We have characterized the regulation of
spermidine transport in yeast and identified some of the genes involved
in its control. Disruption of the SPE2 gene encoding
S-adenosylmethionine decarboxylase, which catalyzes an
essential step in polyamine biosynthesis, up-regulated the initial
velocity of spermidine uptake in wild-type cells as well as in the
polyamine transport-deficient pcp1 mutants. Exogenous spermidine rapidly inactivated spermidine transport with a half-life of
10-15 min via a process that did not require de novo
protein synthesis but was accelerated by cycloheximide addition.
Conversely, reactivation of spermidine influx upon polyamine
deprivation required active protein synthesis. The stability of
polyamine carrier activity was increased 2-fold in polyamine-depleted
spe2 deletion mutants, indicating that endogenous
polyamines also contribute to the down-regulation of spermidine
transport. Ligand-mediated repression of spermidine transport was
delayed in end3 and end4 mutants that are
deficient in the initial steps of the endocytic pathway, and spermidine uptake activity was increased 4- to 5-fold in end3
mutants relative to parental cells, although the stability of the
transport system was similar in both strains. Disruption of the
NPR1 gene, which encodes a putative Ser/Thr protein kinase
essential for the reactivation of several nitrogen permeases, resulted
in a 3-fold decrease in spermidine transport in
NH4+-rich media but did not prevent its
down-regulation by spermidine. The defect in spermidine transport was
more pronounced in NH4+- than proline-grown
npr1 cells, suggesting that NPR1 protects against nitrogen catabolite repression of polyamine uptake activity. These results suggest that (a) the polyamine carrier is an
unstable protein subject to down-regulation by spermidine via a process involving ligand inactivation followed by endocytosis and that (b) NPR1 expression fully prevents
nitrogen catabolite repression of polyamine transport, unlike the role
predicted for that gene by the inactivation/reactivation model proposed
for other nitrogen permeases.
 |
INTRODUCTION |
Putrescine and the polyamines spermidine and spermine are
ubiquitous molecules required for macromolecular biosynthesis (1, 2),
for posttranslational maturation of the essential protein eukaryotic
initiation factor-5A (3), and for the control of various ion channel
activities (4). Intracellular polyamine pools are actively regulated by
de novo synthesis, degradation, excretion, and import from
extracellular sources. Polyamine-specific carriers are widely
distributed in prokaryotes and eukaryotes (5-7) and can replenish
polyamine pools upon inhibition of the biosynthetic enzymes (8-10).
Mammalian polyamine transport activity is also acutely controlled by
cell cycle events (11, 12) and hormonal stimulation (5, 9, 10, 13, 14).
Polyamine transport is a saturable, carrier-mediated and
energy-dependent process (5, 15, 16). Although its
physiological properties have been extensively studied, the molecular
characteristics of the diamine and polyamine carrier proteins have only
been elucidated in prokaryotes (7, 17-19).
In the yeast Saccharomyces cerevisiae, exogenous polyamines
are internalized by specific, saturable plasma membrane uptake system(s) (20-24). Recently, we (22) and others (24) have identified the PTK2 (= STK2) gene, which encodes a putative
Ser/Thr protein kinase, as a major determinant of high affinity
polyamine transport in yeast. The closely homologous PTK1
(=STK1) gene, which directs a lower affinity, low capacity
polyamine transport system, has also been cloned and characterized (22,
23). However, the exact role of these kinases in the regulation of
polyamine transport as well as the molecular identity of the putrescine
and polyamine carrier(s) in yeast have not yet been determined.
In mammalian cells, polyamine transport activity is tightly regulated
by negative feedback mechanisms that depend on intracellular polyamine
levels. A marked induction of polyamine transport activity is triggered
by polyamine depletion, such as that caused by inhibition of ornithine
decarboxylase or S-adenosylmethionine decarboxylase (AdoMetDC)1 (5, 6, 8-10,
25-28), and rapidly repressed by the addition of exogenous polyamines
(9, 26, 29-31). The expression of ornithine decarboxylase antizyme,
which is translationally regulated by polyamine levels (32), plays an
important role in the acute down-regulation of mammalian polyamine
uptake (29-31). However, an exhaustive search in the
Saccharomyces Genome Data Base has disclosed no
antizyme-like gene in yeast, and little is known on the regulation of
diamine and polyamine transport in lower eukaryotes. A
Ca2+-sensitive gene product has been reported to negatively
regulate putrescine transport in Neurospora crassa, although
its molecular identity is as yet unknown (33).
More recently, we reported that spermidine transport in yeast is
subject to negative regulation by substrate availability (22). The aim
of the present study was to gain insight into the components and
mechanisms responsible for the regulation of polyamine transport in
yeast. By using various mutant strains, we now provide evidence that
spermidine transport is mediated by an unstable carrier protein that is
down-regulated by intracellular polyamines of both endo- or exogenous
origins via a mechanism involving ligand-mediated inactivation and
endocytosis. In addition, we show that the NPR1 gene, which
encodes a Ser/Thr protein kinase structurally related to Ptk1p and
Ptk2p, and is required for the derepression of various nitrogen
permeases upon transfer to poor nitrogen sources (34, 35), plays an
important role in the expression of high affinity spermidine uptake
activity under conditions (e.g. high
[NH4+]) that were previously thought
to down-regulate Npr1p activity.
 |
MATERIALS AND METHODS |
Strains, Media, and Plasmids--
The yeast strains used in this
study are listed in Table ns;1900t1} I and were
routinely grown in YPD medium (1% yeast extract, 2% peptone, 2%
D-glucose). The minimal medium used to study the influence
of nitrogen sources on polyamine transport was made of 2%
D-glucose and 0.17% amino acid-free yeast nitrogen base (Difco) without (NH4)2SO4 and
supplemented with the appropriate essential amino acids. This medium
was supplemented with either 10 mM
(NH4)2SO4 (minimal
NH4+ medium) or 9 mM
L-proline (minimal proline medium) as sole
nitrogen-containing compounds. Polyamine-free YNB medium (H medium; 2%
D-glucose, 0.17% amino acid-free yeast nitrogen base, 38 mM (NH4)2SO4, and essential amino acids) and agar plates were prepared as described (36).
Yeast transformations were carried out using the lithium acetate method
(37). The single copy YCp-PTK2 expression vector carrying the
3.4-kilobase pair BamHI fragment encompassing the full-length PTK2 gene cloned into YCp50 has been previously
described (22). The PTK2 gene, including 9 and 70 bp
adjacent to the 5
- and 3
-ends of the coding sequence, was isolated as
a HpaI-HindIII fragment from the
PTK2-encoding insert of YCp-PTK2 and cloned into the
PvuII and HindIII restriction sites of the
pYES2.0 multicopy expression vector (Invitrogen). The wild-type strain
RH144-3D and its RH266-1D (end3) and RH268-1C
(end4) mutant derivatives were kindly provided by Dr. Howard
Riezman (University of Basel, Switzerland). The npr1
(21994b) and npi1 (27038a) mutants and parental strain
(33346c), and the pMV33 plasmid carrying the NPR1 gene as a
5.2-kilobase pair insert in the multicopy pFL1 plasmid (38), were
generous gifts of Dr. Bruno André (Université Libre de
Bruxelles, Belgium).
Isolation of a Polyamine Transport-deficient
Mutant--
Wild-type strain DBY747 was mutagenized with methyl
methanesulfonate (0.1%, v/v) as described (39). The mutagenized cell suspensions were plated on YPD agar plates containing 20 mM
methylglyoxal bis(guanylhydrazone) (MGBG), a cytotoxic polyamine analog
that shares the polyamine transport system in mammalian cells (5, 40).
MGBG-resistant colonies thus obtained were screened for spermidine
uptake activity (cf. below), and one polyamine
transport-deficient isolate (DRY405) was selected for further
characterization. Diploidy tests for dominance of the genetic defect
present in DRY405 cells were carried out as described previously
(22).
Isolation of AdoMetDC-deficient Mutants--
The spe2
mutants carrying a chromosomal deletion of the SPE2 gene
encoding AdoMetDC were obtained by a one-step disruption technique
(36). Wild-type yeast strain DBY747 and its polyamine transport-deficient derived strain DRY405 (=pcp1) were
transformed with a 2829-bp NcoI-SphI fragment
encompassing the SPE2 gene region in which the first 833 bp
of the SPE2 coding sequence have been replaced with a
1970-bp HpaI-NarI fragment from YEp351 containing the full-length LEU2 gene (36), and Leu+
transformants were selected. Deletion-insertion in the chromosomal SPE2 gene in the clones thus obtained
(DBY747spe2
=PCP1spe2
and
DRY405spe2
= pcp1spe2
) was confirmed by PCR
(data not shown).
Polyamine Analysis--
For intracellular polyamine
determination, cells were washed three times with ice-cold 100 mM sodium citrate (pH 5.5), and after centrifugation, cell
pellets were resuspended in 0.5 ml of 5% (w/v) trichloroacetic acid
and stored at
20 °C until chromatographic analysis. After thawing,
cell lysates were sonicated in an ultrasonic water bath for 3 cycles of
45 s in an ice-water slurry and microcentrifuged for 5 min at room
temperature. Supernatants were filtered through 0.45-µm cellulose
acetate filters, and polyamine concentrations were determined using ion
pairing reversed-phase high pressure liquid chromatography with
post-column derivatization with o-phthaldialdehyde (41).
Protein pellets were redissolved in 0.2 N NaOH and
quantitated by the method of Bradford (42).
Spermidine and Citrulline Uptake Assays--
[terminal
methylene-3H]Spermidine trihydrochloride (2.65 × 104 Ci/mol) was obtained from NEN Life Science Products
(Lachine, Quebec, Canada), and
L-[ureido-14C]citrulline (57.8 Ci/mol) was purchased from Amersham Corp. Spermidine uptake assays were
performed as described previously (22), using 10 µM
[3H]spermidine (50 Ci/mol) as substrate, unless otherwise
indicated. Analysis of spermidine uptake characteristics was carried
out according to Michaelis-Menten kinetics as described (22).
Citrulline transport was measured by incubating 100 µl of yeast cell
suspension (5 × 107 cells/ml) in citrate/glucose
buffer (22) containing 20 µM
L-[14C]citrulline. At predetermined times,
the reaction was stopped by adding 1 ml of ice-cold stop buffer
(citrate/glucose buffer containing 2 mM unlabeled
L-citrulline). Cell suspensions were layered on cellulose
acetate filters (pore size = 0.45 µm) held on a manifold filter
vacuum apparatus pre-washed twice with ice-cold stop buffer, and mild
vacuum was applied. Filters were washed 3 times with stop buffer, and
radioactivity on the filters was then measured by liquid scintillation
spectrometry.
 |
RESULTS |
Isolation of Polyamine Transport-deficient Yeast
Mutants--
Previously characterized polyamine transport-deficient
yeast mutants from this laboratory (22) and other laboratories (23, 24)
share mutations in the PTK2 gene as a common genetic defect. Such mutants are most likely regulatory mutants, since PTK2
encodes a putative Ser/Thr protein kinase of unknown function and is
devoid of transmembrane domains (22, 24). To generate polyamine
transport mutants of a different genotype, wild-type DBY747 cells were
chemically mutagenized and selected for their ability to grow in the
presence of 20 mM MGBG, a toxic polyamine analog (22, 40).
One isolate from this selection (DRY405) exhibited a marked defect in
spermidine transport (Fig. 1A)
as well as a complete lack of specific putrescine uptake (data not
shown). Kinetic analysis showed that the deficiency in spermidine
uptake activity resulted from a
50-fold increase in
Km with no change in the Vmax
for transport (Fig. 1B), consistent with a structural
mutation in the polyamine permease gene. Moreover, spermidine transport
deficiency and growth sensitivity to MGBG could not be restored in
DRY405 cells after transformation with the cloned PTK2 and
NPR1 genes that are both required for normal polyamine
transport (cf. below)
(22).2 Taken together, these
observations suggest that DRY405 cells might express a dysfunctional
polyamine carrier, and the locus of the defective gene was tentatively
called PCP1 (for polyamine carrier
protein). The pcp1 mutation was found to be
recessive, as diploids produced in crosses with the EI457 strain were
not resistant to 20 mM MGBG (data not shown).

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Fig. 1.
Kinetic characteristics of the polyamine
transport defect in pcp1 mutants. A, time course
of [3H]spermidine uptake in wild-type DBY747 cells
(wt) and polyamine transport-deficient DRY405
(pcp1, ) mutants. Cells were grown to mid-exponential
stage in minimal medium containing 100 µM spermidine to
down-regulate spermidine uptake activity, and specific
[3H]spermidine uptake was then determined. B,
saturation characteristics of [3H]spermidine uptake in
wild-type ( ) and pcp1 ( ) cells. Cells were grown to
mid-exponential stage in YPD medium, and the initial velocity of
[3H]spermidine uptake was then determined for 1 min at
increasing substrate concentrations. Data represent the means ± S.D. for triplicate determinations from representative
experiments.
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Spermidine Transport Is under Feedback Regulation by Intra- and
Extracellular Polyamines--
In a previous study, we determined that
preincubation with extracellular spermidine leads to a profound
repression of spermidine but not putrescine uptake activity in yeast
(22). To assess the relative contribution of endo- and exogenous
polyamines in the control of spermidine uptake activity, the
SPE2 gene encoding AdoMetDC, which provides aminopropyl
group donors for both spermidine and spermine synthesis, was deleted by
a one-step disruption method (36) in the wild-type DBY747 strain as
well as in the pcp1 mutants. The resulting
spe2-5::LEU2 mutants can be depleted from
intracellular polyamines after incubation for a few generation times in
polyamine-free medium (cf.
Table ns;1900t2} II) (36). The effect of
intracellular polyamine depletion and of exogenous spermidine on
spermidine transport was then examined. After incubation in the
presence of spermidine, a condition known to repress polyamine
transport (22), spermidine uptake activity was identical in wild-type and PCP1spe2
cells (Table II). However, deletion of
SPE2 up-regulated spermidine uptake velocity following
incubation in polyamine-free medium suggesting that endogenous
polyamines negatively regulate spermidine transport. On the other hand,
suppression of polyamine biosynthesis slightly enhanced the rate of
spermidine accumulation in pcp1 mutants grown in the
presence of spermidine (data not shown), although the initial velocity
of uptake remained unaffected (Table II). Moreover, both the initial
uptake velocity and rate of spermidine accumulation were markedly
enhanced in pcp1spe2
double mutants grown in
polyamine-free medium. Thus, the pcp1 mutation still allows
the polyamine-regulated expression of a functional permease with a
strongly impaired affinity.
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Table II
Effect of exogenous spermidine on intracellular putrescine and
polyamine contents and initial velocity of spermidine uptake in
AdoMetDC- and polyamine transport-deficient yeast strains
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An elevated initial velocity of spermidine transport was generally
correlated with a lower spermidine content, either after preincubation
in polyamine-free medium or disruption of the AdoMetDC gene (Table II).
SPE2 deletion also resulted in putrescine accumulation, as
expected from the blockade in polyamine biosynthesis and from the
relief of negative feedback inhibition of ornithine decarboxylase expression (20, 43). The latter phenomenon also likely accounts for the
decrease in putrescine levels accompanying accumulation of exogenous
spermidine. Moreover, intracellular spermidine levels in cells with
wild-type polyamine transport activity (PCP1) grown in the
presence of spermidine reached a similar value whether endogenous
biosynthesis was functional (SPE2) or not (spe2),
suggesting that the size of the spermidine pool is adjusted by a common
mechanism when an extracellular source of the polyamine is available.
However, incubation with exogenous spermidine failed to significantly
enlarge (p > 0.05) spermidine content in
pcp1SPE2 cells, despite a detectable reduction in putrescine
content. In fact, although the size and composition of the putrescine
and polyamine pools were not affected by the pcp1 mutation,
steady-state spermidine accumulation was 3-6-fold lower in these
mutants upon SPE2 inactivation, consistent with a chronic
repression of a defective spermidine uptake system by low levels of
accumulated spermidine. Putrescine addition had no effect on spermidine
transport velocity (data not shown), indicating that fluctuations in
putrescine content resulting from the different incubation conditions
and genetic backgrounds did not influence spermidine uptake activity.
These data suggest that influx of exogenous spermidine strongly
down-regulates spermidine transport, whereas endogenous levels of
polyamines, but not putrescine, also exert a significant, but much less
stringent negative control of spermidine transport. Moreover, marked
down-regulation of polyamine uptake activity by exogenous spermidine
was accompanied with only minor (e.g. in
pcp1spe2
cells) or even without detectable changes (e.g. in pcp1SPE2 cells) in total spermidine
content, suggesting that only a minor fraction of the total spermidine
pool is required for feedback transport inhibition.
The latter assumption was further confirmed by determining the
concentrations of spermidine required to repress polyamine uptake
activity (Fig. 2). Following overnight
preincubation with increasing concentrations of spermidine, subsequent
spermidine uptake activity was inactivated with an IC50 = 2.4 ± 0.7 µM, i.e. a value close to the
Km of spermidine for transport in S. cerevisiae (4 to 5 µM) (20, 22). This observation is
again consistent with a close connection between substrate
internalization and the mechanism of transport inactivation triggered
by spermidine.

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Fig. 2.
Dependence of spermidine-induced
down-regulation of polyamine uptake activity on exogenous spermidine
concentration. Wild-type (DBY747) cells were grown to
mid-exponential stage ( 16 h) in the presence of the indicated
spermidine concentrations. The initial velocity of
[3H]spermidine uptake was then determined for a 2-min
period using 10 µM substrate. Data are the mean ± S.D. of triplicate determinations.
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Down-regulation of Spermidine Transport Does Not Require de Novo
Protein Synthesis--
Substrate-mediated down-regulation of
spermidine transport could either result from decreased synthesis,
increased turnover, or posttranslational inactivation of the polyamine
permease. Thus, the effect of exogenous spermidine on the half-life
(t0.5) of polyamine uptake activity was examined
in wild-type cells in the presence of 10 µg/ml cycloheximide (CHX).
Spermidine (100 µM) was added to cells preincubated under
polyamine-free conditions to fully derepress spermidine transport, and
the rate of decay of spermidine transport activity was then determined.
As shown in Fig. 3A,
spermidine triggered an immediate and rapid loss of spermidine uptake
activity, with a t0.5
10-15 min, until a new equilibrium value was reached after 1 h. After CHX addition,
spermidine uptake activity decayed much more slowly, with a
35%
decrease in velocity observed after 3 h. Furthermore, protein
biosynthesis inhibition enhanced the extent of substrate-induced
inactivation of spermidine transport without affecting the
t0.5 of the decay process. Down-regulation of
spermidine uptake was not prevented when 5 mM
NaN3 was added prior to spermidine addition (data not shown), demonstrating that ligand-mediated inactivation of spermidine transport did not directly depend on metabolic energy.

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Fig. 3.
Effect of spermidine and protein synthesis
inhibition on the time course of down- and up-regulation of polyamine
transport. A, DBY747 (wild-type) cells were grown to
mid-exponential stage in polyamine-free (H) medium (A),
washed, and transferred (S to S+,
t = 0) to medium supplemented with 100 µM
spermidine (Spd, ), 10 µg/ml cycloheximide
(CHX, ), or both agents ( ).
[3H]Spermidine uptake velocity was measured for a 20-min
period at the indicated times. B, same as in A,
except that cells were preincubated in medium containing 100 µM spermidine and then transferred at t = 0 (S+ to S ) to polyamine-free medium ( ) or
to medium containing CHX in the absence ( ) or presence ( ) of 100 µM spermidine. Spermidine transport remained constant in
control cells kept under initial conditions (S and
S+ for A and B, respectively; data
not shown). Data represent the mean ± S.D. of triplicate
determinations from representative experiments.
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The time course of derepression of spermidine transport was examined by
preincubating wild-type cells in the presence of spermidine and then
transferring cultures to spermidine-free conditions in the presence or
absence of CHX. Spermidine uptake activity increased in a
time-dependent manner following transfer to polyamine-free medium, with a 2.5-fold increase observed after 3 h, and a
significant derepression already detectable 30 min after the onset of
polyamine deprivation (Fig. 3B). Derepression of spermidine
uptake absolutely depended on de novo protein biosynthesis
since it was completely abolished by CHX. Moreover, a continuous supply
of exogenous spermidine was required to fully destabilize the transport
system, as shown by the slower rate of decay of spermidine uptake
activity noted upon transfer to spermidine-free conditions in the
presence of CHX.
Intracellular Polyamine Depletion Stabilizes the Spermidine
Transport System--
Since endogenous polyamine pools also exert a
down-regulatory effect on polyamine transport, we determined the decay
of spermidine uptake activity after CHX addition in spe2
mutants preincubated in polyamine-free medium. As shown in Fig.
4, the spermidine transport system was
more stable in the spe2
mutants than in the parental strain. Thus, the increase in spermidine transport activity observed upon depletion of endogenous polyamines is clearly associated with a
decreased rate of its inactivation, in a manner similar to deprivation
of exogenous polyamines.

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Fig. 4.
Spermidine depletion stabilizes the polyamine
transport system. DBY747 (wild-type, wt; ) and their
spe2 knockout derivatives ( ) were grown to
mid-exponential stage in polyamine-free medium. At time = 0, CHX
(10 µg/ml) was added to the medium, and [3H]spermidine
uptake was measured for a 10-min period at the indicated times. Data
are expressed as a percentage of initial activities and represent the
mean ± S.D. of triplicate determinations.
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Polyamine-induced Down-regulation of Spermidine Transport Partly
Depends on Endocytosis--
Several plasma membrane transporters in
yeast are cleared from the cell surface by endocytic internalization
(43-48). The END3 (49) and END4 genes (50, 51)
encode proteins that are involved in actin cytoskeleton organization,
and their expression is required at early steps of the endocytic
pathway for the internalization of several membrane proteins (52). To
assess whether endocytosis is involved in the inactivation and/or
degradation of the polyamine carrier, we analyzed the stability of this
transporter in end3 and end4 mutants that are
thermosensitive for growth (52). Whereas the end3 mutants
also exhibit a defect in endocytosis at the permissive temperature
(25 °C), the genetic lesion of end4 cells is more stringently temperature-dependent (52).
At the permissive temperature, spermidine transport under fully
derepressing (i.e. polyamine-free) conditions was 4- to
5-fold higher in end3 cells than in the parental strain
(Fig. 5). As expected, no significant
difference in spermidine uptake was observed between end4
mutants and wild-type cells at 25 °C, and spermidine transport
velocity in wild-type cells was considerably lower than at 30 °C,
consistent with a requirement for metabolic energy for the uptake
process. To assess the effect of impaired endocytosis on the turnover
of the polyamine uptake system, we then determined the
t0.5 of spermidine transport inactivation in
end3 and end4 mutants grown at 25 °C under
fully derepressing conditions and then transferred to a non-permissive
temperature (37 °C) in the presence of CHX. Fig.
6A shows that following
deprivation of exogenous polyamines, the initial rate of decay of
spermidine uptake activity was similar in end3 mutants and
in the parental strain (t0.5 = 60 ± 12 and
55 ± 9 min, respectively) but was notably accelerated in
end4 mutants (t0.5 = 21 ± 6 min). Thus, the increased spermidine uptake activity observed in
end3 mutants is clearly not associated with a lower rate of
inactivation of the polyamine transport system, and in fact, the defect
in endocytosis present in the end4 mutants decreases the
stability of this carrier.

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Fig. 5.
Spermidine uptake activity is up-regulated by
the end3 mutation at a permissive temperature. The
wild-type RH144-3D strain (END3END4, ) and its
end3 (end3END4, ) and end4
(END3end4, ) mutant derivatives were grown to
mid-exponential stage at 25 °C in polyamine-free medium, after which
the time course of [3H]spermidine uptake was determined.
Data are the mean ± S.D. of triplicate determinations.
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Fig. 6.
Stability and spermidine-induced
down-regulation of the spermidine transport system in
endocytosis-deficient mutants. Wild-type (RH144-3D) strain
(END3END4, ), and its end3
(end3END4, ) and end4 (END3end4,
)-derived mutants were grown to mid-exponential stage at 25 °C in
polyamine-free medium and then resuspended for 10 min in medium
pre-warmed to 37 °C. A, at time = 0, CHX (10 µg/ml) was added to the medium, and [3H]spermidine
uptake velocity was measured for 10-min periods at the indicated times.
B, at time = 0, spermidine (100 µM) was
added to the medium; after 10 min, spermidine was removed, and cells were transferred back to spermidine-free medium pre-warmed to 37 °C
and containing 10 µg/ml CHX. [3H]Spermidine uptake
velocity was measured for 10-min periods at the indicated times.
Results are expressed as a percentage of initial transport activity and
represent the mean ± S.D. of triplicate determinations.
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It has been shown that substrate-mediated inactivation and degradation
of yeast plasma membrane carriers such as the copper transporter Ctr1p
(53) occur via a mechanism that does not require prior endocytic
internalization. To determine whether spermidine can inactivate the
polyamine carrier system independently from endocytosis, we first
deprived end3 and end4 mutants and wild-type cells from exogenous polyamines at 25 °C, then transferred cultures for 10 min into spermidine-containing medium at the non-permissive temperature to initiate the inactivation process, and finally transferred cells to polyamine-free medium in the presence of CHX while
continuously monitoring the time course of spermidine transport
velocity. Spermidine rapidly triggered the decay of spermidine uptake
activity in the parental strain, and spermidine removal slowed down but
did not prevent the time-dependent inactivation of
spermidine transport (Fig. 6B). Moreover, although the
initial rate of inactivation of spermidine transport was significantly lower in the end3 and end4 mutants immediately
after spermidine addition, a steady decay of spermidine transport
activity was still observed following removal of the polyamine, at a
rate comparable with that of wild-type cells. These results suggest
that spermidine internalization rapidly initiates an irreversible
process of polyamine transport inactivation that does not require prior
endocytosis of the polyamine permease nor continuous protein
synthesis.
The NPR1 Gene Is Involved in the Regulation of Spermidine Transport
Activity--
The NPR1 gene encodes a putative Ser/Thr
protein kinase that plays an important role in the regulated expression
of several amino acid permeases (34, 35, 38, 54), as well as of the NH4+ transporter Mep1p (34, 55, 56),
which are under nitrogen catabolite repression (NCR). According to one
model (34, 35), NPR1 expression is required to counteract
the constitutive inactivation/degradation of nitrogen permeases by the
Npi1p ubiquitin-protein ligase in cells growing on poor nitrogen
sources (e.g. low NH4+,
proline, etc.) via a posttranslational mechanism (57). Interestingly, the PTK1 and PTK2 genes that are required for
expression of di- and polyamine transport activity (22, 24) belong to
the same homology class of Ser/Thr protein kinases as NPR1
(58). The role of Npr1p as a reactivator of amino acid transport and
the similarities between the predicted primary structures of Npr1p, Ptk1p, and Ptk2p prompted us to determine whether Npr1p might play a
role in the derepression of spermidine uptake activity observed upon
deprivation of exogenous polyamines.
As shown in Fig. 7A,
spermidine uptake activity was decreased by 2- to 3-fold in
npr1 mutants as compared with the parental strain after
preincubation under polyamine-free conditions. Moreover, exogenous
spermidine down-regulated spermidine transport in both wild-type and
npr1 cells. The defect in spermidine transport in npr1 mutants was clearly related to the lack of
NPR1 expression, as spermidine transport activity was nearly
restored to wild-type levels upon transformation with a multicopy
NPR1 expression vector (Fig. 7B). Thus, these
data clearly demonstrate that Npr1p expression is indeed involved in
the regulated expression of polyamine transport activity but not in the
ligand-dependent repression/derepression of spermidine
transport.

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Fig. 7.
NPR1 gene expression is required for
spermidine transport. A, effect of the npr1
mutation on spermidine uptake activity and its down-regulation by
spermidine. Wild-type cells (33346c strain; wt, , )
and their npr1-derived mutant cells ( , ) were grown to
mid-exponential stage in H medium (containing 38 mM
(NH4)2SO4) in the absence
(open symbols) or presence (solid symbols) of 100 µM spermidine (Spd), after which the time
course of [3H]spermidine uptake was determined.
B, restoration of spermidine transport by the pMV33
NPR1 expression plasmid. Wild-type (wt, ),
npr1 mutants ( ) and npr1 cells transformed
with the multicopy pMV33 expression plasmid ( ) were grown to
mid-exponential stage in polyamine-free medium, and the velocity of
[3H]spermidine transport was then determined. Data
represent the mean ± S.D. of triplicate determinations.
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Polyamine Transport Activity Is Not Subject to NCR--
An
unexpected finding from the above experiments was that the lack of
NPR1 expression in a
NH4+-rich medium (i.e. 38 mM (NH4)2SO4) strongly
depressed spermidine transport activity, although under the same
conditions, Gap1p activity is reduced to almost undetectable levels in
an NPR1-independent manner (34, 35, 54). We indeed verified
that Gap1p activity (as measured by the rate of
[14C]citrulline transport) was dramatically up-regulated
upon transfer from NH4+ to proline as
sole nitrogen sources for growth (data not shown). We next determined
the effect of NCR on spermidine uptake activity in wild-type and
npr1 cells preincubated in polyamine-free minimal medium
containing either NH4+ or proline as
sole nitrogen sources. Spermidine transport in wild-type cells was
clearly not repressed by growth on NH4+
and was in fact reproducibly higher than that measured in proline-grown cells (Fig. 8). Interestingly, the
differential effect of the npr1 mutation on spermidine
uptake was much more pronounced in NH4+-
than proline-grown cells. These results indicate that the spermidine transport system is resistant to NCR and that NPR1
expression might in fact protect this transporter against an
NH4+-dependent inactivating
mechanism.

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Fig. 8.
Effect of the nitrogen source on spermidine
uptake activity in wild-type and npr1 cells.
npr1 mutants ( , ) and their parental strain
(wt, , ) were grown to mid-exponential stage in
minimal medium containing either 9 mM L-proline
(Pro) or 10 mM
(NH4)2SO4 as sole nitrogen sources.
The time course of specific [3H]spermidine uptake was
then determined. Data are the mean ± S.D. of triplicate
determinations.
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One such inactivating mechanism could obviously be the action of the
ubiquitin-protein ligase Npi1p, which targets several yeast membrane
transporters for degradation through the endocytosis-vacuolar pathway
(34, 57, 59-61). Although NPI1 is an essential gene (57),
partly Npi1p-deficient mutants exhibit reduced rates of endocytosis and
ubiquitination of target proteins such as the uracil permease Fur4p
(60) and a pleiotropic up-regulation of amino acid permease activity
under NH4+-repressing conditions (34,
61). Thus, if spermidine transport is under
NH4+-induced repression by Npi1p in a
manner similar to Gap1p, polyamine uptake activity should be
up-regulated in npi1 mutants. However, there was no
difference in spermidine transport between wild-type and
npi1 cells under conditions (i.e. high
[NH4+]) that lead to a marked
derepression of Gap1p activity in NPI1-deficient cells (Fig.
9) (34, 61). Moreover, the ability of
spermidine to down-regulate polyamine transport was not decreased by a
deficiency in NPI1 expression, suggesting that Npi1p is not
a major determinant in the spermidine-mediated inactivation of the
polyamine permease.

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Fig. 9.
The steady-state level and stability of
spermidine uptake activity is not affected by a deficiency in
NPI1 expression. Wild-type cells (33346c strain;
wt, ) and their npi1 mutant derivatives ( )
were grown to mid-exponential stage in polyamine-free (H) medium
containing 38 mM
(NH4)2SO4. Cells were then
transferred to medium containing 100 µM spermidine, and
the velocity of [3H]spermidine uptake was then determined
for 10-min periods at the times indicated. Data represent the mean ± S.D. of triplicate determinations.
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DISCUSSION |
Like for mammalian polyamine transport (5, 6, 8-10, 25-31), high
affinity spermidine uptake activity in yeast is clearly under negative
control by polyamines of both endogenous and exogenous origins.
However, extracellular spermidine quantitatively exerts a more
stringent feedback inhibition on its own high affinity transport than
the endogenous pool. Likewise, deprivation of exogenous polyamines had
a disproportionately large stimulatory effect on spermidine transport
as compared with polyamine depletion due to a genetic block in
endogenous synthesis, as evidenced in pcp1spe2
double
mutants. The weaker feedback inhibition of spermidine transport exerted
by endogenous polyamines could be due to sequestration of de
novo synthesized polyamines in the vacuole, as well as to binding
to macromolecular polyanions. Although subcellular polyamine compartmentalization has yet to be investigated in S. cerevisiae, it has been estimated that at least 25% of total
cellular spermidine is confined to the vacuole in N. crassa
(62), and a vacuolar, H+-ATPase-dependent
active polyamine transport system has been described in yeast (63).
Furthermore, due to their mixed polycationic/aliphatic character,
intracellular polyamines may largely exist as complexes bound to
nucleic acids and free nucleotides, a factor that could reduce their
free fraction to less than 10% (64). Thus, the more efficient
down-regulation exerted by exogenous spermidine might indicate that the
underlying mechanism involves a rapid and direct allosteric interaction
with the polyamine permease, without mixing with the endogenous pool.
This interpretation would account for the closely similar values of the
Km of spermidine for high affinity uptake and the
IC50 of exogenous spermidine for transport repression. The
present data cannot rule out the possibility that the regulatory site
responsible for carrier destabilization by spermidine is identical to
the substrate binding site or lies in a different, extracellular domain
of the polyamine permease. However, the fact that intra- and
extracellular polyamines affect polyamine permease stability in a
qualitatively similar manner suggests that the allosteric site for
ligand inactivation might lie on the endofacial side of the
carrier.
In mammalian cells, polyamine depletion up-regulates polyamine uptake
as a result of decreased antizyme expression (26, 29-31), as well as
via antizyme- and protein synthesis-independent mechanisms (9). Because
antizyme is a short-lived negative regulator of polyamine uptake,
studies using CHX have not yielded clear estimates of the
t0.5 of the mammalian polyamine transporter(s) due to the relief of uptake activity that accompanies antizyme depletion. In contrast, the present data strongly suggest that the
yeast polyamine permease is a rather unstable protein and that no
antizyme-like system participates to its feedback inhibition, as
predicted by the lack of sequences homologous to either bacterial and
mammalian antizyme in the yeast genome.
Some features of the rapid repression elicited by spermidine on
polyamine transport are reminiscent of the ligand inactivation mechanism recently proposed for the down-regulation of the inositol permease Itr1p (45) and the copper transporter Ctr1p by their substrate
(53). The rate of degradation of these carriers is dramatically
enhanced upon exposure to their substrate via a feedback mechanism that
likely involves direct ligand binding to an allosteric site on the exo-
or endofacial side of the carrier (45, 53). However,
spermidine-mediated down-regulation of the polyamine permease exhibits
features that are intermediary between the ligand inactivation
mechanisms respectively described for the Itr1p and Ctr1p carriers.
Endocytosis has been shown to form an integral part of the
inactivation/degradation mechanism elicited by inositol binding to
Itrp1, since end3 and end4 mutants are spared
from ligand-mediated inactivation of Itr1p after removal of inositol from the medium (45). On the other hand, Ctr1p undergoes copper-induced degradation while still inserted in the plasma membrane and does not
require endocytic internalization for its down-regulation (53). In
contrast, we have observed that spermidine-mediated inactivation of
polyamine transport is only delayed by defective endocytosis,
suggesting that spermidine triggers the destabilization process
independently from internalization of the polyamine permease and that
ligand inactivation might in fact accelerate the endocytic degradation
of the transporter. Moreover, deficient endocytosis did not decrease
the basal rate of turnover of the polyamine uptake system, the
t0.5 of spermidine uptake activity being even
shorter in end4 mutants. A role for endocytosis in the
regulation of polyamine transport is nonetheless suggested by the fact
that spermidine uptake was strongly up-regulated in end3
mutants grown at the permissive temperature. The marked instability of
the yeast polyamine uptake system would predict that a high rate of
insertion and endocytic removal of carrier molecules into and from the
plasma membrane is required to maintain adequate transport activity. Since the t0.5 of the transport system was not
affected by the end3 mutation, increased spermidine uptake
activity in end3 cells could result from defective recycling
of functional polyamine permease molecules at the endocytic
internalization step, resulting in a steady-state increase in the
number of active spermidine carriers in the plasma membrane.
Our results are thus consistent with a model where spermidine controls
the rate of degradation of the polyamine permease via a
posttranslational, ligand inactivation mechanism that destabilizes the
carrier. The polyamine permease appears to turn over rapidly, and
up-regulation of spermidine transport clearly depends on de novo protein biosynthesis, either because derepression reflects an
increase in the number of polyamine permease molecules or because a
spermidine-repressed, essential reactivator is induced following polyamine deprivation. Upon its binding to an allosteric site, spermidine accelerates the rate of endocytic internalization of functional transporters, apparently without an involvement of the Npi1p
ubiquitin-protein ligase that controls the endocytic degradation of
several yeast transporters (57, 60). The observed kinetics of
repression/derepression of polyamine transport is consistent with a
predominantly posttranslational control, as is the case for the
regulation of yeast ornithine decarboxylase by spermidine (20, 43).
Without appropriate molecular tools, we cannot currently discriminate
between the possibilities that ligand inactivation of the polyamine
permease involves its proteolytic degradation, conformational changes
that irreversibly inhibit its activity as a result of allosteric
interaction, and/or spermidine-regulated posttranslational
modifications.
In connection with the latter hypothesis, we have explored the
possibility that polyamine transport might be regulated by the
NPR1-dependent inactivation/reactivation system
governing the posttranslational control of several nitrogen permeases
(34, 35). We have indeed found that NPR1 expression promotes
spermidine transport, but its inactivation does not abolish high
affinity spermidine uptake nor prevent its ligand-induced
down-regulation, as noted for ptk2::lacZ
disruption mutants (22). However, inactivation of both PTK1
and PTK2 was sufficient to fully suppress high affinity spermidine transport and feedback transport repression (22). Thus,
although Npr1p, Ptk1p, and Ptk2p all encode putative, homologous Ser/Thr protein kinases, Npr1p action clearly requires functional PTK1 and/or PTK2 genes. Surprisingly, spermidine
transport was strongly depressed in npr1 mutants under
conditions (i.e. high [NH4+]) where Npr1p activity has been
predicted to be down-regulated by NCR (34, 35). In fact, the
npr1 mutation does not further decrease the already low
activity of the Gap1p or Put4p permeases in
NH4+-rich media, although it
up-regulates their expression at the mRNA level (34, 38, 54, 65,
66). Moreover, our data clearly demonstrate that the spermidine
transport system is not subject to NCR, and if anything,
NH4+ would rather be a weak inducer of
spermidine transport.
Obviously, the model of inactivation/reactivation of nitrogen permeases
by NCR as originally proposed (34, 65) is not compatible with the
observed mechanism of regulation of polyamine transport. In that model,
NPR1-regulated permeases such as Gap1p are constitutively
degraded via a ubiquitin-dependent pathway involving the
NPI1 and NPI2 genes, and rich nitrogen sources
such as NH4+ and L-glutamine
down-regulate Npr1p expression at a posttranscriptional level (34, 35,
38). This level of regulation by nitrogen catabolites was suggested to
involve the increased turnover of Npr1p, which, like Ptk1p and Ptk2p
(22), has strong PEST sequences (67) that might signal its rapid
degradation. However, no report is yet available on the regulation of
Npr1p protein levels nor catalytic activity by NCR. Our data show that
NH4+ certainly does not impair
NPR1 expression as pertaining to spermidine transport
regulation. If Npr1p activity per se is not down-regulated by NH4+, reactivation of nitrogen
permeases by NPR1 should thus involve some downstream,
NCR-antagonized target of Npr1p. The simplest case would be that
NPR1-dependent permeases require phosphorylation for their activity and/or stability and that NCR involves the posttranslational activation of a phosphatase directed to
phosphorylated, active permeases. In support of this hypothesis,
L-glutamine down-regulates Gap1p through its rapid
dephosphorylation, a modification that inactivates the permease and
promotes its degradation (68). There are precedents for a role of
Ser/Thr protein phosphatases in the control of membrane transport in
yeast. For instance, protein phosphatase 2B (or calcineurin) causes the
switch of the Trk1p K+ transporter from a low to a high
affinity mode of K+ transport upon Na+ stress
(69).
Although spermidine transport is regulated by Npr1p, it is clearly
resistant to NCR and is not inactivated via an
NPI1-catalyzed pathway. The Mep1p permease responsible for
low affinity, high capacity NH4+ uptake
is another NPR1-dependent,
NPI1-independent nitrogen permease but is nonetheless
subject to NCR (55, 56). Thus, there is no obligatory link between
NPR1, NCR, and the NPI1-dependent
pathway of ubiquitination/degradation in the control of nitrogen
permease activity. That Npr1p deficiency had a substantially larger
effect on spermidine transport in NH4+-
than proline-based medium suggests that the putative kinase could in
fact protect the polyamine transport system against NCR. Why would
Npr1p expression confer resistance of spermidine transport to NCR but
not for other Npr1p-dependent permeases? One possibility is
that Npr1p regulates the expression and/or activity of intermediary factors specifically targeting the spermidine uptake system such as
Ptk1p and Ptk2p, and that NPR1 function requires intact
PTK1 and PTK2 genes. We have indeed found that
ptk2::lacZ disruption mutants exhibit phenotypic
responses similar to npr1 cells in response to nitrogen
sources.2 In fact, any of the Ser/Thr protein kinases known
to affect spermidine transport could either activate the polyamine
permease through phosphorylation, transcriptional induction, and/or
inactivation of a putative permease phosphatase. The ongoing molecular
identification of the PCP1 gene should help to elucidate the
unique features of spermidine transport regulation in yeast.
We acknowledge the expert assistance of
Sylvie Pilote for high pressure liquid chromatography polyamine
analysis and of Guy Chouinard in the construction of the
pYES-PTK2 expression vector and in complementation analysis
of the pcp1 mutants. We are grateful to Dr. Celia W. Tabor
for the generous gift of the SPE2 gene knock-out construct
and to Dr. Howard Reizman for providing the end3 and end4 mutant strains. We are grateful to Dr. Bruno
André for providing the npi1 and npr1
mutants and the pMV33 plasmid as well as for helpful discussions during
the preparation of this manuscript.