Journal of Experimental Botany, Vol. 51, No. 346, pp. 955-960,
May 2000
© 2000 Oxford University Press
A method for expression cloning of transporter genes by screening yeast for uptake of radiolabelled substrate
Plant Biochemistry Laboratory, The Royal Veterinary and Agricultural University, and Centre of Molecular Plant Physiology (PlaCe), 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark
Received 16 November 1999; Accepted 22 December 1999
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Abstract |
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A method has been developed for the cloning of plasma membrane transporters by screening yeast transformed with a cDNA library for the accumulation of radiolabelled substrate. The applicability of the method is demonstrated by cloning the amino acid permease AAP1. A yeast mutant defective in proline uptake was transformed with an Arabidopsis thaliana cDNA library and plated on medium supplemented with L-[U-14C]proline. Yeast colonies accumulating radiolabelled proline were identified by autoradiography. The plasmids of these colonies were reintroduced into the yeast mutant and restoration of proline uptake was confirmed by L-[U-14C]proline uptake measurements. Whereas cloning of transporters by functional complementation requires that the substrate taken up is metabolized by yeast to promote growth, the method described here can be used to isolate transporters of substrates which are not metabolized. The method has great potential for the isolation of transporters of various substrates such as secondary plant products.
Key words: Transporter, uptake, expression screening, yeast.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Since the first reported cloning of plant transporter genes by functional complementation of yeast (Anderson et al., 1992
; Riesmeier et al., 1992), the method has been widely applied to isolate transport proteins from plants (for reviews see Rentsch et al., 1998; Tanner and Caspari, 1996). Cloning of transporters by functional complementation requires a yeast strain which is able to grow on a given medium only if it expresses the transporter of interest introduced by transformation with a cDNA library. The medium is supplemented with the substrate of interest. Typically, the substrates have been essential nutrients such as nitrogen sources, for example amino acids (Frommer et al., 1993; Hsu et al., 1993) and carbon sources, for example, sucrose (Riesmeier et al., 1992). It is a prerequisite for functional complementation that the substrate taken up can be metabolized by the yeast to promote growth. However, many transporter substrates, for example, plant-specific natural products, may not be metabolized by yeast. Such substrates may be transported into the yeast cell by a heterologously expressed transporter and accumulate as xenobiotics in the vacuole without promoting yeast growth (Rea et al., 1998). In some cases, yeast can be engineered to metabolize a given substrate. As an example, a plant sucrose transporter was cloned by functional complementation of an invertase-deficient yeast mutant engineered with a plant sucrose synthase (Riesmeier et al., 1992).
The present paper describes a method for the cloning of plasma membrane transporters in yeast. The method is based on monitoring the accumulation of radiolabelled substrate in the yeast cells expressing the transporter of interest. The applicability of the method is demonstrated by isolation of a yeast expressing the amino acid permease AAP1 (Frommer et al., 1993) by screening a proline-uptake-deficient yeast strain transformed with an Arabidopsis thaliana (L.) Heynh cDNA library on medium containing radiolabelled proline.
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Materials and methods |
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Strains and plasmids
The yeast strain used was S. cerevisiae 22574d (Mat
ura3-1 gap1-1 put4-1 uga4-1; Jauniaux and Grenson, 1990). Plasmids used were pFL61 (Minet et al., 1992) and pFL61 carrying the AAP1 cDNA under the control of the PGK promoter (Frommer et al., 1993).
Colony lifts and detection
Yeast colonies were transferred to nylon membranes (Boehringer Mannheim) after 2 d growth at 28 °C on medium supplemented with 0.25 µCi ml-1 L-[U-14C]proline by placing a membrane onto a plate for 2 min (first lift), 5 min (second lift) and 30 min (third lift), respectively. For quantification of the amount of radioactivity accumulated by the colonies, 0.2 µl of standard dilutions of L-[U-14C]proline were applied to nylon membranes. The membranes were dried for 2 h at room temperature and analysed by STORM 840 phosphor imager (Molecular Dynamics, USA) after 18 h exposure to a 14C-sensitive screen.
Expression screening
Strain 22574d was transformed with a cDNA library made from A. thaliana seedlings in pFL61 (Minet et al., 1992). Transformants were selected on SD drop-out medium (Ausubel et al., 1999; amino acid composition: arg, asp, his, ile, leu, lys, met, phe, thr, trp, tyr) and washed from the plates after 48 h incubation at 28 °C. A total of 60 000 cfu was replated with a density of 10 000 cfu per plate on 9 cm plates of SD drop-out medium supplemented with 0.25 µCi ml-1 L-[U-14C]proline (248 mCi mmol-1, Amersham). After incubation for 48 h at 28 °C two colony lifts were done from each plate. Colonies which corresponded to radiolabelled spots on the membranes were excised from the plates, resuspended in liquid SD medium and taken through a secondary screening. The yeast mutant 22574d was retransformed with plasmid DNA isolated from single colonies corresponding to radiolabelled spots and analysed by L-[U-14C]proline uptake measurements.
DNA manipulations
Yeast plasmid DNA was prepared as described previously (Robzyk and Kassir, 1992). Sequencing was performed on an ALF-Express (Pharmacia) using Thermo Sequence Fluorescent-labelled Primer cycle sequencing kit (7-deaza dGTP) (Amersham). Sequence computer analysis was done with programs of the GCG (Genetics Computer Group, Madison, WI) software package.
Uptake measurements
For uptake measurements, cells were pelleted from a yeast culture (OD600 0.5–0.8) and resuspended in an equal volume of buffer (50 mM MES/TRIS pH 4.5). Uptake of L-[U-14C]proline was measured (according to Smith et al., 1995) with 113 µM L-proline (24.8 mCi mmol-1, Amersham) and an incubation time of 15 min.
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Results |
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The Saccharomyces cerevisiae strain 22574d carries mutations in the general amino acid, proline and
-aminobutyric acid permease genes. It is unable to grow on medium containing proline as the sole nitrogen source due to the lack of low- and high-affinity proline uptake systems (Jauniaux et al., 1987; Jauniaux and Grenson, 1990). AAP1, a broad specificity amino acid permease from A. thaliana (Frommer et al., 1993), is able to restore growth of the yeast mutant on proline medium. To test whether the accumulation of L-[U-14C]proline or its metabolites can be monitored in yeast colonies, the plasmid pFL61 carrying the AAP1 cDNA was transformed (Frommer et al., 1993; Minet et al., 1992) into 22574d and transformants were plated on SD medium supplemented with L-[U-14C]proline. Colonies were transferred to nylon membranes and analysed for accumulation of radioactivity (Fig. 1). Colonies containing the AAP1 cDNA appeared as distinct spots on autoradiograms of the three nylon membrane lifts prepared. Control colonies containing empty vector were barely detectable (Fig. 1). Quantification of the signals shows that the transfer of colonies to the nylon membranes is most efficient for the first membrane lift. From the total amount of radioactivity detected on the nylon membranes of all three colony lifts, it is estimated that colonies harbouring AAP1 accumulate ten times more radioactivity than colonies harbouring empty vector (Fig. 1).
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The applicability of the method for cloning of transporter proteins was investigated in yeast strain 22574d transformed with an A. thaliana expression library in vector pFL61 (Minet et al., 1992
). Transformants were screened for restored proline uptake on SD medium containing L-[U-14C]proline. Four radiolabelled spots were detected on autoradiograms of nylon membranes representing a total of 60 000 colonies. The labelling was present on both membrane lifts. Several radiolabelled colonies derived from each of the four positives were isolated by a secondary screening. Single radiolabelled colonies harbouring plasmids with inserts bigger than 1000 bp were analysed further. Plasmids of six radiolabelled colonies were reintroduced into 22574d, and uptake measurements in retransformed 22574d were used to discriminate between true and false positives (Table 1). L-[U-14C]proline uptake was found in two clones derived from two different positives of the primary screening. Sequence analysis showed that both plasmids contained the full length AAP1 cDNA. 22574d retransformed with the other plasmids did not accumulate L-[U-14C]proline (Table 1). Sequence analysis showed that these plasmids carried inserts with similarity to plasma membrane intrinsic proteins, photosystem II oxygen-evolving complex protein and catalase.
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Discussion |
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Cloning of transporter proteins by functional complementation in yeast is based on the principle that yeast growth can be made dependent on the uptake of the substrate of the transporter of interest. Several prerequisites must be fulfilled to achieve a sufficient complementation system: (1) the yeast strain must not have a functional uptake system for the substrate of the transporter to be cloned; (2) the yeast must not be able to extracellularly metabolize the substrate into compounds which can be taken up; (3) the yeast must be able to metabolize the substrate intracellularly and to use the substrate or its hydrolysis products as nutrient; and (4) the substrate and its hydrolysis products must not be toxic for the yeast. Prerequisite (3) may be difficult to fulfil as many transporter substrates, for example, plant-specific natural products may not be metabolized by yeast. If such a substrate is transported into the cell, it may be recognized as a xenobiotic and stored in the vacuole without promoting yeast growth (Rea et al., 1998
). Alternatively, the yeast can be genetically engineered to metabolize the substrate taken up as done for cloning of the spinach sucrose transporter (Riesmeier et al., 1992). This requires, however, that the metabolizing enzymes have been cloned and that they can be expressed in yeast. As an example, it has been attempted to clone a transporter of the natural products glucosinolates which are thiohydroximate-N-sulphates containing a thioglucose moiety by functional complementation using glucosinolates as S-source. It was found that yeast is not able to metabolize glucosinolates (S Chen and U Wittstock, unpublished results). Genetic engineering of yeast with either sulphatase or thioglucosidase activity was unsuccessful. Several sulphatases of bacterial origin were active towards synthetic substrates, but not towards glucosinolates, and the sulphatase from Helix pomatia which is known to hydrolyse glucosinolates could not be functionally expressed in yeast (Wittstock et al., 2000). Functional expression of a plant thioglucosidase inhibited yeast growth (Chen and Halkier, 1999). Thus it appears that cloning of a glucosinolate transporter by functional complementation of yeast is not feasible.
To circumvent this problem, a new method for expression cloning of transporter proteins in yeast has been developed. The method is based on the detection of intracellularly accumulated transporter substrate. In the current study, a radiolabelled substrate has been used, but other labelling principles such as fluorescence labelling might be applied. In addition, the substrate may be ions. Using the mutant yeast strain 22574d in combination with either a plasmid containing the AAP1 cDNA or an A. thaliana cDNA library we have demonstrated that 22574d colonies harbouring AAP1 can be detected by directly monitoring the accumulation of radiolabelled proline. A non-exhaustive screening of 60 000 cfu of the A. thaliana expression library by this method resulted in the isolation of two colonies harbouring AAP1. As AAP1 was the first amino acid transporter isolated from this library by complementation of yeast (Frommer et al., 1993), this shows that the efficiency of the uptake-based screening method described here is comparable to the efficiency of yeast complementation. Besides its general applicability, the method makes it possible to use yeast expression screening for cloning of transporters with substrates which are not metabolized by yeast.
As in yeast complementation, the prerequisites for the uptake-based strategy are that the given yeast strain does not have an uptake system for the substrate of the transporter of interest, that the yeast is not able to extracellularly metabolize the substrate into compounds which can be taken up, and that the substrate is not toxic for yeast. Additional prerequisites are that labelled substrate of adequate specific activity is available and that the transport activity is high enough to enable intracellular accumulation of detectable amounts of substrate. Although AAP1 expressed in yeast has high affinity for proline, 15 other proteogenic amino acids compete with proline for uptake by AAP1, a number of them with high efficiency (Frommer et al., 1993; Fischer et al., 1995). As the SD medium used in this study contained an amino acid drop-out mixture, non-labelled amino acids and L-[U-14C]proline have competed for uptake by AAP1. Accordingly, it can be expected that transport activity approximately 10-fold lower than the transport activity of AAP1 can be detected by this method if competitors are absent.
Extracellular metabolism of the substrate due to secretion of endogenous yeast enzymes lowers the efficiency of both functional complementation and the uptake-based method described here. The use of a yeast mutant deficient in such enzyme activities circumvents this problem. In the uptake-based method, such a yeast mutant can be used directly for screening. For functional complementation, however, metabolic activity restricted to the intracellular space has to be reintroduced into the respective mutant prior to complementation. For cloning of the spinach sucrose transporter by complementation, an invertase-deficient yeast strain was engineered with a truncated yeast invertase lacking the signal peptide (Riesmeier et al., 1992). The engineered strain, however, was able to grow on sucrose medium, although at a reduced level, probably due to leakage of invertase. In a second approach, a potato sucrose synthetase, which needs UDP as cosubstrate for cleavage of sucrose and, therefore, does not function extracellularly, was introduced into the invertase-deficient mutant (Riesmeier et al., 1992). It is a great advantage of the method presented here that genetic engineering in order to achieve metabolizing activity restricted to the interior of the cell is not needed.
Uptake of radiolabelled substrate has previously been used as a selection criterion in expression cloning of transporters in Escherichia coli (Krause et al., 1985) and Xenopus laevis oocytes (Hediger et al., 1987). The screening for the ADP/ATP translocator from Rickettsia prowazekii in E. coli transformed with a R. prowazekii cosmid library was performed in liquid cultures of individual clones (Krause et al., 1985). This limits the applicability of the method to cloning of high abundant cDNAs. Another limitation arises from the fact that there are no examples of functional expression of eukaryotic plasma membrane transporters in E. coli. The uptake-based screening as it is demonstrated in the present paper allows an efficient high-throughput screening for clones expressing eukaryotic transporter proteins in an easy-to-handle yeast system.
In summary, the method presented here can, in principle, be used to clone any plasma membrane transporter in yeast provided that labelled substrate is available. Whereas cloning of transporters by functional complementation requires that the substrate taken up is metabolized by yeast to promote growth, the method described here can be used to isolate transporters of substrates which are not metabolized. In particular, the method has great potential for the isolation of the many as yet unidentified transporters of, for example, secondary plant products.
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Acknowledgments |
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We would like to thank Professor Bruno Andre (Université Libre de Bruxelles, Belgium) for providing the yeast mutant, Professor Wolf B Frommer (University of Tübingen, Germany) for the AAP1 cDNA clone and Professor François Lacroute (Centre National de la Recherche Scientifique, Gif sur Yvette, France) for the Arabidopsis cDNA library. Dr Peter K Busk is thanked for valuable discussions. Financial support from the National Danish Research Foundation (UW) and the National Danish Research Council (SC) is gratefully acknowledged.
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Notes |
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1 Ute Wittstock and Sixue Chen contributed equally to the study.
2 To whom correspondence should be addressed. Fax: +45 3528 3333. E-mail: halkier{at}biobase.dk
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