Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation

The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy. When expressed from a multi-copy vector, TPO1 was located mainly on the plasma membrane, but with some localization on the vacuolar membrane. Polyamine transport by TPO1 was dependent on pH. Uptake of spermidine and spermine occurred at alkaline pH (pH 8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (pH 5.0). This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli. Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable with the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0. However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) exists mainly as spermidine (or spermine)-ribosome complex in cells. The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1. Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.     

AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae

Polyamines play essential functions in many aspects of cell biology. Plasma membrane transport systems for the specific uptake of polyamines exist in most eukaryotic cells but have been very recently identified at the molecular level only in the parasite Leishmania. We now report that the high affinity polyamine permease in Saccharomyces cerevisiae is identical to Agp2p, a member of the yeast amino acid transporter family that was previously identified as a carnitine transporter. Deletion of AGP2 dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines, and transformation with an AGP2 expression vector restored polyamine transport in agp2delta mutants. Yeast mutants deficient in polyamine biosynthesis required >10-fold higher concentrations of exogenous putrescine to restore cell proliferation upon deletion of the AGP2 gene. Disruption of END3, a gene required for an early step of endocytosis, increased the abundance of Agp2p, an effect that was paralleled by a marked up-regulation of spermidine transport velocity. Thus, AGP2 encodes the first eukaryotic permease that preferentially uses spermidine over putrescine as a high affinity substrate and plays a central role in the uptake of polyamines in yeast.     

Identification of the TPO1 gene in yeast, and its human orthologue TETRAN, which cause resistance to NSAIDs

Non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, have serious gastrointestinal side effects. Since their direct cytotoxicity was suggested to be involved in this side effect, we here tried to identify NSAID-resistant genes. We screened for Saccharomyces cerevisiae genes whose overexpression causes indomethacin resistance and identified the TPO1 gene, which encodes a major facilitator superfamily transporter. Its overexpression or deletion made yeast cells resistant or sensitive, respectively, to some NSAIDs. A BLAST search identified the possible human orthologue of Tpo1p, tetracycline transporter-like protein (TETRAN), whose overexpression in cultured human cells caused resistance to some NSAIDs, suggesting that TETRAN is an efflux pump for some NSAIDs.     

Inward potassium channel in guard cells as a target for polyamine regulation of stomatal movements

A number of studies show that environmental stress conditions such as drought, high salt, and air pollutants increase polyamine levels in plant cells. However, little is understood about the physiological function of elevated polyamine levels. We report here that polyamines regulate the voltage-dependent inward K(+) channel in the plasma membrane of guard cells and modulate stomatal aperture, a plant "sensor" to environmental changes. All natural polyamines, including spermidine, spermine, cadaverine, and putrescine, strongly inhibited opening and induced closure of stomata. Whole-cell patch-clamp analysis showed that intracellular application of polyamines inhibited the inward K(+) current across the plasma membrane of guard cells. Single-channel recording analysis indicated that polyamine regulation of the K(+) channel requires unknown cytoplasmic factors. In an effort to identify the target channel at the molecular level, we found that spermidine inhibited the inward K(+) current carried by KAT1 channel that was functionally expressed in a plant cell model. These findings suggest that polyamines target KAT1-like inward K(+) channels in guard cells and modulate stomatal movements, providing a link between stress conditions, polyamine levels, and stomatal regulation.     

Identification and characterization of a diamine exporter in colon epithelial cells

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N(1)-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.     

Saccharomyces cerevisiae lacking Btn1p modulate vacuolar ATPase activity to regulate pH imbalance in the vacuole

The vacuolar H(+)-ATPase (V-ATPase) along with ion channels and transporters maintains vacuolar pH. V-ATPase ATP hydrolysis is coupled with proton transport and establishes an electrochemical gradient between the cytosol and vacuolar lumen for coupled transport of metabolites. Btn1p, the yeast homolog to human CLN3 that is defective in Batten disease, localizes to the vacuole. We previously reported that Btn1p is required for vacuolar pH maintenance and ATP-dependent vacuolar arginine transport. We report that extracellular pH alters both V-ATPase activity and proton transport into the vacuole of wild-type Saccharomyces cerevisiae. V-ATPase activity is modulated through the assembly and disassembly of the V(0) and V(1) V-ATPase subunits located in the vacuolar membrane and on the cytosolic side of the vacuolar membrane, respectively. V-ATPase assembly is increased in yeast cells grown in high extracellular pH. In addition, at elevated extracellular pH, S. cerevisiae lacking BTN1 (btn1-Delta), have decreased V-ATPase activity while proton transport into the vacuole remains similar to that for wild type. Thus, coupling of V-ATPase activity and proton transport in btn1-Delta is altered. We show that down-regulation of V-ATPase activity compensates the vacuolar pH imbalance for btn1-Delta at early growth phases. We therefore propose that Btn1p is required for tight regulation of vacuolar pH to maintain the vacuolar luminal content and optimal activity of this organelle and that disruption in Btn1p function leads to a modulation of V-ATPase activity to maintain cellular pH homeostasis and vacuolar luminal content.     

Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae

It has been reported that GAP1 and AGP2 catalyze the uptake of polyamines together with amino acids in Saccharomyces cerevisiae. We have looked for polyamine-preferential uptake proteins in S. cerevisiae. DUR3 catalyzed the uptake of polyamines together with urea, and SAM3 was found to catalyze the uptake of polyamines together with S-adenosylmethionine, glutamic acid, and lysine. Polyamine uptake was greatly decreased in both DUR3- and SAM3-deficient cells. The K(m) values for putrescine and spermidine of DUR3 were 479 and 21.2 mum, respectively, and those of SAM3 were 433 and 20.7 mum, respectively. Polyamine stimulation of cell growth of a polyamine requiring mutant, which is deficient in ornithine decarboxylase, was not influenced by the disruption of GAP1 and AGP2, but it was diminished by the disruption of DUR3 and SAM3. Furthermore, the polyamine stimulation of cell growth of a polyamine-requiring mutant was completely inhibited by the disruption of both DUR3 and SAM3. The results indicate that DUR3 and SAM3 are major polyamine uptake proteins in yeast. We previously reported that polyamine transport protein kinase 2 regulates polyamine transport. It was found that DUR3 (but not SAM3) was activated by phosphorylation of Thr(250), Ser(251), and Thr(684) by polyamine transport protein kinase 2.     

Metabolism of polyamines in NaCl-resistant cell lines from Nicotiana sylvestris

The polyamine titers in three cell lines of Nicotiana sylvestris were compared: Type 1, rapidly adapting to NaCl; Type 2, constantly resistant to NaCl; Type 3, a saltsensitive wild strain. During short-term cultivation in MS medium in the presence of 170 mM NaCl (1 passage, 14 d) the changes in polyamine titer in cell suspensions of type 1 (in a slightly adapted state) and non-adapted wild strain (type 3) showed a considerable increase in spermidine and spermine and a decrease in putrescine. After prolonged adaptation to NaCl (20 passages) the putrescine content in the cells of type 1 and of type 2 was increased at the expense of the polyamines. This suggests that the pattern of polyamine titer varies under short- and long-term adaptation to NaCl. The inverse ratio between growth processes and changes in polyamine and proline level indicates that polyamines fulfil primarily a protective and osmorepulatory function in plant cells under NaCl stress.     

Histamine prevents polyamine accumulation in mouse C57.1 mast cell cultures.

The effects of histamine on polyamine uptake and metabolism was studied in a mouse mast cell line (C57.1), as a cell model in which both biogenic amines are important for maintaining cell function and viability. Results obtained after incubations with exogenous histamine indicated that histamine prevents polyamine accumulation by affecting polyamine uptake. A plasma membrane transport system for polyamines has been also studied in mast cells. It seems to be a Na(+)-dependent uptake with high affinity for both spermine and spermidine and lower affinity for putrescine and agmatine. Polyamine uptake was reduced in both cells treated with exogenous histamine and histamine-preloaded cells. However, ornithine decarboxylase activity and cell proliferation were not affected by histamine. Incubation with histamine enhanced the spermidine/spermine acetyl transferase induction caused by N(1)-ethyl-N(11)-[(cyclopropyl)methyl]-4,8-diazaundecane, suggesting that polyamine acetylation could be another mechanism by which histamine prevents polyamine accumulation in C57.1 mast cells.     

Multidrug resistance modifies polyamines uptake in P388 murine lymphoma cells: experimental and modeling approach.

Polyamines (putrescine, spermidine and spermine) are ubiquitous compounds, essential for cell growth. This paper compares the polyamine transport between sensitive P388 murine lymphoma cells and two multidrug resistant P388 sublines with the assistance of an experimental model. This new model allows the characterisation of the whole polyamines uptake and efflux. Three parameters are identified by the model: two rate constants (K+ for the uptake and K- for the efflux) which are considered as physical constants specific to the transport of one polyamine in one cell type, and Ci(o) which represents the initial intracellular concentration. This model well describes our experimental results of polyamine transport across the P388 cell plasma membrane. Multidrug resistant P388 cells exhibit spermine uptake significantly higher than that of sensitive cells when on the opposite, putrescine enters more rapidly into the sensitive P388 cells. In conclusion, comparison of polyamine transport between sensitive and multidrug resistant P388 phenotypes shows large and significant differences.     

Screening for modulators of spermine tolerance identifies Sky1, the SR protein kinase of Saccharomyces cerevisiae, as a regulator of polyamine transport and ion homeostasis

Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants of Saccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Δ cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity of PPZ1 but not of ENA1.     

Tpo1‐mediated spermine and spermidine export controls cell cycle delay and times antioxidant protein expression during the oxidative stress response

Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H₂O₂) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H₂O₂ tolerance. Thus, central components of the stress response are timed through Tpo1‐controlled polyamine export.     

Intracellular polyamine pools, oligopeptide-binding protein A expression, and resistance to aminoglycosides in Escherichia coli

The role of intracellular free polyamine (putrescine and spermidine) pools in multiple resistance to aminoglycoside antibiotics was investigated among in vitro selected kanamycin-resistant Escherichia coli J53 mutants expressing diminished oligopeptide-binding protein (OppA) levels and/or defective ornithine decarboxylase (ODC) activity. The results suggest that diminished OppA content, but not defective ODC activity expression, increased the relative concentration of free spermidine as compared to the wild type strain. Moreover, by adding exogenous polyamines or polyamine synthesis inhibitors to cultures with different mutant strains, a direct relationship between the intracellular OppA levels and resistance to kanamycin was revealed. Collectively these results further suggest a complex relation among OppA expression, aminoglycoside resistance and polyamine metabolism.     

The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.     

Agp2, a Member of the Yeast Amino Acid Permease Family,Positively Regulates Polyamine Transport at the Transcriptional Level

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.     

Polyamine transport system mediates agmatine transport in mammalian cells

Agmatine is a biogenic amine with the capacity toregulate a number of nonreceptor-mediated functions in mammalian cells, including intracellular polyamine content and nitric oxide generation. We observed avid incorporation of agmatine into several mammalian celllines and herein characterize agmatine transport in mammalian cells. Intransformed NIH/3T3 cells, agmatine uptake is energy dependent with asaturable component indicative of carrier-mediated transport. Transportdisplays an apparent Michaelis-Menten constant of 2.5 µM and amaximal velocity of 280 pmol · min¹ · mg¹ proteinand requires a membrane potential across the plasma membrane foruptake. Competition with polyamines, but not cationic molecules thatutilize the y⁺ system transporter, suppresses agmatineuptake. Altering polyamine transporter activity results in parallelchanges in polyamine and agmatine uptake. Furthermore, agmatine uptakeis abrogated in a polyamine transport-deficient human carcinoma cellline. These lines of evidence demonstrate that agmatine utilizes, and is dependent on, the polyamine transporter for cellular uptake. Thefact that this transport system is associated with proliferation couldbe of consequence to the antiproliferative effects of agmatine.