CURRENT MICROBIOLOGY Vol. 43 (2001), pp. 93–95 DOI: 10.1007/s002840010267

Current Microbiology An International Journal © Springer-Verlag New York Inc. 2001

The Saccharomyces cerevisiae MLF6/YPL244C Gene, Which Encodes a Possible Yeast Homologue of Mammalian UDP-Galactose Transporter, Confers Resistance to the Immunosuppressive Drug, Leflunomide Hiro-aki Fujimura Laboratory of Advanced Technology, Discovery Research Laboratories, Nippon Hoechst Marion Roussel, 1-3-2 Minamidai, Kawagoe 350-1165, Japan Received: 13 October 2000 / Accepted: 4 January 2001

Abstract. The immunosuppressant leflunomide (LFM) inhibits the growth of cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of eukaryotic microorganism, Saccharomyces cerevisiae. As a first step to elucidate the molecular mechanism of LFM action in human, yeast gene which suppresses the anti-proliferative effect when in increased copy number was cloned and designated MLF6 for multicopy suppressor of LFM sensitivity. DNA sequencing analysis revealed that the MLF6 gene is identical to the YPL244C gene which encodes a possible yeast homologue of human UDP-galactose transporter. The disruption of the MLF6 gene increased the sensitivity of yeast cells to the drug.

The immunosuppressant leflunomide (LFM), an isoxazol derivative, is a novel anti-inflammatory and immunosuppressive drug that has been shown to be effective in preventing and treating the autoimmune diseases in animals and the reaction leading to organ transplantation rejection [1]. Many of the immunosuppressive effects of LFM can be attributed to its inhibitory effect on the activity of many cytokines most likely through receptor expression and signal transduction [1]. The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin inhibit the intermediate steps in signal transduction leading to T-cell activation [11]. CsA and FK506 block the activation of quiescent T cells (transition of the G0 to the G1 of cell cycle) by interfering with the T cell receptor-mediated signal transduction pathway [2,17]. Rapamycin inhibits subsequent progression from G1 to S phase of the cell cycle by blocking the response to the lymphokine IL-2 [2]. The yeast Saccharomyces cerevisiae has been a model organism to elucidate the molecular mechanism of the action of many drugs including immunosuppressants. CsA and FK506 inhibit recovery from ␣-mating factor arrest

through interaction strikingly similar to those involved in the inhibition of T-cell activation [12]. Rapamycin arrests the S. cerevisiae cell cycle in G1 phase by binding to a highly conserved FK506-binding protein (FKBP)[9] and then by inhibiting TOR1, yeast homologue of phosphatidylinositol-3-phosphate kinase [13]. FK506 also blocks the import of amino acids in yeast cells [16]. My earlier study showed that S. cerevisiae was susceptible to LFM [6]. I report here the cloning of the MLF6 gene which confers the LFM resistance in Saccharomyces cerevisiae when in increased copy number and suggest a possibility that LFM could inhibit the transport of UDP-galactose by affecting Mlf6 protein.

Correspondence to: Dr. Hiro-aki Fujimura; email: Fujimura.Hiroaki@ organon.co.jp

Isolation of the MLF6 gene. A YEp213-based yeast genomic library, kindly given by A. Sakai (Mitsubishi Kasei Institute of Life Sciences)

Materials and Methods Yeast strains and culture conditions. The S. cerevisiae strains used in this study were: DH17-1C (MATa ura3 leu2 his4 trp1 gal2 lfs1)[6,7] and XF106 (MATa/MAT␣ ura3/ura3 leu2/leu2 trp1/⫹ ⫹/his4). Cultivation and maintenance of cells were carried out in YEPD medium (1% yeast extract, 2% peptone, and 2% glucose) as described previously [18]. Selective medium for drug sensitive test was SD medium (2% glucose, 0.67% yeast nitrogen base w/o amino acids) supplemented with bases and amino acids [18] and containing 50 ␮g of LFM per ml. Media were solidified by addition of 2% agar.

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Fig. 1. Subcloning analysis of the MLF6 gene. The restriction map of the original MLF6 isolate (p17J3) is shown on the top line. A bar indicating the relative scale in kilobase pairs is noted on the left-hand side of the figure. Restriction sites are abbreviated as: E. EcoRI; H, HindIII; S, SalI; X, XbaI. The thick horizontal lines represent DNA fragments present in the various subclones. The relative resistance phenotype of transformants harboring plasmids in sensitive S. cerevisiae strain (DH17-1C) is shown by photographs. Phenotype was tested based on their ability to grow in SD minimal agar medium in the absence (⫺Lef) or presence (⫹Lef) of 50 ␮g of LFM per ml.

was introduced into DH17-1C by high efficiency transformation method [8]. Leu⫹ transformants were selected on minimal medium and tested for LFM resistance by replica-plating on the leucine deficient medium containing 50 ␮g of LFM per ml. Plasmid which conferred LFM resistance was recovered as described previously [5] and reintroduced into DH17-1C for confirmation of LFM resistance. Plasmid preparation was made from drug-resistant transformants and transformed into Escherichia coli, and DNA was amplified and isolated as described above [15]. Isolate was analyzed by restriction digestion and subsequent gel electrophoresis. Plasmids. Plasmid p17J3 is an original isolate. By filling-in the XbaI site with p17J3, p17J3⌬X was constructed By inserting the 1.6-kb HindIII fragment of p17J3 into the HindIII site of pUC18, pMLF6-H1 was constructed. By inserting the 2.7-kb EcoRI-XbaI fragment of p17J3 into the EcoRI and XbaI sites of pUC18, pMLF6-EX was constructed. By inserting the 1.1-kb HindIII fragment containing the URA3 gene into the HindIII site of pMLF6-EX, pMLF6-EX1⌬URA3 was constructed. YEpMLF6-SX was constructed by inserting the 4.1-kb SalI-XbaI fragment of p17J3 into the SalI site of YEp213. For DNA sequencing, plasmids pMLF6-H1 and pMLF6-EX were used as templates for determination of DNA sequence and analyzed. Sequencing was performed using an ALF automatic DNA sequencer (Pharmacia Co.). Construction of the mlf6 disruptants. Disruption of the MLF6 gene was carried out by the one-step method [14]. pMLF6-EX1⌬URA3 was digested with EcoRI and XbaI and introduced into XF106 by genetic transformation [10]. Micromanipulation and dissection of asci were done according to Sherman et al. [18].

Results and Discussion The method for cloning a gene which suppresses the anti-proliferative activity of LFM relies on an increased dosage of a wild-type gene product to overcome the anti-proliferative effect of the drug [3,4,19]. The LFM supersensitive lfs1 strain DH17-1C was transformed with genomic library constructed in the multicopy plasmid YEp213. Leu⫹ transformants were grown on SD-leu medium and replica-plated on the same selective medium containing LFM (50 ␮g/ml). Approximately 20, 000 Leu⫹ clones were examined for resistance to LFM,

and one Leu⫹ transformant showed resistance to LFM. Upon amplification in E. coli, plasmid reconstituted the resistance to LFM (50 ␮g/ml). The gene was designated as MLF6 (multicopy suppressor of leflunomide). Restriction mapping of the plasmid indicated that the insert was approximately 4.5-kb in length (Fig. 1). To identify the chromosomal location of the insert cloned in plasmid p17J3, the subcloned fragments were sequenced. The insert was found to contain the three open reading frames (ORFs) of chromosome XVI (Fig. 1). To show which gene corresponds to the MLF6 gene, constructed plasmids were introduced into yeast cells and examined for their activity to confer LFM resistance. The 2.7-kb SalI-XbaI fragment of p17J3 failed to confer the LFM resistance. Deletion of the XbaI site which is located within the ORF of the YPL244C gene was found to destroy the ability of the MLF6 gene to confer LFM resistance (Fig. 1). Thus, I concluded that the MLF6 gene corresponds to the YPL244C gene. The YPL244C gene codes for a protein of 339 amino acids. To analyze the biological function of the MLF6 gene, the deduced protein sequence was analyzed. As shown in Fig. 2, the percent amino acid identity between Mlf6 protein and human GDP-galactose transporter related isozyme was 26%, and the percent similarity was 47%, suggesting that Mlf6 protein may be a yeast homologue of UDP-galactose transporter related isozyme and function to transport GDP-galactose in yeast cells. To explore the function further, one-step gene disruption was performed. Diploid strain XF106 (ura3/ura3) was transformed with DNA fragment containing the mlf6::URA3 gene, and resultant ura⫹ transformant was sporulated and the spores were dissected. Every ascus produced four viable spores, two ura⫹ and two ura⫺, indicating that the MLF6 gene is not essential for cell growth. The effect of the disruption on sensitivity to LFM was investi-

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Literature Cited

Fig. 2. Amino acid sequence alignment of the MLF6/YPL244C gene product (upper row) and human UDP-galactose transporter related isozyme 1 (lower row). The percent amino acid identity between the sequences is 26%.

Fig. 3. Drug sensitivity of the mlf6 disruption mutants. The MLF6 and mlf6 segregants were grown on an agar medium in the absence (⫺Lef) or presence (⫹Lef) of leflunomide (50 ␮g/ml), followed by photography.

gated. As shown in Fig. 3, the disruption of the MLF6 gene caused an increase in sensitivity to LFM. At present the physiological function of Mlf6 is unknown. However, the present finding may suggest that part of the immunosuppressive effect of LFM could be attributed to the inhibition of glycosylation through blocking the transport of UDP-galactose in human. Elucidation of the direct interaction between Mlf6 protein and LFM may uncover a possible in vivo function of the yeast MLF6 gene product and the human UDP-galactose transporter. ACKNOWLEDGMENT The author thanks Dr. R. Bartlett for critical reading of the manuscript prior to publication.

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