JXB Advance Access originally published online on March 21, 2006
Journal of Experimental Botany 2006 57(7):1563-1571; doi:10.1093/jxb/erj150
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RESEARCH PAPER |
Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp. PCC6803 and Arabidopsis thaliana
School of Biological and Biomedical Sciences, University of Durham, Science Laboratories, South Road, Durham DH1 3LE, UK
*To whom correspondence should be addressed. E-mail: a.p.brown{at}durham.ac.uk
Received 4 October 2005; Accepted 6 February 2006
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Abstract |
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 Top Abstract IntroductionThe TAP strategyFatty acid biosynthesis in...TAP tagging of FAS...Future perspectivesReferences |
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De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following enzyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual proteins determined. There are several lines of biochemical evidence indicating that FAS enzymes form a multi-protein complex and both in vitro and in vivo strategies can be used to investigate the association and interactions between them. To investigate protein interactions in vivo, tandem affinity purification-tagged FAS components are being used to purify complexes from both Arabidopsis thaliana and Synechocystis PCC6803. Here, the development of the tandem affinity purification method, its modification, and its use in plants is described and the experimental results achieved so far are reported.
Key words: Arabidopsis, fatty acid synthase, protein–protein interactions, Synechocystis, tandem affinity purification, TAP tag
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Introduction |
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TopAbstractIntroductionThe TAP strategyFatty acid biosynthesis in...TAP tagging of FAS...Future perspectivesReferences |
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Determination of the localization and organization of proteins within a cell is becoming of great importance in biological research. Analysis of both the subcellular localization of proteins and identification of proteins interacting in vivo are now fundamental approaches that utilize proteomic technologies. Methods developed initially in yeast and animal systems are now routinely applied to plants and considerable efforts have been made to define the protein complements of purified plant organelles. Comprehensive data-sets are available for plastids and thylakoids (Baginsky and Gruissem, 2004; Friso et al., 2004; Kleffman et al., 2004), and sub-proteome analyses of many other plant subcellular compartments such as the plasma membrane, nucleus, and endo-membrane system are well underway (Bae et al., 2003; Alexandersson et al., 2004; Dunkley et al., 2004). In addition to the analysis of large numbers of proteins present in such fractions, mass-spectroscopic techniques can be used to identify the components of protein complexes isolated by affinity chromatography methods. The tandem affinity purification (TAP) strategy is one of the original methods used to isolate such complexes and in this article the emergence of this technique is reviewed, its use and modification for experiments with plant material are discussed, and current experiments in which TAP tags are linked to fatty acid biosynthetic enzymes are described.
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The TAP strategy |
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TopAbstractIntroductionThe TAP strategyFatty acid biosynthesis in...TAP tagging of FAS...Future perspectivesReferences |
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The purpose of the TAP method is to purify native protein complexes from cell lysates. Affinity chromatography is highly suitable for this and proteins of interest are fused with a TAP tag containing affinity peptides before stable or transient expression in host cells. At least two affinity steps are used to isolate the bait protein together with its interacting partners. Column elutions are performed under non-denaturing conditions and typically include a protease cleavage step. Utilizing sequential affinity purifications gives higher yields and lower backgrounds than procedures with a single epitope tag (Shevchenko et al., 2002).
The TAP strategy was developed in Saccharomyces cerevisiae for the analysis of ribo-nucleoprotein complexes (Rigaut et al., 1999; Puig et al., 2001). Initial experiments investigated the utility of a number of different affinity tags and discovered only calmodulin-binding peptide (CBP) and tandem immunoglobulin G (IgG)-binding domains from Staphylococcus aureus protein A (ProtA) efficiently recovered a fusion protein from complex protein mixtures. Accordingly both of these sequences were used in the TAP tag. Elution of ProtA from IgG columns requires low pH, which would destroy protein complexes and a tobacco etch virus (TEV) protease cleavage site was therefore included in the tag for elution from the IgG column. Bait proteins were constructed as C-terminal fusion proteins with CBP–TEV site–ProtA tags. Homologous recombination into the yeast genome meant that tagged proteins were under the control of native promoters, and correct expression levels were maintained in the cell—an important consideration if only normal interacting partners are to be identified (Swaffield et al., 1996).
Purification of protein complexes from these cells required application of the cell lysate to an IgG column, column washing, cleavage with TEV protease, binding to calmodulin in the presence of calcium, column washing, and finally elution with EGTA (Fig. 1). After concentration of the sample and analysis by SDS–PAGE, the identity of proteins in the final eluate was determined by mass spectrometry methods. The efficacy of the TAP technique was shown by identification of known components of snRNP (small nuclear ribo-nucleoprotein) complexes and the method quickly became established as an alternative to the yeast two-hybrid method for analysis of interacting proteins. It has several advantages over the two-hybrid system, including analysis of multiple proteins in a complex and indication of the stoichiometry between components. In the initial study, 
5% of the C-terminally tagged proteins tested produced growth defects or were lethal. This was presumably due to inactivation of protein function or the inability to form correct complexes due to the addition of the 20 kDa TAP tag. Methods were therefore developed for N-terminal tagging (as ProtA–TEV site–CBP–bait constructs), which allowed functional expression of some proteins that were inactive as C-terminal fusions (Puig et al., 2001).
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The relatively trivial molecular and genetic methods required to construct C-terminal TAP-tagged ORFs (open reading frames) in yeast under the control of their native promoters has enabled systematic analysis of protein interactions in yeast (Gavin et al., 2002). Global protein expression in this organism has also been analysed using an anti-TAP-tag antibody against a library of 6234 TAP-tagged ORF fusions (Ghaemmaghami et al., 2003). Similar large-scale TAP-tagging and protein interaction studies have been performed in Escherichia coli, engineered to express the enzymes required for site-specific recombination of fusion TAP-tags into the chromosome (Butland et al., 2005). The TAP strategy described above, using sequential purification steps and protease cleavage elution after the first step, has proved applicable to a diverse range of organisms in addition to bacteria and yeast, including insect and mammalian cells (Forler et al., 2003; Knuesel et al., 2003) and Arabidopsis plants (Rubio et al., 2005).
Diversity of TAP tags
TAP of protein complexes utilizes two sequential affinity tags to decrease contamination from proteins interacting non-specifically with either the tagged protein or chromatographic matrices. As the technique has been exploited, alternative TAP-tags containing different affinity peptides and protease cleavage sites have been developed. The original ProtA–TEV cleavage–CBP TAP tag is clearly effective and has allowed isolation of many protein complexes, but alterations have been made to overcome potential problems with its constituents.
One of these is the size of the tag, which might be expected to impair the function of the tagged protein or affect complex formation with other proteins. There are many interaction- and immuno-affinity tags that can be used instead of CBP and ProtA, ranging in size from 5 to 51 amino acids (Terpe, 2003). Immuno-affinity tags such as the FLAG® or haemagglutinin epitopes have commonly been used as replacement components. Examples of smaller TAP constructs are the sequential peptide affinity (SPA) tag made by substitution of the 3x FLAG epitope (22 amino acids) for the ProtA (137 amino acids) domain (Zeghouf et al., 2004) or replacement of CBP with a spacer and single FLAG sequence (Knuesel et al., 2003). In practice, however, it appears that the majority of proteins tagged with the classical TAP tag are functional in yeast (Gavin et al., 2002), and even small proteins such as acyl-carrier protein (<10 kDa) have been used successfully for complex isolation when linked with this 20 kDa tag (Gully et al., 2003).
Tandem affinity tags that do not contain CBP domains may be useful for the isolation of active complexes containing metal-binding proteins, as chelating agents are required for elution from calmodulin. This has been addressed by replacement of the CBP with a 9x myc plus 6x His sequence during construction of the alternative tandem affinity purification (TAPa) tag (Rubio et al., 2005). TAPa fusion tags also contain a rhinovirus 3C protease cleavage site instead of the TEV one, as the former is more active at lower temperatures and the cleavage step from IgG can be performed at 4 °C rather than 16 °C, which may stabilize some complexes. Another tri-functional purification tag, 65 amino acids in size and containing CBP, a 6x His sequence and three copies of the haemagglutinin epitope has been used to identify interacting proteins in S. cerevisiae (Honey et al., 2001).
The necessity for smaller or altered TAP tags for isolation of a specific protein in native complexes might need to be determined on an empirical basis. Testing the functionality of tagged proteins by complementation of knockout mutants (Tzafrir et al., 2004) may indicate whether a chosen TAP tag stands a chance of success, but factors such as the expression level of tagged constructs, the amounts of starting material from which complexes are isolated and whether the bait is N- or C-terminally tagged may also be critical in determining the success of TAP experiments.
Tandem affinity purification in plants
TAP of protein complexes from plants has not been widely reported to date. The classical TAP tag was first used for protein complex analysis in plants after fusion with tomato glycoproteins conferring resistance to fungal pathogens (Rivas et al., 2002a, b), although it was used as an epitope tag for western blotting and pull-down experiments rather than for sequential affinity purification. Purification of a protein complex by sequential affinity steps was first demonstrated to be effective in plants using a TAP-tagged synthetic transcription factor, which was transiently expressed in Nicotiana benthamiana leaves (Rohila et al., 2004). The original TAP tag (Rigaut et al., 1999) was modified in a number of ways during the course of this study to make a better tag for plant analysis. Cryptic splice sites, polyadenylation sites, and duplicated regions in the DNA were altered while maintaining a virtually identical protein sequence, and an observed nuclear localization sequence in the CBP peptide was removed (without affecting calmodulin binding) by substitution of basic amino acids. These modified TAPi sequences can be used as either C- or N-terminal tags and are available in vectors incorporating GATEWAYTM (Invitrogen) recombination sites for expression of fusion proteins under control of the cauliflower mosaic virus 35S promoter (Rohila et al., 2004).
TAP of a protein complex from stably transformed Arabidopsis was demonstrated using the TAPa tag linked to a subunit of the COP9 signalosome by Rubio et al. (2005). These authors also conducted a large-scale assessment of the TAPa system, in which 30 proteins were expressed in Arabidopsis, the majority as both N- and C-terminal fusions. TAPa fusion proteins were detected in 36 out of 43 tested lines and, for the majority of genes, expression was independent of the orientation of the tag, although two genes were expressed only with the tag at one end and not the other. Complementation of mutant lines with a number of tagged proteins showed either no, partial, or complete rescue of mutant phenotypes and indicates that the expression and function of TAP-tagged proteins (and hence their utility in purification) are determined by inherent properties of the protein under study, rather than the tag itself.
TAP-tagged fusion proteins contain useful epitopes for immuno-precipitation and detection and are valuable tools for the analysis of protein interactions even without following a standard TAP purification protocol. For example, utilization of the TAPa sequence as an immuno-precipitation tag, in which proteins are bound to IgG beads or sepharose followed by elution with 3C protease treatment, has recently been reported (Figueroa et al., 2005; Sang et al., 2005).
The TAPa and other TAP plant transformation vectors are available from Arabidopsis stock centres, and web searching shows that many laboratories around the world are starting to use TAP vectors to investigate protein interactions in plants. To date the number of published articles using this technique in plants is limited but this is likely to change over the next few years.
Single-step affinity purification of protein complexes
While TAP enabled purification of the COP9 signalosome from Arabidopsis (Rubio et al., 2005), single-step affinity methods might have some advantages if they are sufficiently selective. The utility of the StrepII tag for single-step protein purification from plants has been investigated recently and a direct comparison made to results obtained with a ProtA/CBP TAP tag (Witte et al., 2004). Proteins tagged with the StrepII tag, which structurally mimics biotin, are purified using the StrepTactin matrix (IBA GmbH, Göttingen, Germany) from which proteins are eluted with biotin or desthiobiotin. The addition of avidin to the plant extracts prevents binding of naturally occurring biotinylated proteins to StrepTactin (Witte et al., 2004). In this study, the eight-amino-acid StrepII epitope (WSHPQFEK) was linked to the bait protein as part of a 23-amino-acid C-terminal extension and purifications performed after transient expression in N. benthamiana or stable expression in Arabidopsis. The results showed that the final protein yields were similar between the StrepII and TAP systems and the same potential interacting protein co-purified with the tagged bait using both methods.
From the limited data available to date, it appears that purification results from plants using either the Strep-tag or TAP systems are equivalent and, although the Strep-tag system has a shorter and more convenient purification protocol, its use does not enable isolation of different interacting proteins that might otherwise be lost during TAP purification. A derivative of the StrepII tag, containing duplicate copies of the above eight-amino-acid epitope, has recently been shown to be effective in isolating protein complexes from mammalian cells (Junttila et al., 2005).
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Fatty acid biosynthesis in plants |
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TopAbstractIntroductionThe TAP strategyFatty acid biosynthesis in...TAP tagging of FAS...Future perspectivesReferences |
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Fatty acid biosynthesis in plants occurs in plastids and is catalysed by two enzyme systems, acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS) (Harwood, 1996, 2005). ACCase and FAS contain several catalytic components and they can both be classified as either type I, composed of multi-functional proteins in which the components are linked on one or two polypeptide chains, or type II, which contain separate proteins that are thought to associate to form complexes for efficient substrate channelling (Roughan and Ohlrogge, 1996). The majority of plant plastidial ACCase enzymes are type II and contain four proteins (Fig. 2) but members of the Graminaceae have a multifunctional type I enzyme in the plastid instead (Nikolau et al., 2003). Plastids contain a type II FAS which requires the activity of seven separate enzymes to synthesize the most common biosynthetic product, stearic acid. Together with a thioesterase, needed to release fatty acids from the acyl-carrier protein on which they are synthesized, and including acetyl-CoA synthetase, the classic incorporation of acetate to palmitic and oleic acids in plant plastids involves at least 14 proteins (Fig. 2).
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The organization, stoichiometry, and nature of the interactions between these proteins are not known in detail at present, although there is strong biochemical evidence for complex formation between some of them. Interactions between the components of type II ACCases have been extensively studied and, in addition to co-purification and co-immunoprecipitation data (Roesler et al., 1996), more recent immunological analysis has shown that at least 50% of the ACCase subunits in pea leaves form a complex that is associated with the chloroplast envelope membrane through non-ionic interactions (Thelen and Ohlrogge, 2002). Co-purification of proteins with thioesterase through four chromatographic steps has also been observed (Hellyer et al., 1992). The interacting proteins were subsequently identified as enoyl-ACP reductase (ENR) and beta-hydroxyacyl-ACP dehydratase, indicating strong interaction between at least three components of the fatty acid biosynthetic pathway.
The association of enzymes that catalyse sequential reactions into complexes has been described for a number of metabolic pathways. The formation of such metabolons, in which biochemical intermediates are transferred between enzymes without diffusion into the bulk phase of the cell, is thought to greatly increase reaction rates and provide possibilities for greater regulation of biochemical activity. Examples where this phenomenon has been demonstrated in plants include the cysteine synthase complex and the Calvin cycle (Winkel, 2004). Evidence that such metabolic channelling occurs during plant fatty acid biosynthesis has been obtained from experiments using permeabilized plastid preparations treated with hypotonic medium (Roughan and Ohlrogge, 1996). Under these conditions 50% of the soluble protein was lost from the plastid stroma and the envelope membranes became permeable to added acetyl- or malonyl-CoA, ATP, and pyridine nucleotides. In assays following the incorporation of labelled acetate into long chain fatty acids, the addition of unlabelled acetyl- or malonyl-CoA had no effect on the measured rate of fatty acid synthesis. In addition, radiolabelled acetyl- or malonyl-CoA were not incorporated into newly synthesized fatty acids. These data demonstrate that exogenous CoAs were excluded from the biosynthetic pathway and, similarly, addition of ATP or NADH did not alter biosynthetic rates, indicating that a pool of these substrates is sequestered away from the bulk stroma, possibly in FAS complexes. Measurement in chloroplasts of the substrate concentrations for several FAS enzymes also showed that they are well below the observed Km values for individual enzymes, suggesting that metabolon formation, leading to localized high substrate concentrations, is important to achieve detected rates of fatty acid synthesis (Roughan, 1997).
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TAP tagging of FAS components |
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TopAbstractIntroductionThe TAP strategyFatty acid biosynthesis in...TAP tagging of FAS...Future perspectivesReferences |
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The available data suggest that separate proteins in plant type-II ACCase and FAS systems are organized in macromolecular complexes, channelling substrates from two-carbon acetate and acetyl-CoA to long chain fatty acids. The exact nature of the protein interactions leading to these complexes is the focus of a research project in our laboratory and, in addition to in vitro interaction analysis with purified FAS enzymes and a co-immunoprecipitation approach, the strategy of TAP tagging FAS components is being used. The aim of these experiments is to purify complexes from organisms expressing tagged FAS enzymes and identify proteins interacting with them following sequential affinity purification. This strategy has been used in E. coli with tagged acyl carrier protein (ACP) (Gully et al., 2003; Butland et al., 2005) and, among the co-purified proteins were beta-ketoacyl-ACP synthases I and II, beta-ketoacyl-ACP reductase (ß-KR), and beta-hydroxyacyl-ACP dehydratase, all key enzymes in fatty acid biosynthesis. It is desirable to extend these studies into other species containing a type-II FAS, and experiments are currently underway in both Arabidopsis thaliana and Synechocystis PCC6803 using the modified ProtA–TEV protease–CBP TAPi tag described by Rohila et al. (2004).
Synechocystis PCC6803
Synechocystis PCC6803 is a photosynthetic cyanobacterium that contains a type-II dissociable FAS system and has a number of properties that make it an attractive model organism for this project.
First, the genome has been sequenced (Kaneko et al., 1996). The total genome of this relatively simple organism contains 3168 genes and orthologues of plant ACCase, and FAS genes can be identified easily. The availability of fully annotated databases for this organism (http://www.kazusa.or.jp/cyano/cyano.html) should facilitate identification of interacting proteins after TAP purification followed by mass spectrometric analysis.
Secondly, Synechocystis PCC6803 is easily transformable and homologous recombination occurs, inserting DNA fragments at specific gene loci. Constructs for recombination into the Synechocystis genome contain 500 bp genomic sequences flanking an antibiotic-resistance selectable marker. If one of these flanking sequences is the C-terminal end of a target protein fused with a TAP tag, then recombination results in a transformant in which the bait protein is under control of its native promoter (Fig. 3). Expression of tagged proteins under the control of their normal promoters may help reduce non-native complex formation with proteins such as chaperones, which might interact with highly abundant over-expressed bait proteins. This strategy is identical to that used for the large-scale TAP-tagging studies in S. cerevisiae and E. coli described above.
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Thirdly, Synechocystis PCC6803 is polyploid, containing 8–10 copies of the genome per cell. This enables an assessment of the functionality of a tagged protein to be made. Selection of initial transformants on low concentrations of antibiotics allows growth of cells containing a mixture of both tagged and non-tagged target genes. After confirmation by PCR that insertion of the TAP-tagged gene into the chromosome has occurred at the correct site, further growth with increasing antibiotic concentrations selects for cells that are fully segregated at the tagged locus and contain increased copies of the selectable marker. If these cannot be obtained, and higher antibiotic concentrations are lethal, then it is likely that the tagged protein is non-functional, either because the TAP tag prevents correct conformational folding or it hinders necessary interactions with other macromolecules.
To date transformation constructs have been made for Synechocystis carboxyltransferase 
, carboxyltransferase ß, and ENR proteins by cloning of genomic fragments generated by PCR and a TAPi tag amplified from the CTAPi plant transformation vector. Additional FAS component constructs are now being made utilizing splice overlap extension PCR to make the constructs more rapidly. Synechocystis PCC6803 containing a fully segregated C-terminal TAP-tagged carboxyltransferase ß has been obtained (Fig. 4) and these cells grow at equivalent rates to wild-type cells. This demonstrates that carboxyltransferase ß fused with the 20 kDa TAP tag is still functional. After lysis of cells in a bead beater (BioSpec Products), TAP tagged carboxyltransferase ß can be detected via western blot (Fig. 5A), and 80% of the tagged protein is associated with a membrane fraction (Fig. 5B). Detergent treatment of the membranes with dodecyl maltoside and digitonin, but not Triton X-100, extracted some of the tagged carboxyltransferase ß into the supernatant. Localization of a proportion of carboxyltransferase ß into a Triton-insoluble membrane fraction is similar to that observed in pea plastids (Thelen and Ohlrogge, 2002), although the functional significance of the membrane-bound and soluble fractions is unclear at present.
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TAP purification procedures performed with a soluble protein extract from PCC6803 containing tagged carboxyltransferase ß have to date resulted in the isolation of small amounts of the tagged bait protein. The expressed TAPi tag is therefore functional in Synechocystis, and experiments are underway to optimize cell lysis procedures and determine the Synechocystis culture volumes required for efficient protein recovery.
Arabidopsis thaliana
Stable transformants of Arabidopsis expressing TAP-tagged FAS components are being made with the TAPi plant transformation vectors. FAS enzymes are located in the chloroplast and their coding sequences contain targeting peptides at the N-terminus. Tagging of plastidial proteins therefore requires insertion of a signal sequence into the NTAPi vector and, although such a vector will be used, initial experiments were designed with the CTAPi plasmid. ENR and ß-KR were the first target proteins selected for TAP tagging, and PCR primers were designed to amplify coding sequences for the full-length, unprocessed proteins from cDNA libraries. Coding sequences were cloned into the entry vector pENTR4 (Invitrogen) before transfer into the CTAPi vector using the GATEWAYTM system. Transformation of Arabidopsis was by floral dip using Agrobacterium GV3101 pMP90 (Koncz and Schell, 1986).
Transformants containing tagged ß-KR sequences have been obtained and T1 plants express different amounts of TAP-tagged protein in their leaves (Fig. 6). TAP-fusion proteins in CTAPi are under the control of the strong cauliflower mosaic virus 35S promoter, and the variable amounts of TAP-tagged protein observed presumably reflect positional effects due to T-DNA insertion sites. Western analysis with a ß-KR antibody should enable the selection of lines in which tagged protein expression is broadly similar to the wild-type protein, minimizing artefacts caused by over-expression of the bait protein.
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Future perspectives |
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TopAbstractIntroductionThe TAP strategyFatty acid biosynthesis in...TAP tagging of FAS...Future perspectivesReferences |
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Determining the organization and interaction between the enzymes required for fatty acid biosynthesis is important for a complete understanding of this important metabolic pathway and may give new insight into the mechanism by which it is regulated. A number of different approaches are being used to investigate interactions between FAS and ACCase components, including TAP-tagging strategies in both Arabidopsis and Synechocystis PCC6803. TAP of protein complexes from Synechocystis has not to our knowledge been reported to date, but the properties of this organism described earlier and the relative speed with which experimental lines can be generated may make it a useful system for TAP experiments.
The expression level of affinity-tagged proteins is an important consideration in such studies. In Arabidopsis, it is aimed to select lines which contain equivalent amounts of tagged and native target proteins, and the generation of Synechocystis strains by homologous recombination should ensure that bait proteins are at normal concentrations within the cell. Complete segregation of a gene linked to a TAP tag in Synechocystis provides a good indication that the tagged protein is functional and interacts with proteins in the correct way. The possibility that only non-tagged proteins bind to their correct partners in cells containing both tagged and normal target proteins (as will be the case in Arabidopsis) is thus avoided. The inability to isolate fully segregated tagged loci in Synechocystis may be due to a non-functional FAS enzyme or loss of expression of downstream genes in an operon. Many of the FAS and ACCase genes are adjacent to hypothetical or known protein-coding sequences, but the lethality of preventing expression of downstream genes by insertion of the spectinomycin cassette will need to be determined in most cases by attempting to make the tagged strain. Systematic TAP-tagging of fatty acid biosynthetic genes at both the N- and C-terminus, followed by recombination and segregation for tagged loci, will enable the isolation of useful Synechocystis strains for analysis of FAS and ACCase interactions.
One such strain, expressing TAP-tagged carboxyltransferase ß has been isolated and the majority of tagged protein is associated with a membrane fraction. Carboxyltransferase subunits were chosen for initial experiments as they interact strongly with each other and, hence, should at least co-purify during TAP purification and might provide complementary data if protein complexes can be isolated using them. Purification of plasma and thylakoid membranes will make it possible to determine whether the localization of membrane-associated CT-ß reflects that seen in pea chloroplasts. The structural relationship between FAS and ACCase systems is unclear at present, although it has been suggested that a protein complex containing both systems and catalysing the conversion of acetate to C18 fatty acids is associated with membranes (Roughan and Ohlrogge, 1996; Roughan, 1997). TAP purification using the soluble cellular fraction and detergent-extracted membrane proteins may give an indication if FAS and ACCase form a complex. Purification of such potentially large complexes might require the addition of protein cross-linking reagents (Schmitt-Ulms et al., 2004; Vasilescu et al., 2004; Sinz, 2005), and Synechocystis cultures will provide a good source of material for optimization of such procedures.
TAP purification of protein complexes from plants has been reported and the application of this method to plants demonstrated for cases where the interacting components were predicted or known (Rohila et al., 2004; Rubio et al., 2005). It remains to be seen if the method will become routinely effective for the discovery of new protein interactions and the efficacy of the method may depend on which protein in a complex is labelled. One-step purification tags such as the StrepII tag offer an alternative to the TAP tag and this has been linked to both ENR and ß-KR for expression in Arabidopsis and direct comparison of the two methods. Whatever the method of purification chosen, it is important that control experiments, such as purification from lines expressing the TAP-tag unlinked to a bait protein, are carried out. For the isolation of potentially large complexes capable of synthesizing fatty acids, attention will have to be paid to the methods for chloroplast isolation and lysis, and the effects of cross-linking reagents carefully studied. In addition to providing material for attempts to purify protein complexes, the availability of Arabidopsis and Synechocystis lines expressing tagged FAS and ACCase genes will prove useful in more conventional co-immunoprecipitation and co-purification approaches to studying protein–protein interactions.
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Abbreviations |
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ACCase, acetyl-CoA carboxylase; ACP, acyl carrier protein; CBP, calmodulin-binding peptide; CoA, Coenzyme A; ENR, enoyl-ACP reductase; FAS, fatty acid synthase; IgG, immunoglobulin G; ProtA, Ig G-binding domain from Staphylococcus aureus protein A; ß-KR, beta-ketoacyl-ACP reductase; TAP, tandem affinity purification; TAPa, alternative tandem affinity purification; TEV, tobacco etch virus.
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