, respectively, allowing easy confirmation of expression through immunoblot.
to verify whether ninja can connect ear-lacking jazs with tpl, as previously proposed  <cit> , we performed a y3h assay in which we expressed ninja, under control of a constitutive promoter , as a bridging protein.
hereby we used the multisite gatewaytm vector pmg <dig>  that carries the ura <dig> auxotrophic marker that is often still available in yeast strains used for y2h.
as a c-terminal tag we used the nls-flag-his tag.
only when ninja is co-expressed, yeast growth was observed on selective –his medium, indicating that interaction between jaz <dig> and tpl requires the involvement of ninja .
CONCLUSIONS
we have successfully constructed a set of three-segment multisite gatewaytm destination vectors for s. cerevisiae.
our findings make high-throughput recombinatorial cloning of multiple genetic segments in one single reaction accessible in one of the most widely used experimental model systems in molecular biology.
the availability of different auxotrophic markers in this vector set, together with the large amount of existing compatible building blocks for multisite gatewaytm cloning already available in several research groups, creates a versatile utility for these vectors.
in addition, we have cloned two constitutive and one inducible yeast promoter in appropriate pentr vectors and constructed three novel epitope tags, each including a nls, which are suitable for interaction studies in yeast.
the usefulness of the multisite gatewaytm vectors was demonstrated in a y3h assay with which we corroborated the hypothesis that ninja connects the jaz proteins with the co-repressor tpl.
this trimeric complex mediates repression of jasmonate responsive genes in the absence of the hormone  <cit> .
implementation of the vector set presented in this article, together with the cloning of more promoters and  tags according to personal experimental needs, will facilitate gene functional studies and contribute to the high-throughput versatile expression of heterologous  proteins in yeast.

 the model organism saccharomyces cerevisiae has contributed greatly to our current understanding on eukaryotic genes, their products, and their functions. decades of study have resulted in an extensive knowledge on yeast physiology, genetics, and the molecular functions and interactions of its proteins. furthermore, this unicellular eukaryotic system is well suited for the study of basic cellular processes which are often conserved in higher eukaryotes. because of its ease for genetic modification and fast growth, yeast became the system of choice for in vivo protein analyses from other eukaryotes. s. cerevisiae was used, for example, to perform proteome-wide analysis of the human protein-protein interaction networks, to systematically analyze protein-dna interaction networks of the nematode c. elegans, and to produce high-value bioactive plant secondary metabolites through metabolic engineering approaches. large scale genomics approaches to uncover protein function are adopted more and more in the current era of systems biology research. to cope with the large amounts of constructs needed, scientists make use of high-throughput cloning technologies, such as the gatewaytm technology, which is based on the site-specific recombination system from the bacteriophage lambda that facilitates the integration of the phage’s dna into the escherichia coli chromosome. dna segments that are flanked by the appropriate recombination sites in a standard vector can easily be transferred to a compatible vector for functional analysis. multisite gatewaytm uses modified recombination sites to allow the combination of multiple dna segments in one single in vitro recombination reaction. the segments are joined in a pdest in a predefined order and orientation, maintaining the reading frame and with low risk for mutations. three-segment multisite gatewaytm makes it possible to easily make any combination of a promoter, gene and tag without the need of redesigning new destination vectors for each new experimental approach. the ability to choose a promoter allows varying temporal, spatial, and quantitative control of gene expression, while different possible tags enable the inclusion of fluorescent protein tags for localisation or epitope tags for detection or purification, or the creation of protein chimeras. the flexibility introduced by the multisite gatewaytm technology is illustrated by the large amount of “building blocks” already made available as pentr clones by several research groups. existing pentr collections include orfeomes from multiple prokaryotic and eukaryotic organisms. single-segment pdest vectors are available for virtually all commonly used systems, such as drosophila. moreover, for plants an extensive repertoire of pdest for two- and three-segment multisite gatewaytm has been established. three-segment multisite gatewaytm destination vectors are also available for gram-positive bacteria. in s. cerevisiae, single-segment pdest vectors are available for yeast two-hybrid screens, and two-segment multisite gatewaytm vectors have been described. an extensive set of single-fragment gatewaytm vectors was constructed, allowing n-terminal fusions with four fluorescent tags, and c-terminal fusions with five different fluorescent tags, an affinity tag and an epitope tag under the control of the inducible gal or constitutive gpd promoter. alternatively, tags that are not present in this vector set can be fused to the gene of interest through 2-step pcr fusion before performing the bp reaction. however, to our knowledge no three-segment multisite gatewaytm pdest vectors exist for s. cerevisiae to date. as a consequence, a large number of commonly used protein tags already available as pentr clones are not readily applicable in the organism that - together with e. coli - is the workhorse of molecular biology. in this paper we present a set of eleven functionally validated three-segment multisite gatewaytm pdest vectors for use in s. cerevisiae. the vector set features the four most commonly used auxotrophic markers for selection of yeast transformants combined with three different replication mechanisms. the presence of the cyc terminator in these vectors allows construction of any promoter::orf:tag combination. in addition, a number of useful entry clones harbouring commonly used yeast promoters and entry clones with protein tags to be used with these pdests are presented and validated. these vectors have been appended to our collection of ‘gateway™ vectors for functional studies’ and can be ordered through the website http://gateway.psb.ugent.be/. finally, we illustrate the applicability of this vector set by confirming the formation of a ternary complex between the jasmonate zim-domain, the novel interactor of jaz and the topless proteins, with ninja acting as a bridging protein. this complex was previously shown to be involved in jasmonate signalling in the model plant arabidopsis.  construction of multisite gatewaytm vectors the multisite gatewaytm cassette of pkm43gw flanked by attb and attb sites, was pcr amplified adding xhoi and saci restriction sites. the amplification product was subsequently cloned into the xhoi and saci sites present in the backbones of the vectors of the pag-series. this step replaced the original gatewaytm cassette and the eukaryotic promoter from the pag vectors, while maintaining the cyc terminator. ligation products were transformed into ccdb resistant e. coli cells. the pag vectors contain an ampicillin resistance gene for selection in e. coli, one of four different auxotrophic selection markers and one of three different replication determinants thus giving rise to a multisite gatewaytm vector set for expression in s. cerevisiae. we did not succeed in creating the integrating vector with the his autotrophic marker. *pmgxyz where mg indicates the presence of the multisite gatewaytm cassette; x stands for the presence or absence of replication determinants on the vector; y stands for the replication determinant: integrating, cen or 2 μ; z indicates the auxotrophic selection marker: his, trp, leu or ura. testing the versatility of the system to illustrate the flexibility of this vector collection, we took advantage of a few “building blocks” for multisite gatewaytm cloning that were available in-house as entry clones. one of the assets of multisite gatewaytm destination vectors is that any promoter of interest can be used, provided it is available as an entry clone flanked by attl and attr sites. to achieve this, we cloned two constitutively active promoters and a galactose-inducible promoter into pdonr p4-p1r. the inducibility of the gal promoter was verified by expression in yeast of a heterologous arabidopsis gene fused to a nls-flag-his tag under the control of the gal promoter using pmg. another advantage of the multisite gatewaytm system is that several different tag sequences are readily available and can easily be introduced to acquire translational fusions, since the att sites do not disturb the reading frame. for proof-of-concept, we cloned different plant genes in a c-terminal translational fusion with either a v c-myc, or flag-his tag. to make these constructs compatible with protein interaction studies in yeast, we additionally fused each epitope tag with a nuclear localization signal derived from sv thereby creating the nls-3xv nls-3xc-myc, and nls-3xflag-6xhis tags, respectively. such constructs allow avoiding false negative experimental outcomes that result from protein localisation in different cellular compartments, for instance. tagged plant proteins were expressed in yeast under the control of the constitutive gpd or adh promoters in different vectors from our set. total protein extracts were obtained and the expressed proteins were visualized through immunoblot analysis. the flag-his tag is particularly suitable when protein purification under denaturing conditions is needed, while, at the same time, low background detection on immunoblots is desired. to express c-terminal translational fusions by means of our promoters, tags and destination vectors, entry vectors containing the goi should be cloned with a start codon and without a stop codon, and the manufacturer’s guidelines regarding the reading frame should be adopted. organisms for which orfeome collections contain vectors that match these criteria include several bacterial collections, human and mouse, plants, viruses and yeasts. caution is advised since some orfeome collections contain entry clones both with and without stop codon. orfeome collections that comprise only goi’s provided with a stop codon, as is the case for some bacterial and viral orfeomes are not compatible with our vectors. other orfeomes in which the goi is cloned without a start and without a stop codon, are not compatible with our current promoters but can be used for expression of c-terminal translational fusions with our vectors provided the promoter is cloned followed by a start codon. since this orfeome list is non-exhaustive and, as described above, different approaches are used when cloning the goi, the compatibility of available pentr collections should always be corroborated either in silico before assembling the different sequences into expression vectors or through epitope tag detection in immunoblots. finally, the destination vector set presented here also allows expression of n-terminal fusions, provided the att sites flanking the different fusion components are adapted accordingly. value of the vector set the value of this vector set was exemplified by a yeast three-hybrid experiment, in which we investigated the formation of the ternary protein complex by the arabidopsis jaz ninja, and tpl proteins. we have proposed recently that the adaptor protein ninja bridges the jaz proteins to tpl proteins and thereby forms a repressor complex that blocks the cellular programs regulated by the jasmonates, ubiquitous plant hormones that regulate various aspects of plant growth, development, and survival. tpl interacts with the ethylene responsive factor–associated amphiphilic repression motif that is present in ninja but absent in most jaz proteins, including jaz some jaz proteins however, i.e. jaz to jaz contain ear motifs themselves and are capable of direct interaction with tpl. the n-terminal domain of tpl contains the lise and cthl domains and was previously shown to be essential for binding the ear motif in aux/iaa proteins. therefore we cloned this part as a bait protein for y2h. in agreement with the proposed models, the interaction of tpl-n with ninja was confirmed but tpl-n could not interact with jaz. commonly used y2h vectors such as pgadt and pgbkt are designed such that the bait and prey fusion proteins are targeted to the same subcellular compartment and are equipped with an epitope tag, ha and c-myc, respectively, allowing easy confirmation of expression through immunoblot. to verify whether ninja can connect ear-lacking jazs with tpl, as previously proposed, we performed a y3h assay in which we expressed ninja, under control of a constitutive promoter, as a bridging protein. hereby we used the multisite gatewaytm vector pmg that carries the ura auxotrophic marker that is often still available in yeast strains used for y2h. as a c-terminal tag we used the nls-flag-his tag. only when ninja is co-expressed, yeast growth was observed on selective –his medium, indicating that interaction between jaz and tpl requires the involvement of ninja.  we have successfully constructed a set of three-segment multisite gatewaytm destination vectors for s. cerevisiae. our findings make high-throughput recombinatorial cloning of multiple genetic segments in one single reaction accessible in one of the most widely used experimental model systems in molecular biology. the availability of different auxotrophic markers in this vector set, together with the large amount of existing compatible building blocks for multisite gatewaytm cloning already available in several research groups, creates a versatile utility for these vectors. in addition, we have cloned two constitutive and one inducible yeast promoter in appropriate pentr vectors and constructed three novel epitope tags, each including a nls, which are suitable for interaction studies in yeast. the usefulness of the multisite gatewaytm vectors was demonstrated in a y3h assay with which we corroborated the hypothesis that ninja connects the jaz proteins with the co-repressor tpl. this trimeric complex mediates repression of jasmonate responsive genes in the absence of the hormone. implementation of the vector set presented in this article, together with the cloning of more promoters and tags according to personal experimental needs, will facilitate gene functional studies and contribute to the high-throughput versatile expression of heterologous proteins in yeast. 
here, we present a set of three-fragment multisite gatewaytm destination vectors that have been developed for gene expression in s. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. to date, this technology has not been accessible in the yeast saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors. our vectors make multisite gatewaytm cloning accessible in s. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of proteins in one of the most widely used model organisms for molecular biology research. the multisite gatewaytm system allows combining multiple genetic fragments such as promoter, orf and epitope tag in one single reaction. as an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the jaz, ninja and topless proteins. gateway cloningmultisitesaccharomyces cerevisiaeyeastvectorfusion proteinepitope tagjasmonatearabidopsis thaliana recombinatorial cloning using the gatewaytm technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire orfeomes in gatewaytm compatible vectors.
 recombinatorial cloning using the gatewaytm technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire orfeomes in gatewaytm compatible vectors. the multisite gatewaytm system allows combining multiple genetic fragments such as promoter, orf and epitope tag in one single reaction. to date, this technology has not been accessible in the yeast saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.  here, we present a set of three-fragment multisite gatewaytm destination vectors that have been developed for gene expression in s. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. as an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the jaz, ninja and topless proteins.  our vectors make multisite gatewaytm cloning accessible in s. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of proteins in one of the most widely used model organisms for molecular biology research. gateway cloningmultisitesaccharomyces cerevisiaeyeastvectorfusion proteinepitope tagjasmonatearabidopsis thaliana 