Isolation and characterization of Saccharomyces cerevisiae mutants deficient in S-adenosylmethionine decarboxylase, spermidine, and spermine.

Four mutants were isolated from Saccharomyces cerevisiae that are deficient in S-adenosylmethionine decarboxylase (spe2). All four mutants are chromosomal and fall into a single complementation group tightly linked to arg1. Since one of the mutants contained a temperature-sensitive activity, this complementation group defines the structural gene. Mutants totally lacking enzymic activity did not contain spermidine or spermine and had a greatly increased doubling time when grown in the absence of these two polyamines. Addition of 10(-6) M spermidine or 10(-5) M spermine, but not putrescine or cadaverine, restored the doubling time to that of the wild type. Diploids formed from a cross of two mutants completely deficient in spermidine and spermine were unable to sporulate in the absence of added spermidine or spermine. We obtained evidence that arg1 was not located on any of the 17 known chromosomes, and therefore we postulate that arg1 and spe2 are located on a new 18th chromosome.     

Construction of an Escherichia coli strain unable to synthesize putrescine, spermidine, or cadaverine: characterization of two genes controlling lysine decarboxylase.

We have previously described a polyamine-deficient strain of Escherichia coli that contained deletions in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). Although this strain completely lacked putrescine and spermidine, it was still able to grow at a slow rate indefinitely on amine-deficient media. However, these cells contained some cadaverine (1,5-diaminopentane). To rule out the possibility that the presence of cadaverine permitted the growth of this strain, we isolated a mutant (cadA) that is deficient in cadaverine biosynthesis, namely, a mutant lacking lysine decarboxylase, and transduced this cadA gene into the delta (speA-speB) delta speC delta D strain. The resultant strain had essentially no cadaverine but showed the same phenotypic characteristics as the parent. Thus, these results confirm our previous findings that the polyamines are not essential for the growth of E. coli or for the replication of bacteriophages T4 and T7. We have mapped the cadA gene at 92 min; the gene order is mel cadA groE ampA purA. A regulatory gene for lysine decarboxylase (cadR) was also obtained and mapped at 46 min; the gene order is his cdd cadR fpk gyrA.     

Cyclic AMP may not be involved in catabolite repression in Saccharomyces cerevisiae: evidence from mutants unable to synthesize it

Yeast cells with a nonsense adenylate cyclase mutation, cyr1-3, required cyclic AMP for growth. This phenotype was suppressed by the byc1 mutation; however, cyr1-3 bcy1 cells produced no detectable level of adenylate cyclase or cyclic AMP. On induction, the bcy1 and cyr1-3 bcy1 mutant cells produced the same levels of galactokinase and alpha-D-glucosidase as did the wild-type cells and fourfold-higher levels of invertase. Since galactokinase synthesis was severely repressed by glucose in the constitutive GAL81 mutants, irrespective of the cyr1-3 bcy1 genotype, cyclic AMP may not be involved in catabolite repression.     

The roles of the polyamines, putrescine, spermidine, and spermine in normal and malignant tissues

Despite the initial aversion to polyamine research which can be attributed to their peculiar nomenclature and to the erroneous idea that polyamines are products of bacterial decay, it appears that these ubiquitous amines play important roles in the physiological regulation of growth. Many of the definitive roles are yet to be elucidated, and these areas offer promise to biochemists. The evolution of the multifaceted ramifications of polyamines is not unlike that for cyclic AMP, which has profound effects at the cellular level. Further, we are at a stage at which basic knowledge of the roles of polyamines is becoming clinically relevant. We should begin to measure polyamines routinely to obtain clinical parameters which might allow for more efficacious treatment of cancer. No longer can the discussion of polyamines in biochemical textbooks be limited to a page and a half or no discussion at all, and no longer can well-informed scientists afford to neglect the importance and the far-reaching applications of polyamine research.     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

The modulation of the induction of ornithine decarboxylase by spermine,spermidine and diamines

Extremely low concentrations of putrescine, spermidine and spermine added to the extracellular medium of cultures of mammalian cells inhibit the induction of ornithine decarboxylase activity despite 100- to 1,000-fold greater intracellular polyamine concentrations. The diamines, 1,2-diaminoethane, 1,3-diaminopropane, 1,5-diaminopentane, 1,7-diaminoheptane, 1,10-diaminodecane, 1,12-diaminododecane also inhibit ornithine decarboxylase at all concentrations tested (greater than 10^?6 M). In contrast, 10^?6 M to 10 ^?3 M 1,8-diaminooctane, the alkyl analog of spermidine, enhances ornithine decarboxylase activity. The concentraton of putrescine required to inhibit the activity of ornithine decarboxylase by 50% is a characteristic of each cell line; however, it varies by as much as 1,000-fold among the five cell lines we have tested (L1210 leukemic, H35 hepatoma, N18 neuroblastoma, W256 carcinosarcoma and 3T3 fibroblasts). The antizyme to ornithine decarboxylase can be induced in all these cells by high (di)(poly)amine concentrations. Based on these and other experiments we suggest a working hypothesis: that the polyamines regulate ornithine decarboxylase activity through two different sites that may be interrelated; a sensitive membrane-mediated site that responds to minute fluctuations of extracellular polyamine levels and a coarse site which may be intracellular or membrane associated that responds to larger fluctuations of intracellular polyamine levels. The consequences of such a control mechanism operating within the whole organism are discussed.     

Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae

It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.     

Contents of putrescine, spermidine and spermine in tissue of the human brain glial tumors

It is shown that in the tissue of the human brain glial tumours the content of putrescine depends on the degree of the tumour malignization. In malignant gliomas (glioblastomas), as compared to the benign (astrocytomas), the content of putrescine is significantly higher. The content of spermidine in glial tumours of a malignancy different degree is twice as high as the level of this polyamine in the brain grey matter, and it is twice as low as in the white matter. The content of spermine in the brain glial tumours does not differ essentially from its level in the brain tissue.     

Expression of a heterologous S-adenosylmethionine decarboxylase cDNA in plants demonstrates that changes in S-adenosyl-L-methionine decarboxylase activity determine levels of the higher polyamines spermidine and spermine

We posed the question of whether steady-state levels of the higher polyamines spermidine and spermine in plants can be influenced by overexpression of a heterologous cDNA involved in the later steps of the pathway, in the absence of any further manipulation of the two synthases that are also involved in their biosynthesis. Transgenic rice (Oryza sativa) plants engineered with the heterologous Datura stramonium S-adenosylmethionine decarboxylase (samdc) cDNA exhibited accumulation of the transgene steady-state mRNA. Transgene expression did not affect expression of the orthologous samdc gene. Significant increases in SAMDC activity translated to a direct increase in the level of spermidine, but not spermine, in leaves. Seeds recovered from a number of plants exhibited significant increases in spermidine and spermine levels. We demonstrate that overexpression of the D. stramonium samdc cDNA in transgenic rice is sufficient for accumulation of spermidine in leaves and spermidine and spermine in seeds. These findings suggest that increases in enzyme activity in one of the two components of the later parts of the pathway leading to the higher polyamines is sufficient to alter their levels mostly in seeds and, to some extent, in vegetative tissue such as leaves. Implications of our results on the design of rational approaches for the modulation of the polyamine pathway in plants are discussed in the general framework of metabolic pathway engineering.     

Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., J?nne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.     

Evidence that spermine, spermidine, and putrescine are transported electrophoretically in mitochondria by a specific polyamine uniporter.

We present evidence that polyamine uptake into rat liver mitochondria is mediated by a specific polyamine uniporter. Polyamine transport is not mediated by the ornithine, lysine, or Ca2+ transporters of mitochondria. Polyamine transport is a saturable process, with apparent Km values of 0.13 mM for spermine, 0.26 mM for spermidine, and 1 mM for putrescine. These substrates are mutually competitive inhibitors, indicating a common transport system. Polyamine transport is strictly dependent on membrane potential and insensitive to medium pH, showing that these polycations are transported electrophoretically. Spermine, spermidine, and putrescine are taken up by rat liver mitochondria at rates that increase with increasing valence of the transported species. The activation enthalpies for transport were 24, 32, and 59 kJ/mol for putrescine, spermidine, and spermine, respectively. These values, which amount to about 12 kJ/mol per charge transferred, may be compared to a value of 76 kJ/mol observed for monovalent tetraethylammonium cation. Flux-voltage analysis is consistent with the hypothesis that the mitochondrial polyamine transporter catalyzes transport via a channel mechanism.     

Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. Translational inhibition of synthesis and acceleration of degradation of the enzyme by putrescine, spermidine, and spermine

We have recently isolated, without using any inhibitors, a mutant of Chinese hamster ovary cell line which greatly overproduces ornithine decarboxylase in serum-free culture. Addition of polyamines (putrescine, spermidine, or spermine, 10 microM) or ornithine (1 mM), the precursor of polyamines, to the culture medium of these cells caused a rapid and extensive decay of ornithine decarboxylase activity. At the same time the activity of S-adenosylmethionine decarboxylase showed a less pronounced decrease. Notably, the polyamine concentrations used were optimal for growth of the cells and caused no perturbation of general protein synthesis. Spermidine and spermine appeared to be the principal regulatory amines for both enzymes, but also putrescine, if accumulated at high levels in the cells, was capable of suppressing ornithine decarboxylase activity. The amount of ornithine decarboxylase protein (as measured by radioimmunoassay) declined somewhat more slowly than the enzyme activity, but no more than 10% of the loss of activity could be ascribed to post-translational modifications or inhibitor interaction. Some evidence for inactivation through ornithine decarboxylase-antizyme complex formation was obtained. Gel electrophoretic determinations of the [35S]methionine-labeled ornithine decarboxylase revealed a rapid reduction in the synthesis and acceleration in the degradation of the enzyme after polyamine additions. No decrease in the amounts of the two ornithine decarboxylase-mRNA species, hybridizable to a specific cDNA, was detected, suggesting that polyamines depressed ornithine decarboxylase synthesis by selectively inhibiting translation of the message.     

SAM1, the structural gene for one of the S-adenosylmethionine synthetases in Saccharomyces cerevisiae. Sequence and expression

Saccharomyces cerevisiae contains two genes, SAM1 and SAM2, encoding functional S-adenosylmethionine synthetases. The gene SAM1 was isolated by functional complementation of a double mutant of S. cerevisiae, and its identity was confirmed by gene disruption. The cloned gene was used to probe wild type chromosomal DNA, and two regions hybridizing with SAM1 were found, one of which is the SAM1 region. The DNA sequence of SAM1 is reported. The translation product shows a high homology with the one deduced from the sequence of the MetK gene encoding the SAM synthetase of Escherichia coli.     

Thermal denaturation of mononucleosomes in the presence of spermine,spermidine, N1-acetylspermidine,N8-acetylspermidine or putrescine: implications for chromosome structure

Putrescine (a diamine) raises the thermal denaturation temperature of mononucleosomes but produces only minor changes in the overall shape of the thermal denaturation curve. This is similar to the effect of sodium ions and is consistent with nonspecific binding to the DNA of the nucleosome. At very low levels of spermidine or spermine the same simple rise in thermal denaturation temperature is seen but at higher levels (above 1 M for total spermidine concentration) the thermal denaturation curve becomes substantially sharper and the premelt region of the curve diminishes in area. The acetylspermidines display intermediate effects. The change in shape of the thermal denaturation curve was resolved into components (R1 and R2) due to mononucleosomes in their original conformation plus a component (T) induced by the presence of spermidine or spermine. The proportion of component T was substantially reduced with acetylspermidine, compared to equivalent concentrations of spermidine. Hence, we suggest that spermidine acetylationin vivo has the potential to partially destabilise the nucleosome structure, possibly in coordination with histone acetylation.     

WASP suppresses the growth defect of Saccharomyces cerevisiae las17Delta strain in the presence of WIP

Wiskott-Aldrich syndrome is caused by alterations in the Wiskott-Aldrich syndrome protein (WASP) and several of these mutations affect WASP's interaction with WIP (WASP-interacting protein), suggesting that loss of interaction between WASP and WIP is causal to the disease. Las17p is the yeast homologue of WASP and las17Delta strain is unable to grow at 37 degrees C. We show that Human WASP suppresses the growth defect of Saccharomyces cerevisiae las17Delta strain, only in the presence of WIP. WIP mediates cortical localisation of WASP as well as stabilise WASP in yeast cells. Mutations which affected WASP-WIP interaction abolished WASP's ability to suppress the growth defect of las17Delta strain. We have demonstrated that WASP-WIP is an active complex and WASP's ability to suppress the growth defect of las17Delta strain is dependent on the presence of a functional Arp2/3 activating domain of WASP and also the Verprolin domain (V) of WIP.     

Saccharomyces cerevisiae membrane sterol modifications in response to growth in the presence of ethanol.

Membranes isolated from yeasts grown in the presence of ethanol do not display the thermally induced transition in diphenylhexatriene anisotropy that is seen in control cells when they are exposed to ethanol in vitro. The total sterol content of the cells that were exposed to ethanol during growth is reduced, with no steryl esters being detected. A greater proportion of the total sterol pool is ergosterol in cells grown in the presence of alcohol. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase is reduced by ethanol in vitro. Ethanol-exposed cells take up more exogenous sterol under aerobic conditions than do control cells. The presence of ethanol during growth reduces the activity of the plasma membrane enzyme, chitin synthase, as well as increasing the thermosensitivity of this enzyme.     

Excretion of putrescine and spermidine by the protein encoded by YKL174c (TPO5) in Saccharomyces cerevisiae

The properties of the protein encoded by YKL174c (TPO5) were studied. It was found that TPO5 excretes putrescine effectively and spermidine less effectively. Gamma-aminobutyric acid slightly inhibited the excretion of putrescine, but basic amino acids did not affect excretion, suggesting that TPO5 preferentially recognizes polyamines. Accordingly, yeast cells transformed with the plasmid encoding YKL174c (TPO5) were resistant to toxicity caused by 120 mm putrescine or by 3 mm spermidine, and a mutant with a disrupted YKL174c (TPO5) gene was sensitive to toxicity by 90 mm putrescine. The growth of this mutant was faster than that of the wild-type strain. In parallel, there was an increase in putrescine and spermidine content of the YKL174c (TPO5) mutant compared with wild-type. It is noted that TPO5 functions as a suppressor of cell growth by excreting polyamines. The level of YKL174c (TPO5) mRNA was increased by the addition of polyamines to the medium. The degree of induction of the mRNA was spermine > spermidine > putrescine. The subcellular localization of TPO5 was determined by immunostaining of hemagglutinin-tagged TPO5, and it was found on Golgi or post-Golgi secretory vesicles. Excretion of putrescine and spermidine by TPO5 was reduced in cells that have mutations in the secretory or endocytic pathways, indicating that both processes are involved in the excretion of polyamines.