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

Properties of a membrane protein encoded byYLL028w 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 byYLL028w is a transport protein specific for polyamines. When the YLL028w gene was disrupted by inserting theHIS3 gene, the cells became sensitive to polyamines, and spermine uptake activity of the vacuolar membrane vesicles decreased significantly. The accumulated spermine in YLL028wgene-disrupted cells decreased greatly compared with that in wild type cells. The results indicate that a membrane protein encoded byYLL028w (TPO1) is a polyamine transport protein on the vacuolar membrane.

Polyamines, which are essential for cell growth, are regulated by biosynthesis, degradation, and transport (1)(2)(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)(14)(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.
Spermine Transport Assay with Intact Cells and Vacuolar Membrane Vesicles-Yeast cells were harvested during the exponential phase (A 540 ϭ 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 [ 14 C]spermine (37 MBq/mmol) at a final concentration of 100 M, and 0.5-ml aliquots were filtered through cellulose acetate filters (pore size, 0.45 m) 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).

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 M Mg 2ϩ ) 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.
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).
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 poly- amine 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 A 1 , a specific inhibitor of vacuolar H ϩ -ATPase (25), inhibits polyamine uptake of vacuolar membrane vesicles (18). The spermine uptake activity described above was inhibited by bafilomycin A 1 (data not shown). Then, the effect of bafilomycin A 1 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 A 1 . Spermine toxicity in wild type cells was also increased by bafilomycin A 1 (Fig. 3A), but this was not affected as much by bafilomycin A 1 in polyamine transport-deficient cells (Fig. 3B).
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 Ni 2ϩ and Co 2ϩ was not.
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 chromo-some 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 NH 2 -terminal region, in which many serine and threonine residues are included. Thus, the NH 2 -terminal region of the protein may be important for regulation by PTK1.