From the Department of Biochemistry and the Lucille P. Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky 40536-0084
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ABSTRACT |
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Sphingoid long chain bases have many effects on
cells including inhibition or stimulation of growth. The physiological
significance of these effects is unknown in most cases. To begin to
understand how these compounds inhibit growth, we have studied
Saccharomyces cerevisiae cells. Growth of tryptophan
(Trp) auxotrophs was more strongly inhibited by
phytosphingosine (PHS) than was growth of Trp+ strains,
suggesting that PHS diminishes tryptophan uptake and starves cells for
this amino acid. This hypothesis is supported by data showing that
growth inhibition is relieved by increasing concentrations of
tryptophan in the culture medium and by multiple copies of the
TAT2 gene, encoding a high affinity tryptophan transporter. Measurement of tryptophan uptake shows that it is inhibited by PHS.
Finally, PHS treatment induces the general control response, indicating
starvation for amino acids. Multiple copies of TAT2 do not
protect cells against two other cationic lipids, stearylamine, and
sphingosine, indicating that the effect of PHS on tryptophan utilization is specific. Other data demonstrate that PHS reduces uptake
of leucine, histidine, and proline by specific transporters. Our data
suggest that PHS targets proteins in the amino acid transporter family
but not other distantly related membrane transporters, including those
necessary for uptake of adenine and uracil.
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INTRODUCTION |
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The long chain base component of sphingolipids inhibits cell growth under many conditions and is cytotoxic to some cell types, but the mechanisms underlying these phenomenona are not well characterized. Interest in the effect of long chain bases on cells was stimulated by the observation that they are potent inhibitors of protein kinase C (1-3). Since these initial studies, there has been a growing number of reports showing effects of long chain bases on a variety of cells (reviewed in Refs. 4 and 5). Most of these studies have been performed on cultured mammalian cells, and it is not clear if the observed effects of long chain bases actually cause growth inhibition or whether some secondary effect inhibits growth. To try and understand how long chain bases inhibit growth, we have examined the effect of long chain bases on Saccharomyces cerevisiae cells.
Sphingolipids are an essential component of wild type S. cerevisiae cells (6). Synthesis of the long chain base component of sphingolipids is similar to that in mammals and begins with the
condensation of serine and palmitoyl-CoA to yield 3-ketosphinganine (for review, see Ref. 7). The 3-ketosphinganine is converted to the
long chain base sphinganine (dihydrosphingosine) which is
N-fatty-acylated to yield dihydroceramide. Dehydrogenation of dihydroceramide in animals yields ceramide containing the long chain
base sphingosine, which is rapidly converted to sphingolipids by the
addition of polar components to the 1-hydroxyl group. The predominant
ceramides in fungi and plants contain
N--hydroxyfattyacylphytosphingosine (6). Phytosphingosine
(PHS)1 lacks the 4,5-double
bond found in sphingosine and has instead an hydroxyl group at the
4-position. In S. cerevisiae and other fungi, the 1-hydroxyl
of phytoceramide is modified by addition of myo-inositol
phosphate to form inositol phosphoceramide, which is then
mannosylated to yield mannose-inositol phosphoceramide. The final step
in S. cerevisiae sphingolipid synthesis is the addition of
inositol phosphate to mannose-inositol phosphoceramide to yield
the major sphingolipid mannose-(inositol-P)2 ceramide (8-10).
When grown in complex medium having a non-inhibitory concentration of long chain base, S. cerevisiae cells are able to take up the long chain base, by an unknown mechanism that requires the multimembrane-spanning Lcb3 protein (11), and incorporate it into ceramide and other sphingolipids (12). Surprisingly, even the non-biological threo isomer of sphinganine can be incorporated into sphingolipids (12). When present in high enough concentrations, most long chain bases will inhibit growth of S. cerevisiae cells. However, growth inhibition is only seen if the culture density is kept very low, for example, less than 104 cells/ml (13).
After initiating these studies, we observed that growth is more strongly inhibited by long chain bases in strains requiring amino acids (auxotrophic strains), particularly tryptophan and leucine. Amino acid transport in S. cerevisiae is a complex and highly regulated process because amino acids serve not only as building blocks for protein synthesis, but also as a source of nitrogen when a preferred nitrogen source, such as ammonia, glutamine, or asparagine, is unavailable. In the absence of a preferred nitrogen source, for example, when proline or urea are the primary nitrogen source, amino acids are transported by both specific amino acid permeases and by the general amino acid permease (Gap1p; Ref. 14). Gap1p transports all natural L-amino acids found in proteins and also ornithine, citrulline, and several D-amino acids and toxic amino acid analogs (15).
Gap1p activity is repressed when a preferred nitrogen source is available, a phenomenon referred to as nitrogen catabolite repression (16). Repression of Gap1p activity occurs at several levels by complex regulatory mechanisms (14, 17, 18). In contrast, the activity of some, but not all, specific amino acid permeases is constitutive and not repressed by preferred nitrogen sources (19).
We demonstrate here that PHS blocks transport of tryptophan, leucine, histidine, and proline by their specific amino acid transporters. Inhibition of amino acid transport explains why PHS blocks growth of auxotrophic S. cerevisiae strains and why prototrophic strains are resistant to the drug.
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EXPERIMENTAL PROCEDURES |
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Strains, Plasmids, and Culture Conditions-- Strains used in this work are shown in Table I. Plasmids pAS5 and pAS6 carried the TAT1 and TAT2 gene, respectively, on YEp351 and were provided by Michael Hall (Biozentrum, Basel, Switzerland).
Yeast were grown at 30 °C in complex medium (PYED) buffered to pH 5.0 as described (20) or in defined medium (SD) containing 0.17% yeast nitrogen base (U. S. Biological, Swampscott, MA), 2% glucose, and 0.5% ammonium sulfate. Proline (1 mg/ml) was used as a nitrogen source in place of ammonium sulfate where indicated. SD complete medium was supplemented with adenine sulfate, uracil, L-leucine, L-lysine, L-tryptophan, L-histidine, L-arginine, and L-methionine (all at 20 mg/liter); L-isoleucine (30 mg/liter); L-valine (150 mg/liter); and L-threonine (200 mg/liter). SD minimal medium was supplemented with required nutrients only. All media containing PHS also contained 0.05% Tergitol.Assay for Growth Inhibition by PHS-- Cells were grown with aeration in SD minimal medium to a density of about 107/ml. The culture was briefly sonicated, and cells were counted and diluted in PYED or SD complete medium to a density of 2.5 × 104/ml. Following a 4-h incubation at 30 °C, 100-µl aliquots of cells were diluted into a series of wells in a microtiter plate, each well containing 100 µl of PYED or SD complete medium supplemented with PHS at final concentrations of 4, 8, 16, 32, 64, and 128 µM. One well in each series had no PHS as a control. The final concentration of Tergitol in each well was 0.05%. Plates were incubated at 30 °C until the optical density of the culture lacking PHS reached 0.9-1.0 (24 h for PYED and 40 h for SD medium). The contents of each well were mixed, and the absorbance at 600 nm (A600 unit) was measured.
Assay of the General Control Response--
The GCN4
induction assay was performed as described previously (21), except that
YPH252 cells were grown in SD medium (minus uracil and histidine but
containing 0.05% Tergitol as a carrier for PHS) and were transformed
with both pRS313(HIS3) (22) and pB180, a plasmid having the
GCN4 promoter fused to lacZ (23). Under these
growth condition, cells can be starved for lysine, leucine, and
tryptophan. Saturated cultures were diluted, grown overnight with
shaking at 30 °C to an A600 nm of 0.1-0.15, treated with the compounds indicated in Table II for 5 h, and harvested, and cell-free extracts were assayed for -galactosidase activity (24).
Tryptophan Uptake Assay-- The rate of tryptophan uptake by cells was measured as described previously (21) but with modifications. Cells were grown at 27 °C in PYED medium to an A600 of 0.2, washed, and resuspended at an A600 of 0.7 for transport measurements. Cells were incubated 10 min with or without 100 µM PHS at 30 °C, and then uptake was initiated at time zero by addition of L-[5-3H]tryptophan (20-30 Ci/mmol, NEN Life Science Products) to a final concentration of 7-9 µM or 70-90 µM. Samples were filtered (0.25 µm pore size, 25 mm diameter filters; Nucleopore, Pleasanton, CA), air-dried, and analyzed in a liquid scintillation counter. Data are expressed as the percent of radioactivity retained on the filter.
Miscellaneous Methods-- Yeast were transformed using a LiOAc procedure (25). Standard yeast genetic techniques were used (26). Protein concentrations were measured using the Bio-Rad Bradford reagent with bovine serum albumin as the protein standard.
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RESULTS |
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Tryptophan Auxotrophs Are Sensitive to PHS-- An initial screening of S. cerevisiae strains for inhibition of growth by PHS revealed differences in sensitivity. Strains such as YPH250 were inhibited (PHS-sensitive, PHSS), whereas other strains such as SJ19 were not (PHS-resistant, PHSR). Segregation analysis demonstrated a genetic basis for this difference in sensitivity; five four-spored tetrads gave 2:2 segregation for the PHSS:PHSR phenotypes, showing that the phenotypes are due to a single, nuclear gene. Since the PHSR and Trp+ phenotypes co-segregated, the gene responsible for the PHSR phenotype must be TRP1 or a closely linked gene. The PHSR phenotype was dominant to the PHSS phenotype.
TRP1 is most likely the gene responsible for the PHSR phenotype because transformation of strain YPH250 with the centromeric plasmid pRS314 (TRP1) gave PHSR cells. Since pRS314 carries a portion of the GAL3 gene that is linked to TRP1, we verified that cells transformed with a plasmid carrying GAL3 remained sensitive to PHS. We conclude from these data that tryptophan auxotrophs (Trp
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Tryptophan and Enhanced Tryptophan Transport Protect Cells from PHS Inhibition-- Growth inhibition by PHS is most likely due to inhibition of tryptophan transport into cells and consequent starvation for this amino acid. This hypothesis predicts that growth inhibition by PHS should be alleviated by increasing the concentration of tryptophan in the culture medium. This prediction was verified (Fig. 2).
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Tryptophan Accumulation Is Inhibited by PHS-- To determine directly if PHS inhibits tryptophan accumulation, the rate of uptake of radioactive tryptophan by wild type and mutant strains defective in tat1, tat2, or both genes was determined at low (7-9 µM Trp) and high (70-90 µM Trp) tryptophan concentrations in the presence or absence of PHS. Tryptophan accumulation was inhibited 60-70% at a low tryptophan concentration and about 50% at a high concentration in the presence of 100 µM PHS (Fig. 4A).
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The Nitrogen Source Affects PHS Sensitivity-- Nutrient transport, particularly amino acid transport, across the plasma membrane is known to be affected by the nitrogen source present in the culture medium (19). In complex culture medium like the PYED medium used here, amino acids serve in part as a source of nitrogen because other nitrogen sources are limiting. Under these circumstances amino acid transport is mediated by both specific amino acid permeases, such as Tat2p, and also by the general amino acid permease (Gap1p). Thus, it is possible that PHS may be inhibiting other specific amino acid transporters but inhibition would not be seen because the amino acid would be transported also by Gap1p and its transport would not become limiting for growth. In addition, the concentration of amino acids in PYED medium is not defined and the concentration might be high enough to overcome the growth inhibiting affect of PHS.
The effect of the nitrogen source on PHS sensitivity was examined first using defined medium (SD) containing ammonium ions as the nitrogen source. In this growth medium, amino acid uptake is thought to occur through specific transporters and not through Gap1p whose synthesis is repressed by ammonium ions (16). Growth of prototrophic YPH-A cells is barely inhibited under these conditions of nitrogen catabolite repression (Fig. 5A), even at the highest possible PHS concentration, unlike the case in PYED medium (nitrogen depressing conditions) where some inhibition of growth is seen at high PHS concentrations (Fig. 1). These data further support the hypothesis that PHS inhibits growth by blocking the activity of specific amino acid transporters.
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PHS Induces the General Control Response--
Starvation of yeast
cells for amino acids induces a signaling pathway, the general control
response, which increases translation of GCN4 mRNA. The
resulting Gcn4 protein activates transcription of genes encoding the
biosynthetic enzymes necessary for synthesis of many amino acids (32).
If PHS is inhibiting amino acid uptake, then PHS treatment should
induce the general control response. Induction of the general control
response was quantified by measuring -galactosidase activity in
YPH252 cells transformed with a reporter plasmid containing the
GCN4 promoter fused to the lacZ gene (plasmid pB180; Ref. 23).
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DISCUSSION |
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The studies reported here demonstrate that growth inhibition of S. cerevisiae cells by the sphingoid long chain base PHS is due to inhibition of amino acid transporters and subsequent starvation for amino acids. Most data are for tryptophan transport by the Tat2 transporter, other data show that PHS reduces uptake of leucine, histidine, and proline. No interference with uracil or adenine uptake was observed.
Inhibition of tryptophan uptake by PHS was initially indicated by
segregation analysis, which revealed that the sensitive/resistance phenotype was due to a single, nuclear gene and that the resistance phenotype was tightly linked to the TRP1 locus. Because
Trp+ cells such as SJ19, YPH-A (Fig. 1) or YPH250
transformed with a plasmid (pRS314) carrying the TRP1 gene
were resistant, whereas Trp cells such as YPH-D (Fig. 1)
were sensitive to growth inhibition by PHS, we hypothesized that
tryptophan uptake was impaired by PHS. Several types of data support
this hypothesis. First, increasing concentrations of tryptophan in the
culture medium reduce growth inhibition of the Trp
strain
YPH-D by PHS (Fig. 2). Second, growth inhibition by PHS is greatly
reduced by multiple copies of TAT2, encoding a high affinity
tryptophan transporter, and to a lesser extent by multiple copies of
the TAT1, encoding the low affinity tryptophan transporter (Fig. 3A). Third, PHS treatment diminishes the rate of
tryptophan uptake. In PYED medium, most of this uptake is mediated by
the Tat2 transporter (Fig. 4). Finally, the general control response is
induced by PHS treatment (Table II), indicating that cells are being
starved for one or more amino acids.
How might PHS affect Tat2 transport activity? Our data are not consistent with PHS directly inhibiting activity because the rate of tryptophan transport is normal for about 10 min and then it tapers off (Fig. 4). These kinetics are consistent with PHS activating a signal transduction pathway that down-regulates Tat2 activity. PHS is not disrupting the proton gradient necessary for tryptophan transport, because transport of other nutrients, such as uracil, adenine, and vitamins that require a proton gradient, is not blocked.
The Tat2 protein is a member of the major facilitator superfamily (33)
whose members contain 12 membrane-spanning -helices, a hydrophilic N
and C terminus, and 500-600 amino acids. These proteins lack the
nucleotide-binding domain typical of ABC transporters and the conserved
amino acids characteristic of the catalytic site in P-type ATPases. In
S. cerevisiae, the Tat2, Gap1p, Tat1, and other amino acid
transporters belong to a family of closely related proteins that is
distinct from the uracil/allantoin transporters and from the purine
transporter (19, 34, 35). PHS did not inhibit uracil or adenine
transport under nitrogen-repressing (ammonia ions in the culture
medium), partially nitrogen-repressing (PYED medium), or
nitrogen-derepressing (proline in the culture medium) conditions (Figs.
1 and 5), indicating that PHS does not affect these two families of
major facilitator superfamily of membrane-bound transporters.
Under nitrogen-repressing conditions, PHS strongly inhibited growth of
Leu cells and slightly inhibited growth of
His
cells, indicating that uptake of these two amino
acids is diminished by PHS (Fig. 5A). PHS may also inhibit
transport of proline by the Put4 protein since growth of prototrophic
YPH-A cells was more sensitive to PHS under conditions where proline
was the nitrogen source (Fig. 5B) than it was when ammonium
ions were the nitrogen source (Fig. 5A). These results,
along with the phylogenetic relationship of the amino acid
transporters, predict that the activity of most or perhaps all amino
acid transporters in S. cerevisiae is inhibited by PHS.
Long chain bases have numerous effects on mammalian cells (reviewed in Refs. 4 and 5), and many of these are probably specific for differentiated cells and, therefore, have no counterpart in S. cerevisiae cells. Growth inhibition (not cell death) by PHS was the end point in our experiments, and this effect has been seen with mammalian cells but the underlying mechanism(s) is unknown (reviewed in Ref. 4). Based upon our data, long chain bases may inhibit mammalian cell growth by blocking uptake of an essential nutrient, perhaps one transported by a membrane-bound protein.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM41302 (to R. L. L. and R. C. D.).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.: 606-323-6052;
Fax: 606-257-8940; E-mail: bobd{at}pop.uky.edu.
1 The abbreviations used are: PHS, phytosphingosine; Gap1p, a general amino acid transporter; GCN4, gene encoding a transcription activator; PHSR, resistance to PHS; PHSS, sensitivity to PHS; PYED, complex medium; SD, defined medium; TAT1, gene encoding the high affinity tyrosine transporter; TAT2, a gene encoding a high affinity tryptophan transporter.
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REFERENCES |
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