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J Biol Chem, Vol. 274, Issue 6, 3265-3267, February 5, 1999
From the Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
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ABSTRACT |
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 Top
 Abstract
 Introduction
References
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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.
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.
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).
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.
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 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).
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.
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.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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.
, wild type yeast YW5-1B/YEp351; 
, polyamine transport-deficient
mutant YTM22-8; 
, TPO1-transformed yeast
YW5-1B/pYLL028w. D-F, cells were grown in the
presence and absence of polyamine. , wild type yeast YPH499 cultured
without polyamine; 
, 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; 
, TPO1-disrupted mutant
YTH27-1 cultured with polyamine indicated above.
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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 (). 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.
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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). , none; , with 0.3 mM
spermine; , with 0.3 mM spermine and 10 µM
bafilomycin A1; , with 0.3 mM spermine and
50 µM bafilomycin A1.
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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. , wild type
yeast YW5-1B/YEp351; , wild type yeast YW5-1B/YEp351 cultured with
cations; , TPO1-transformed yeast
YW5-1B/pYLL028w; , TPO1-transformed yeast
YW5-1B/pYLL028w cultured with cations.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. A. J. Michael for critical reading of the manuscript.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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Top
Abstract
Introduction
References
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