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INTRODUCTION |
Polyamines are essential compounds occurring in virtually all
prokaryotic and eukaryotic cells (1). Because these compounds are toxic
in higher concentrations, their intracellular content is tightly
regulated by controlling biosynthesis and degradation and also by
transport into intracellular storage compartments or out of the cell.
In the yeast Saccharomyces cerevisiae the polyamines
putrescine, spermidine, and spermine are found. All three compounds are
actively transported into the vacuole by a substrate/nH+
antiport mechanism (2). Four transport proteins, Tpo1p-Tpo4p, have
been postulated to be responsible for the polyamine transport capability of the yeast vacuolar membrane (3, 4). According to their
protein sequences, these four proteins are all members of a family of
multidrug resistance transporters within the major facilitator
superfamily. Tpo1p was identified by its sequence similarity to the
Bacillus subtilis multidrug transporter Blt, which is
involved in spermidine excretion (5). The other transporters of this
group were found on the basis of sequence similarity to Tpo1p (4). The
function of all Tpo proteins was deduced from the phenotype of mutant
strains. Their overexpression led to an increased tolerance for
polyamines added to the growth medium, whereas a respective deletion
rendered the cells more sensitive to polyamines. Evidence for their
participation in vacuolar polyamine uptake, however, was largely indirect.
In the course of a project to identify new vacuolar transporters
belonging to the class of secondary carriers, we were looking for a
well characterized carrier of this type in the yeast vacuolar membrane
to use as a control for localization assays. We chose Tpo1p because of
its sequence similarity to the transport proteins we were interested
in, in particular to Ycr023p. In an analysis of their subcellular
distribution, however, we found that all Tpo transporters are
actually localized in the cytoplasmic membrane. This observation
encouraged us to perform a detailed study on Tpo1p function. The
results obtained suggest that at least Tpo1p participates in
polyamine export out of the cell.
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EXPERIMENTAL PROCEDURES |
Strains and Media--
The haploid S. cerevisiae
strain 23344c (MAT
ura3) was used as genetic background
for all of the experiments. This strain is isogenic with
1278b (6)
and was kindly provided by Bruno André (Brussels, Belgium). The
cells were grown aerobically at 30 °C. Preparation of yeast-rich
(YPD) and synthetic complete minimal media followed standard
recipes (7). Growth assays on solid media were performed using a
modified citrate-buffered yeast minimal medium (8) in which the
Mg2+ content was limited to 50 µM to enhance
polyamine sensitivity (9). For polyamine export experiments and for the
preparation of vacuolar vesicles, a modified CBS medium
described by Verduyn et al. (10) was used, containing 20 g/liter glucose and 5 g/liter NH4SO4. Uracil
auxotrophic strains were grown in the presence of 20 mg/liter uracil.
The CBS medium was buffered with either 1% succinic acid adjusted to
pH 5.8 with NaOH or 50 mM potassium phthalate at pH 5.5
Gene Modifications--
Replacement of the TPO1 gene
by lacZ was done via PCR-based gene targeting using the gene
disruption cassette encoded by plasmid pUG6lacZ (11). The plasmid
contains the lacZ gene from Escherichia coli,
followed by the dominant kanMX marker, and is a derivative of pUG6 described by Güldener et al. (12). The
sequences of the primers used for the amplification of the disruption
cassette were
5'-TTTTTTTTAGTCAAAGAAGCAAGAGAAAACTAGACAGAGACAATGTTCGTACGCTGCAGGTCGAC and
5'-AAAAATGCAAATATAGAAAGAGCATGATTTCTGCTTTTCTTTTTCGCATAGGCCACTAGTGGATCTG.
Genomic HA1 tagging of the
four TPO genes and of the open reading frame YCR023c was
performed by using the plasmid pUG6-HA (13), which encodes three tandem
repeats of a HA epitope followed by kanMX. Via PCR a DNA
molecule was generated, consisting of a 3xHA-kanMX marker
cassette flanked by short regions homologous to the end of the
respective gene. The PCR primers used for this purpose consisted of 45 nucleotides corresponding to the genomic sequence ultimately upstream
and downstream, respectively, of the stop codon of the gene to be
tagged, followed by 20 nucleotides homologous to the pUG6-HA plasmid to
amplify the epitope and the kanMX marker. After
transformation of the 1.7-kb PCR product into strain 23344c and
selection for resistance to G418 (200 mg/l) on YPD agar plates, the
stop codon of the respective gene was usually replaced by
3xHA-kanMX through homologous recombination. All of the gene
modifications were verified by diagnostic PCR. A list of the strains
used and generated in this study is given in Table
I.
Plasmid Constructions--
The yeast expression vector pDR199
was obtained from Wolf Frommer (Tübingen, Germany). It is a
derivative of pDR195 (14), which in turn was generated from YEplac195
(15). The plasmid contains a copy of the PMA1 promoter and
the terminator region of the ADH1 gene. The TPO1
gene was amplified via PCR using genomic DNA of strain 23344c as
template and cloned between promoter and terminator using an
XmaI and an XhoI site. The PCR primers for TPO1 were 5'-GCGTCCCCGGGATGTCGGATCATTCTCCCATT
and 5'-GCGTCCTCGAGTTAAGCGGCGTAAGCATACTT. The underlined
sequences indicate the XmaI and XhoI sites,
respectively. The TPO1-3xHA fusion gene was amplified via
PCR with genomic DNA from strain RK 25 as template using the same 5'
primer as for the amplification of the unmodified TPO1. The
sequence of the 3' primer was
GCGTCCTCGAGTTAGGCGGCGTAGTCAGGAAC, with the XhoI site underlined. The PCR fragment was inserted into pDR199 by using the
XmaI and the XhoI site between PMA1
promoter and ADH1 terminator. Accuracy of the constructs was
verified by sequencing. The plasmids were transformed into yeast
according to Gietz et al. (16).
Sucrose Density Gradient Centrifugation--
Subcellular
localization of triple HA-tagged proteins was determined following the
protocol of Sorin et al. (17). The cells were grown in 600 ml of YPD medium to A600 of 2-3.
NaN3 (10 mM) was added prior to harvesting, and
the culture was chilled on ice. The cells were converted to
spheroplasts by adding lysing enzymes (Sigma) in a concentration of 1 mg/ml in S/K buffer (1.2 M sorbitol, 100 mM
potassium phosphate, pH 7.5) and incubating for 45 min at 30 °C. The
spheroplasts were suspended in 4 ml of lysis buffer (0.3 M
sorbitol, 20 mM triethanolamine acetate, pH 7.2, 1 mM EDTA, supplemented with a commercially available
protease inhibitor mixture (Complete, EDTA-free; Roche Molecular
Biochemicals) and homogenized by 30 strokes of a Wheaton A Dounce
homogenizer. Unlysed cells were removed by centrifugation (800 × g for 3 min at 4 °C), and the supernatant was layered on
top of a noncontinuous gradient ranging from 18 to 54% (w/v) sucrose
in 10 mM Hepes, pH 7.1, 1 mM EDTA (10 steps of
4% difference each). The gradients were centrifuged for 2 h at
40,000 rpm (4 °C) in a Beckmann SW41 Ti rotor and fractionated
manually from top to bottom (12 fractions of 1 ml each). Isolated
fractions were diluted (1:5) in 100 mM Tris-Cl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and the membranes were pelleted by ultracentrifugation (100,000 × g for
2 h at 4 °C). The resulting pellets were incubated on ice (30 min) in 400 µl of Tris buffer (see above) containing 5 M
urea. They were again centrifuged (17,000 × g for 45 min at 4 °C) and finally resuspended in 100 µl of SDS sample buffer.
Immunoblotting--
The proteins were separated by SDS-PAGE
(12%) and electroblotted onto nitrocellulose membranes using a semidry
transfer system. For immunodetection a monoclonal anti-HA (Roche
Molecular Biochemicals), a monoclonal anti-Vph1p (kindly provided by
Patricia M. Kane, Syracuse, NY), and a polyclonal anti-Pma1p (kindly
provided by Bruno André, Brussels, Belgium) were used as first
antibody. The respective secondary antibody coupled to horseradish
peroxidase and the enhanced chemiluminescence detection system
(Invitrogen) was used for visualization.
Polyamine Transport in Intact Yeast Cells--
The cells were
grown overnight in synthetic complete minimal medium, harvested at
A600 = 1.0, washed twice in the same volume of
20 mM Na/Hepes buffer, pH 7.2, containing 10 mM
glucose and resuspended at A600 = 1.0 in the
same buffer. Transport assays were done at 30 °C and started by
adding [14C]spermine to a final concentration of 20 or
100 µM. 100-µl aliquots were filtered at defined time
points through nitrocellulose filters that were preincubated in 100 mM LiCl containing 1 mM spermine. The
radioactivity trapped on the filters was determined in a liquid scintillation counter.
Preparation of Vacuole-derived Vesicles and Transport
Measurements--
The preparation of vacuolar vesicles was done
following a slightly modified protocol of Ohsumi and Anraku (18). The
cells were grown in CBS medium buffered with 50 mM
potassium phthalate, pH 5.5, and supplemented with 10 mM
putrescine and 1 mM of each spermidine and spermine to an
A600 of 2-3. The cells were harvested and
resuspended in 100 mM Tris-sulfate, pH 9.4, 10 mM dithiothreitol to yield a concentration of 0.5 g of
cell fresh weight/ml. The cells were shaken for 10 min at 30 °C,
centrifuged, and resuspended to 0.15 g of cell fresh weight/ml in
SOB (1.2 M sorbitol, 5 mM MES-Tris, pH 6.9).
The spheroplasts were generated by incubation with lysing enzymes
(Sigma) (1 mg/5 × 108 cells) for 30 min at 30 °C.
The spheroplasts were washed twice in SOB and resuspended in buffer A
(12% Ficoll, 10 mM MES-Tris, pH 6.9, 100 µM
MgCl2), protease inhibitors (Complete; Roche Molecular Biochemicals) were added, and the suspension was homogenized by seven
strokes of a Wheaton A Dounce homogenizer. The resulting suspension was
transferred to a centrifuge tube, overlaid with half the volume of
buffer A, and centrifuged at 60,000 × g for 1 h
and 15 min. The white floating layer that appeared after centrifugation was transferred to an ultracentrifuge tube, adjusted with buffer A (see
above) to 6 ml, and overlaid with 6 ml of buffer B (8% Ficoll, 10 mM MES-Tris, pH 6.9, 100 µM
MgCl2). After further centrifugation (60,000 × g for 1 h and 15 min), vacuolar vesicles formed a white layer on top of the solution. They were aspirated, suspended in 100 mM MES-Tris, pH 6.9, 100 mM KCl, 20 µM MgCl2, frozen under liquid nitrogen, and
stored for further use at
80 °C. The enzyme assays were done
according to Roberts et al. (19). Phenylalanine transport
measurements were performed according to Sato et al. (20),
and spermine import was determined after Kakinuma et al. (2).
Export Measurements of Accumulated Spermidine--
The cells
were grown overnight in succinate-buffered CBS medium in the presence
of 10 mM spermidine and harvested at
A600 = 1.3-1.5 by centrifugation. The cell
pellets were washed three times in medium without polyamines but
containing only 1 g/liter NH4SO4 and finally
resuspended in the same medium. The cell suspension was incubated at
30 °C with agitation, and aliquots were taken at defined time
points. To analyze spermidine efflux, the cells were immediately
separated from the medium by centrifugation (11,000 × g for
3 min), and released spermidine was quantified using high performance
liquid chromatography according to Price et al. (21).
Determination of Total Polyamine Content--
Extraction of
total polyamines was performed according to Tomitori et al.
(4). Yeast cells were incubated in 10% trichloroacetic acid at
65 °C for 1 h. Derivatization and quantification by high performance liquid chromatography were done as described above.
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RESULTS |
Tpo1p Is Localized in the Cytoplasmic Membrane--
In the course
of an approach to identify new vacuolar transporters, we determined the
subcellular localization of various putative transport proteins that
have been fused C-terminally to a triple HA epitope tag. A version of
Tpo1p with the same tag was generated as bona fide control,
because we considered it to be an established vacuolar transporter (3,
4). The intracellular localization was determined by sucrose density
gradient centrifugation of membrane preparations isolated from yeast
cells carrying the respective fusion construct as the only gene copy.
Defined fractions were separated by SDS-PAGE and subsequently analyzed
by immunoblotting (Fig. 1). Although the
3xHA-tagged version of Ycr023p co-localized with the 100-kDa subunit of
the vacuolar ATPase (Vph1p), the Tpo1p-3xHA fusion co-localized with
the plasma membrane ATPase (Pma1p), thus raising the question of
whether tagging of Tpo1p leads to mislocalization, although the HA tag
is not known to direct proteins to the plasma membrane.

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Fig. 1.
Subcellular localization of Tpo1p and
Ycr023p. The cell lysates were separated in a sucrose gradient as
described under "Experimental Procedures." The gradient was
fractionated from top (fraction 1) to bottom (fraction
12) and analyzed by SDS-PAGE followed by immunoblotting.
A, Western blot analysis of a fractionation of strain RK 25 expressing a triple-HA-tagged version of Tpo1p. The blot was probed
with anti-HA antibodies (uppermost panel) or with antibodies
raised against the proteins listed on the right.
B, fractionation of a lysate from strain WF 7 expressing a
Ycr023p-3xHA fusion.
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To get further evidence we tested the functionality of the HA fusion of
Tpo1p. Disruption of TPO1 leads to increased polyamine sensitivity of the mutant cells, when grown under
Mg2+-limiting conditions (3). This phenotype should be
rescued by expression of the HA-modified version of the transporter if this fusion is functional. We thus transformed a plasmid encoded version of TPO1-3xHA into a strain in which TPO1
had been replaced by a lacZ-kanMX deletion cassette. The
fused gene on the plasmid was expressed under the control of the
PMA1 promoter. The resulting yeast cells were tested for
spermidine sensitivity in comparison with the wild type and the
tpo1::lacZ strain, each transformed with the empty vector (Fig. 2). We found
that synthesis of the HA-tagged Tpo1p leads to markedly increased
spermidine tolerance. Because the functionality of the HA fusion of
Tpo1p could be demonstrated, we tried to get further evidence for the
Tpo1 protein being located in the plasma membrane, where it should
contribute to polyamine transport.

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Fig. 2.
Functionality of tagged Tpo1p. Cell
growth was assayed on solid synthetic medium in which magnesium content
has been limited to 50 µM. Spermidine was added at a
concentration of 6 mM (right half); the
same strains have also been applied to a plate without spermidine to
ensure uniform growth (left half). The plate without
spermidine was incubated at 30 °C for 2 days, and the plate with
spermidine was incubated for 6 days at the same temperature. The growth
of wild type and tpo1::lacZ cells each
transformed with the empty vector (pDR199) is shown together with the
tpo1::lacZ strain transformed with a
plasmid encoding a Tpo1p-3xHA fusion (pDR199
TPO1-3xHA).
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The Polyamine Uptake Rate of Intact Cells Is Not Influenced by TPO1
Deletion--
Because the localization of Tpo1p in the plasma membrane
was contradictory to the proposed function in vacuolar polyamine transport, we tested first whether polyamine import into cells is
influenced by TPO1 deletion. An earlier report had indicated that a TPO1 deletion strain shows spermine import rates
similar to wild type cells (3), whereas a decreased import rate in TPO1 deletions was reported later (4). We found, however, no difference in spermine uptake activity between the
tpo1::lacZ strain and wild type using
100 µM (Fig. 3) or 20 µM spermine (not shown), suggesting that Tpo1p is not
involved in polyamine import of yeast cells and/or that Tpo2p-Tpo4 may
complement the knockout of Tpo1p.

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Fig. 3.
Spermine uptake of intact yeast cells.
Time course of spermine uptake of wild type ( ) and
tpo1::lacZ cells ( ). The cells have
been grown in synthetic complete minimal medium without added
polyamines. Transport was assayed at a spermine concentration of 100 µM. The mean values from five determinations are given
together with standard deviations.
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Vacuolar Vesicles from a TPO1 Disruption Strain Do Not Show Any
Impairment in Polyamine Uptake--
In view of several reports
describing Tpo1p as vacuolar polyamine importer, we tried to account
for an impairment in vacuolar polyamine uptake after TPO1
deletion. Consequently, we prepared vacuolar vesicles from wild type
cells and tpo1::lacZ mutants, respectively, grown in the presence of 10 mM putrescine, 1 mM spermidine, and 1 mM spermine. Polyamines
were added to the growth medium because a stimulatory impact of
externally added polyamines on TPO1 expression had been
reported recently (4). The polyamine concentrations employed led to a
markedly increased intracellular polyamine level (about 4-fold in the
case of putrescine and spermine, and about 2-fold for spermidine),
which should be high enough to enhance the expression of
TPO1.
We optimized the protocol for the isolation of vacuolar vesicles, which
led to preparations of high purity (Table
II). The ATPase activity of the vesicle
preparation was completely inhibited by the addition of the
V-ATPase-specific inhibitor concanamycin A (not shown), proving that
significant contaminations of plasma membrane and mitochondria were
virtually absent. The vesicles were used to determine the uptake of
amino acids (20) and spermine (2). Because phenylalanine uptake
measurements in particular were highly reproducible in isolated
vacuolar vesicles, we used this carrier activity to normalize the
spermine uptake rate and thus to eliminate possible variations caused
by the vesicle preparation, a strategy that was not employed in the
original report of Tomitori et al. (4), who reported a
slightly decreased import activity for vacuolar vesicles prepared from
a TPO1 deletion strain compared with wild type. But this
correction might be crucial, because we found that although the
absolute values varied slightly for each preparation, the relative
uptake rates measured were essentially the same for the two strains
(Fig. 4). Furthermore, the maximal amount
of accumulated spermine was very similar for the two vesicular preparations when normalized to the maximum of accumulated
phenylalanine. We thus conclude that the TPO1 deletion had
no effect on spermine import into vacuolar vesicles.

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Fig. 4.
Relative spermine uptake of isolated vacuolar
vesicles. Vacuolar vesicles from wild type (filled
bars) and tpo1::lacZ cells
(open bars) have been prepared. The cells have been grown in
phthalate-buffered CBS medium supplemented with 10 mM
putrescine and 1 mM of both spermidine and spermine. Total
uptake and the uptake rate of the vesicles were determined for spermine
and phenylalanine. The ratio of spermine and phenylalanine uptake and
uptake rate, respectively (relative uptake), is shown at the
ordinate.
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Polyamine Export of Growing Yeast Cells--
The absence of the
TPO1 gene causes increased polyamine sensitivity, indicating
a function for Tpo1p in the detoxification of polyamines (Fig. 2 and
Ref. 3). Detoxification in general can be mediated either by transport
into the vacuole or out of the cell across the plasma membrane. The
results presented so far favor the latter possibility. Thus, we
analyzed the putative polyamine efflux mediated by Tpo1p.
Because little is known about polyamine export in budding yeast, we
investigated first whether polyamines might be released into the medium
during growth, as has been reported for other microorganisms (22, 23).
Wild type and tpo1::lacZ cells were grown from A600 = 0.5 to stationary phase in
succinate-buffered CBS medium. Putrescine and spermidine excreted into
the medium were quantified. Spermine could not be determined as a
contaminant in the synthetic growth medium and interfered with the
quantitative HPLC analysis. We found that wild type and mutant cells in
fact excrete putrescine but not spermidine until they reach the diauxic shift (Fig. 5) after ~24 h. The
putrescine release of the two strains was indistinguishable, suggesting
that Tpo1p is not involved in this process.

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Fig. 5.
Putrescine export of growing yeast
cells. Wild type ( ) and
tpo1::lacZ cells ( ) have been grown
in succinate-buffered CBS medium from A600 = 0.5 to stationary phase. A, growth of wild type and
TPO1 disruption cells. B, putrescine content of
the medium.
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Release of Accumulated Spermidine Is Impaired in a tpo1
Mutant--
Although spermidine is the most abundant polyamine in
yeast cells (Ref. 9; see also Fig.
6B), we did not observe
release of this solute under normal growth conditions. To challenge
yeast cells for spermidine export, we increased the intracellular
spermidine content by growing cells in the presence of this compound.
The cells were cultured in the presence of 10 mM spermidine
in succinate-buffered CBS medium under nonlimiting
Mg2+-conditions, where both wild type and mutants are more
tolerant to polyamine stress, and the spermidine concentrations applied are thus not toxic for both strains. During the incubation the total
intracellular spermidine content of yeast cells in the logarithmic growth phase increased significantly from about 8 to 25 nmol/mg cell
dry mass (Fig. 6B). After washing and resuspending in
polyamine-free medium, the accumulated spermidine was released. We
found a significant difference between wild type and tpo1
deletion mutant under these conditions. Spermidine efflux of the
tpo1::lacZ cells was significantly decreased as compared with the wild type (Fig. 6A). The
observed difference in efflux rates was not due to a different
preloading of cells with spermidine, as can be seen in Fig. 6B. To
provide a solid basis for the observed difference in polyamine
excretion, the initial efflux rates of 15 independent cultures of wild
type and mutant cells, respectively, were determined. It could be shown that the spermidine efflux rate of the TPO1 deletion strain
drops to 49 ± 28% of wild type rates (Fig. 6C). These
experiments indicate a function of Tpo1p in spermidine detoxification.
Preloading yeast cells with putrescine unfortunately led to widely
varying results; however, in a series of experiments we did not observe
a statistically significant difference between the wild type and the
TPO1 deletion strain in terms of putrescine efflux activity
(not shown).

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Fig. 6.
TPO1 disruption leads to impaired
release of spermidine into the medium. Wild type and
tpo1::lacZ cells were grown in
succinate-buffered CBS medium in the presence of 10 mM
spermidine. After resuspension in amine-free medium, spermidine efflux
was followed by reverse phase HPLC. A, time course of
spermidine efflux in wild type ( ) and
tpo1::lacZ cells ( ). The graph shows
data from three independent experiments. The error bars
represent standard deviation. B, polyamine content of wild
type cells grown in the absence of added spermidine (filled
bars) as well as of wild type cells (light gray bars)
and tpo1::lacZ cells (dark gray
bars) grown in the presence of 10 mM spermidine. The
bars represent the mean value from three independent cell
cultures. C, initial spermidine efflux rates of wild type,
of tpo1::lacZ cells, and of the same
TPO1 disruption strain transformed with a plasmid expressing
TPO1 under control of the PMA1 promoter (pDR199
TPO1). In the case of the two former strains, the
bars represent the means from 15 independent experiment. The
spermidine efflux rate of the mutant strain yeast was determined four
times. The error bars represent standard deviation.
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Complementation of the Impaired Export Activity--
If the
observed impairment of spermidine efflux was indeed a consequence of
the TPO1 deletion, the introduction of plasmid-borne Tpo1p
should lead to an increase of the spermidine export rate. Therefore, we
constructed a plasmid expressing TPO1 under control of the
PMA1 promoter and transformed this construct into the
tpo1::lacZ strain. Expression of the
plasmid encoded gene was controlled by analyzing polyamine sensitivity
of the resulting yeast, which was in fact highly decreased (not shown).
The initial spermidine export rates of this strain were determined and
gave values in the same range as observed for the wild type (Fig.
6C). When the tpo1::lacZ
strain was transformed with the empty vector, the export rates were in
the same range as observed for the TPO1 deletion without any
plasmid (not shown). The initially accumulated spermidine concentration
was unchanged in both experiments. Because TPO1 was found to
complement the described phenotype, we consider Tpo1p to be a plasma
membrane-bound exporter involved in spermidine export.
Triple HA Fusions of Tpo2p-4p Are Also Located in the Plasma
Membrane--
The fact that Tpo1p was localized in the plasma membrane
raises the question on the location of the other described polyamine transporters in S. cerevisiae, i.e. whether
Tpo2p, Tpo3p, and Tpo4p are located in the plasma membrane as well (4).
We therefore generated three strains bearing 3xHA-tagged
versions of TPO2, TPO3, and TPO4,
respectively, as the only gene copy. Using sucrose density gradient
centrifugation, we fractionated membrane preparations of these strains
and found that all of the TPO gene products localize in the
same fraction, which was identified as plasma membrane fraction before
(Fig. 7; compare Fig. 1). An involvement
of the three yeast polyamine transporters in transport of these solutes across the plasma membrane seems to be likely and will be analyzed in
the future.

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Fig. 7.
Localization of the Tpo transporters.
Cell lysates of yeast strains expressing triple HA-tagged versions of
Tpo1p-Tpo4p, respectively, were separated by sucrose gradient
centrifugation. The gradients were fractionated from top
(fraction 1) to bottom (fraction 12) and analyzed
by SDS-PAGE followed by immunoblotting using anti-HA antibody.
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DISCUSSION |
Subcellular Localization of Tpo1p-4p Revised--
The expression
and subsequent detection of epitope-tagged proteins has turned out to
be a powerful method in unraveling the subcellular localization of
proteins. By applying this approach we found that a triple HA-tagged
version of Ycr023p co-localized with the vacuolar ATPase, using sucrose
gradient centrifugation as the analytical method. Ycr023p is a member
of the multidrug resistance family and most probably functions
in the detoxification of hazardous compounds into the vacuolar lumen.
It has been shown that its deletion confers resistance to allylglycine
(25). Unexpectedly, triple HA-tagged versions of all four polyamine
transporters, Tpo1p-4p, that have been described in S. cerevisiae so far co-localized with the plasma membrane ATPase.
Despite the fact that tags in general might cause mislocalization of
proteins, we decided to carefully re-evaluate whether these
transporters were in fact plasma membrane bound and thus not involved
in vacuolar transport of polyamines as had been described before (4).
Direct localization studies of these proteins had not been performed so
far; their intracellular localization had been deduced indirectly from
physiological experiments with Tpo1p being the best studied protein of
this group (3, 4, 24). We provide experimental evidence for a role of
Tpo1p in transport across the plasma membrane, which is based on the
observations that the HA-tagged protein is functional and that a
TPO1 knockout is not impaired in uptake of polyamines into
vacuolar vesicles. The observation of Tomitori et al. (3) that vesicles prepared from TPO1-overexpressing strains show
an increased spermine import activity is most probably due to
mislocalization of the overproduced protein. In this study we could
show that a strain deleted in TPO1 shows a significant
defect in spermidine secretion under conditions in which the cells are
challenged by incubation in medium containing high levels of
polyamines. We thus conclude that Tpo1p is a plasma membrane-embedded
carrier protein. The arguments that Tpo2p, Tpo3p, and Tpo4p have the
same subcellular location are so far exclusively based on results of sucrose gradient fractionation and need further experimental proof.
Polyamine Excretion in S. cerevisiae--
Export of polyamines,
especially of putrescine as the key metabolite in polyamine
biosynthesis, had been described for some prokaryotic (22) and
eukaryotic organisms (23), as well as for cultured mammalian cells
(26). The biological significance of this process is most probably
related to the fact that although polyamines are pivotal for many
physiological processes, they are toxic when present in high levels, so
that cells had to develop mechanisms for controlling the intracellular
pools (27). Although the regulation of biosynthesis is certainly a
possibility to control nontoxic levels (28), excretion provides an
additional rationale. Taking into account that re-uptake is guaranteed
by the presence of importer systems, a possible loss of precursors can
be avoided. Putrescine export had not been studied in detail in
S. cerevisiae before. When trying to establish a
function of Tpo1p in polyamine export, we therefore asked whether yeast
had developed mechanisms for putrescine homoeostasis similar to other
microorganisms and whether Tpo1p might be involved in that process.
Although we were able to detect significant putrescine export which,
interestingly, was restricted to the fermentative growth phase, Tpo1p
is obviously not involved in this process to a significant extent,
because wild type and the TPO1 disruption strain had equal
export activity. We cannot rule out, however, that redundant
transporters may adjust their putrescine export activities in the
deletion strain to compensate for the missing TPO1 function.
Polyamine Detoxification--
When cells are stressed under
conditions of growth in the presence of high levels of polyamines, a
detoxification mechanism has to exist to overcome toxic intracellular
levels that may arise because of the presence of polyamine importers.
This could in principle be achieved by regulating the uptake activity
and/or the activity of exporters for these solutes. We therefore
preloaded yeast cells with polyamines and followed the efflux after
transfer of cells into polyamine-free medium. Tpo1p was shown not to
contribute significantly to putrescine tolerance (3); thus we
concentrated on the analysis of spermidine export, which was in fact
affected by TPO1 deletion. The decrease in excretion
activity was significant and could be reverted by introducing a
plasmid-encoded copy of TPO1, proving that the phenotype
observed was a direct consequence of the missing TPO1 gene.
The rescued strain showed spermidine export rates comparable with the
wild type (Fig. 6C), which was also the reason for the
polyamine tolerance that we found when testing the functionality of the
TPO1-3xHA construct (Fig. 2). In the latter experiment we
even observed an increased tolerance of the
tpo1::lacZ strain containing
TPO1-3xHA on a plasmid, compared with wild type, which was
most probably due to the experimental conditions applied. Spermidine
tolerance was assayed under magnesium limitation, a condition that
leads to enhanced polyamine uptake (9), thus generating higher
intracellular levels than the 4-fold increase we observed when
challenging the cells for export measurements. Furthermore, it has been
shown that polyamine stress in combination with Mg2+
limitation leads to increased TPO1 mRNA abundance, even in a TPO1-overexpressing strain (4). This might explain the
overcompensation of spermidine sensitivity observed with the plate
assay in contrast to our observation in the spermidine export
measurements, which were performed under Mg2+ excess.
Unfortunately, we were not able to provide appropriate growth medium
completely devoid of a contaminant, which interfered with spermine
analysis by HPLC. The contribution of Tpo1p in spermine export thus
remains an unsettled question.
TPO1 Is Related to Multidrug Resistance--
The data presented so
far favor a function of Tpo1p in detoxification of high intracellular
polyamine levels. This observation is interesting in the context of
other recent publications on the properties of Tpo1p. This transporter
has been reported to confer resistance to a variety of structurally
nonrelated toxic compounds like quinidine and cycloheximide (24),
mycophenolic acid (29), or 2-methyl-4-chlorophenoxyacetic acid and
2,4-dichlorophenoxyacetic acid (30), suggesting an broad substrate
specificity, which is characteristic for multidrug resistance proteins
(5). Furthermore, TPO1 was recently shown to be a target for
the regulators Pdr1p (24) and Pdr3p (30), both of which mediate
multidrug resistance. This further emphasizes the concept that the
primary function of Tpo1p in S. cerevisiae is the
detoxification of hazardous compounds, which includes excess spermidine.