COMMUNICATION
Identification of a Gene for a Polyamine Transport Protein in Yeast*

Hideyuki Tomitori, Keiko Kashiwagi, Kaori Sakata, Yoshimi Kakinuma, and Kazuei IgarashiDagger

From the Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

    ABSTRACT
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Abstract
Introduction
References

Properties of a membrane protein encoded by YLL028w were examined using yeast cells transformed with the gene. The transformed cells became resistant to polyamine toxicity, and the resistance was overcome by bafilomycin A1, an inhibitor of vacuolar H+-ATPase. Although spermine uptake activity of the transformed cells was almost the same as that of wild type cells, the uptake activity of vacuolar membrane vesicles from the transformed cells was higher than that from wild type cells. The transformed cells became resistant to MGBG (methylglyoxal bis(guanylhydrazone)) and paraquat, but not Ni2+ and Co2+, suggesting that the protein encoded by YLL028w is a transport protein specific for polyamines. When the YLL028w gene was disrupted by inserting the HIS3 gene, the cells became sensitive to polyamines, and spermine uptake activity of the vacuolar membrane vesicles decreased significantly. The accumulated spermine in YLL028w gene-disrupted cells decreased greatly compared with that in wild type cells. The results indicate that a membrane protein encoded by YLL028w (TPO1) is a polyamine transport protein on the vacuolar membrane.

    INTRODUCTION
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Abstract
Introduction
References

Polyamines, which are essential for cell growth, are regulated by biosynthesis, degradation, and transport (1-3). As for polyamine transport, properties of three polyamine transport systems were clarified by cloning the genes for these systems from Escherichia coli in our laboratory (4-7). Two of them were spermidine-preferential and putrescine-specific uptake systems, in which ATP was necessary as the energy source. The third system was involved in the excretion of putrescine by a putrescine-ornithine antiporter activity. In eukaryotic cells, the genes for a polyamine transporter have not been reported yet, although the genes for proteins which regulate polyamine transport are known. In yeast, genes encoding protein kinases which stimulate polyamine uptake have been reported (8-10). In animal cells, antizyme, which stimulates the degradation of ornithine decarboxylase (11, 12), also regulates polyamine transport negatively (13-15).

In this study, we looked for the polyamine transport gene in yeast. It has been reported recently that excretion of spermidine can be catalyzed by the Bacillus subtilis multidrug transporter Blt (16). Thus, we searched the amino acid sequence homology between Blt and proteins encoded by yeast genome (17) and detected four candidate genes; we then examined polyamine toxicity and transport activity using yeast transformed with the genes. A membrane protein on vacuoles, which catalyzes proton gradient-dependent polyamine (putrescine, spermidine, and spermine) transport (18), was encoded by one of the genes (YLL028w). This is the first report of the isolation of a gene for a polyamine transporter (TPO1) from eukaryotic cells.

    EXPERIMENTAL PROCEDURES

Yeast Strains and Culture Conditions-- Yeast strain YW5-1B (MATa, trp1, ura3-52, leu2-3, 112) and its polyamine transport-deficient mutant YTM22-8 (8) were grown aerobically at 30 °C in Mg2+-limited CSD (completely synthetic medium) in which 50 µM MgSO4 was supplemented as described previously (19). The gene YLL028w (TPO1) was amplified by polymerase chain reaction (PCR)1 using a 5'-end primer with a SalI site (5'-AAGTCGACGATCGTAGGAATTCCCTAAAGA-3') and a 3'-end primer with a BamHI site (5'-AGGGATCCTTTGTTCTGTAGTTGTGTGTCT-3'). After digestion with SalI and BamHI, the PCR product was inserted into the same restriction sites of plasmid YEp351 (20). The plasmid thus obtained (pYLL028w) was introduced into YW5-1B by the lithium acetate method of Ito et al. (21). A YLL028w (TPO1) gene-disrupted mutant (YTH27-1) of yeast strain YPH499 (MATa, ade2-101, his3-D200, leu2-801, trp1-D63, ura3-52) was generated by one-step gene disruption (22) using the YLL028w::HIS3 PCR product. The YLL028w::HIS3 cassette was constructed by inserting the HIS3 gene (1.8 kilobase pair) into the NcoI and AatI restriction sites of YLL028w. Correct disruption was verified by Southern blot analysis. Yeast strain YPH499 was also transformed by pYLL028w as described above.

Spermine Transport Assay with Intact Cells and Vacuolar Membrane Vesicles-- Yeast cells were harvested during the exponential phase (A540 = 0.5), washed twice with 5 ml of 20 mM Na-Hepes buffer (pH 7.2) containing 10 mM glucose and suspended at 2 mg dry weight/ml in the same buffer, and incubated at 30 °C. The reaction was started by the addition of [14C]spermine (37 MBq/mmol) at a final concentration of 100 µM, and 0.5-ml aliquots were filtered through cellulose acetate filters (pore size, 0.45 µm) at certain intervals (5, 10, and 20 min). The reaction was linear during incubation. The radioactivity trapped on the filters was counted in a liquid scintillation counter. Preparation of vacuolar membrane vesicles and the assay of spermine uptake were performed as described previously (18).

Measurement of Polyamine and Protein Contents-- Polyamine contents were determined with high performance liquid chromatography as described previously (23) after extraction with hot trichloroacetic acid. Protein was determined by the method of Lowry et al. (24).

    RESULTS AND DISCUSSION

Effect of the Membrane Protein Encoded by YLL028w on Polyamine Toxicity-- To identify a gene encoding a protein that catalyzes excretion of polyamines, we searched for amino acid sequence homology between Blt, a protein involved in the excretion of spermidine (16), and membrane proteins encoded by yeast genome (17), and detected four candidate genes, YLL028w, YBR180w, YKR105c, and YCR023c. The activity of proteins encoded by these genes was first tested by polyamine toxicity in yeast.

We reported previously that cell growth of yeast was inhibited by polyamines in magnesium-limited (50 µM Mg2+) synthetic medium (19) and that the inhibition of cell growth did not occur in a polyamine transport-deficient mutant YTM22-8 (8). Thus, if a protein catalyzing excretion of polyamines is encoded by one of the genes, polyamine toxicity in wild type cells should be decreased when transformed by that gene. As shown in Fig. 1, A-C, inhibition of cell growth of wild type yeast was observed by addition of 75 mM putrescine, 3 mM spermidine, or 0.3 mM spermine to the medium. However, cell growth of the polyamine transport-deficient mutant was not inhibited by the polyamines. When wild type yeast was transformed with the YLL028w gene using the multicopy vector YEp351, the transformed cells became resistant to polyamine toxicity. The resistance to polyamine toxicity was not observed in yeast transformed with YBR180w, YKR105c, and YCR023c. These results suggest that a membrane protein encoded by YLL028w is a polyamine-excreting protein on the plasma membrane or a polyamine uptake protein on the vacuolar membrane.


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Fig. 1.   Effect of YLL028w (TPO1) on cell growth in the presence and absence of polyamine. A-C, cell growth in the presence of 75 mM putrescine (PUT), 3 mM spermidine (SPD), or 0.3 mM spermine (SPM) was followed by measuring A540. open circle , wild type yeast YW5-1B/YEp351; , polyamine transport-deficient mutant YTM22-8; black-triangle, TPO1-transformed yeast YW5-1B/pYLL028w. D-F, cells were grown in the presence and absence of polyamine. open circle , wild type yeast YPH499 cultured without polyamine; bullet , wild type yeast YPH499 cultured with 65 mM putrescine (PUT), 1.5 mM spermidine (SPD), or 0.2 mM spermine (SPM); , TPO1-disrupted mutant YTH27-1 cultured without polyamine; black-square, TPO1-disrupted mutant YTH27-1 cultured with polyamine indicated above.

The above results were confirmed by making the YLL028w gene-disrupted mutant. The toxicity of 65 mM putrescine, 1.5 mM spermidine, or 0.2 mM spermine was much stronger in the mutant than in wild type yeast (Fig. 1, D-F).

Properties of the Membrane Protein Encoded by YLL028w (TPO1)-- Polyamine contents in wild type yeast cells and in YLL028w (TPO1, a gene for a transporter of polyamines)-transformed or -disrupted cells were compared. When TPO1 was transformed into YW5-1B cells, the accumulation of spermine in the cells cultured in the presence of 0.3 mM spermine increased significantly compared with that in wild type cells (Figs. 2A, 1 and 2). When TPO1 was disrupted in YPH499 cells, the final accumulation of spermine in the mutant YTH27-1 was much lower than that in wild type cells (Figs. 2A, 3 and 4).


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Fig. 2.   Polyamine contents (A) and spermine uptake activities of intact cells (B) and of vacuolar membrane vesicles (C) of yeast. Those were measured using YW5-1B (wild type), its TPO1-transformed yeast, YPH499 (wild type), and its TPO1-transformed or -disrupted mutant. For measurement of polyamines (A), cells were cultured in the presence and absence of spermine (0.2 or 0.3 mM) and harvested at A540 = 0.3 () or A540 = 1.2 (black-square). Data are shown as the average of duplicate experiments. For measurement of spermine uptake (B and C), cells were harvested at A540 = 0.5. Spermine uptake of intact cells was shown as the activity per dry weight of the cells. ATP-dependent spermine uptake activity in vacuolar membrane vesicles was shown as the value with ATP minus that without ATP. Values are mean ± S.D. of triplicate experiments.

Spermine uptake activities in wild type cells or TPO1-transformed or -disrupted cells were nearly equal (Fig. 2B). We then measured spermine uptake activity of vacuolar membrane vesicles, since the existence of proton potential-dependent polyamine transport system has been reported (18). As shown in Fig. 2C, vacuolar spermine uptake activity of TPO1-transformed cells was higher than that of wild type cells. Furthermore, the spermine uptake activity of vacuolar membrane vesicles prepared from TPO1-disrupted mutant YTH27-1 was lower than that of wild type cells. It is known that bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase (25), inhibits polyamine uptake of vacuolar membrane vesicles (18). The spermine uptake activity described above was inhibited by bafilomycin A1 (data not shown). Then, the effect of bafilomycin A1 on spermine toxicity was examined. As shown in Fig. 3C, attenuation of spermine toxicity by the membrane protein encoded by TPO1 was abolished by bafilomycin A1. Spermine toxicity in wild type cells was also increased by bafilomycin A1 (Fig. 3A), but this was not affected as much by bafilomycin A1 in polyamine transport-deficient cells (Fig. 3B).


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Fig. 3.   Effect of bafilomycin A1 on cell growth of wild type yeast (A), its polyamine transport-deficient mutant (B), and YLL028w (TPO1)-transformed yeast (C). open circle , none; bullet , with 0.3 mM spermine; black-square, with 0.3 mM spermine and 10 µM bafilomycin A1; black-triangle, with 0.3 mM spermine and 50 µM bafilomycin A1.

Substrate specificity of the membrane protein encoded by YLL028w was examined next (Fig. 4). Although toxicity of polyamine analogues, MGBG and paraquat, was attenuated by the protein, toxicity of Ni2+ and Co2+ was not.


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Fig. 4.   Effect of cations (1 mM MGBG (A), 0.5 mM paraquat (B), 0.05 mM Ni2+ (C), and 0.05 mM Co2+ (D)) on cell growth of wild type yeast and YLL028w (TPO1)-transformed yeast. open circle , wild type yeast YW5-1B/YEp351; bullet , wild type yeast YW5-1B/YEp351 cultured with cations; , TPO1-transformed yeast YW5-1B/pYLL028w; black-square, TPO1-transformed yeast YW5-1B/pYLL028w cultured with cations.

The results, taken together, indicate that a membrane protein encoded by YLL028w (TPO1) is a polyamine transport protein on the vacuolar membrane. However, the change of spermine uptake activity of vacuolar membrane vesicles in TPO1-transformed or -disrupted cells was small, suggesting that there is at least one more polyamine transport protein on the vacuolar membrane.

Characteristics of the Membrane Protein Encoded by YLL028w (TPO1)-- The YLL028w (TPO1) is located on chromosome XII and encodes a membrane protein consisting of 586 amino acid residues (17). The protein has 12 putative transmembrane segments, and three glutamic acids, which may interact with polyamines, are located in similar positions to those of PotE, a putrescine excreting protein in E. coli (6). Polyamine transport in yeast is positively regulated by protein kinases (PTK1 and PTK2) (8-10). The activity of our polyamine transport-deficient mutant YTM22-8 was recovered by PTK2 in a single-copy vector (9) and by PTK1 in a multicopy vector (8). Thus, we hypothesize that polyamine transport on the plasma membrane is regulated by PTK2 and that on the vacuolar membrane by PTK1. When amino acid sequences of the yeast membrane protein and PotE were compared, the yeast protein possessed a longer hydrophilic NH2-terminal region, in which many serine and threonine residues are included. Thus, the NH2-terminal region of the protein may be important for regulation by PTK1.

    ACKNOWLEDGEMENT

We are grateful to Dr. A. J. Michael for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan and by Yamanouchi Foundation for Research on Metabolic Disorders, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-43-290-2897; Fax: 81-43-290-2900; E-mail: iga16077{at}p.chiba-u.ac.jp.

The abbreviations used are: PCR, polymerase chain reaction; MGBG, methylglyoxal bis(guanylhydrazone).
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