Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

J. Plant Physiol. 161. 3 – 14 (2004) http://www.elsevier-deutschland.de/jplhp

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity Christine Theiss1, Peter Bohley1, Hans Bisswanger1, Jürgen Voigt1, 2 * 1

Physiologisch-Chemisches Institut der Universität Tübingen, Hoppe-Seyler-Straße 4, D-72076 Tübingen, Germany

2

Institut für Pflanzenbiochemie der Universität Tübingen, Corrensstraße 41, D-72076 Tübingen, Germany

Received December 2, 2002 · Accepted June 1, 2003

Summary Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation. but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels. Key words: Chlamydomonas reinhardtii – ornithine decarboxylase – polyamine uptake – putrescine – spermidine – spermine Abbreviations: DFMO = difluoromethyl ornithine. – ODC = ornithine decarboxylase

Introduction In Chlamydomonas reinhardtii, as in other organisms, polyamines (putrescine, spermidine, and/or spermine) are indispensable for cell growth (Tabor and Tabor 1984, Marton and Pegg 1995, Theiss et al. 2002). Elevated polyamine levels * E-mail corresponding author: [email protected]

have been found in proliferating plant and mammalian cells as well as in cancer cells (Hibshoosh et al. 1991, Auvinen et al. 1992, Moshier et al. 1993, Daoudi and Biondi 1995, Marton and Pegg 1995, Ben Hayyim et al. 1996, Fowler et al. 1996, Cvikrova et al. 1999). In mammalian cells, spermidine is required as a cosubstrate for the activation of polypeptide chain initiation factor eIF-5A by post-translational hyposinylation of a lysine side chain (Jakus et al. 1993). Therefore, pub0176-1617/04/161/01-3 $ 30.00/0

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lished effects of inhibitors of polyamine synthesis on cell division could be referred to a decrease in the rate of protein biosynthesis and cell growth (Koza and Herbst 1992, Fredlund et al. 1995). However, in synchronized cultures of Scenedesmus obliquus and C. reinhardtii, an increase in the polyamine level was measured prior to the transition to the cell division phase (Kotzabasis and Senger 1994, Theiss et al. 2002). Furthermore, cell divisions were found to be preceded by an increase in the activity of ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme of polyamine synthesis (Cohen et al. 1982 a, b, 1984, Fredlund et al. 1995, Voigt and Bohley 2000, Theiss et al. 2002). As recently reported, this increase in the polyamine level is essential for the transition to the cell division phase (Theiss et al. 2002). The intracellular polyamine level can be modulated either by exogenous polyamines or by application of inhibitors of polyamine biosynthesis. The uptake of polyamines is dependent on the absence or presence of specific polyamine transporters (Seiler et al. 1996, Le Quesne et al. 1996, Dot et al. 2000, Cabella et al. 2001, Satriano et al. 2001). All the eukaryotic and prokaryotic organisms studied so far are able to synthesize polyamines with the exception of some pathogens, which obtain these compounds from their hosts (Tabor and Tabor 1984, Marton and Pegg 1995, Le Quesne and Fairlamb 1996). The three commonly occurring polyamines (putrescine, spermidine, and spermine) are synthesized from ornithine and/or arginine, putrescine being the first polyamine in these biosynthetic pathways (Slocum 1991). Spermidine and spermine are generated from putrescine by the addition of aminopropyl groups derived from decarboxylated S-adenosyl methionine (Slocum 1991). In the unicellular green alga C. reinhardtii, as in animals and most fungi, the rate-limiting step in putrescine synthesis is the decarboxylation of ornithine by ornithine decarboxylase (ODC; for references, see Marton and Pegg 1995). The ODC activity is regulated both at the translational level (Tabor and Tabor 1984, Marton and Pegg 1995) and via controlled, ATP-dependent proteolysis by the 26S proteasome, at least in mammalian and yeast (Saccharomyces cerevisiae) cells (Bercovich et al. 1989, Mamroud-Kidron and Kahana 1994). ODC has a rather short half-life, varying between 30 and 200 min (Voigt et al. 2000, Voigt and Bohley 2000) with the exception of Trypanosoma brucei (t1/2 = 6 h; Phillips et al. 1987). For mammalian cells, it has been reported that degradation of ODC is not mediated by ubiquitination (Bercovich et al. 1989) but by binding of ODC monomers to an inhibitor protein named antizyme (Murakami and Hayashi 1985, Hayashi and Murakami 1995, Hayashi et al. 1996). At least two different pathways of degradation of ODC in mammalian cells are known as yet, a constitutive and a polyaminedependent pathway (Li and Coffino 1993). The C-terminal domain is necessary in both cases and sufficient to make an ODC molecule constitutively unstable. Surface hydrophobicity and PEST sequences (sequences with high proportions of Pro, Glu/Asp, Ser, and Thr, and lacking basic amino acid residues) are actually considered as signal structures for the

rapid and selective degradation of specific proteins by the proteasome (Bohley 1996, Rechsteiner and Rogers 1996). Both uptake of exogenous polyamines and polyamine biosynthesis are regulated by the intracellular polyamine concentration (Hayashi and Murakami 1995, Seiler et al. 1996). In mammalian cells, one type of regulation of both ODC and the high-affinity polyamine transport activities is mediated by a small labile protein called antizyme (Hayashi and Murakami 1995). In the present study, we have investigated the uptake of exogenous polyamines by the unicellular green alga C. reinhardtii and their effects on synthesis and inactivation of ODC.

Materials and Methods Strains and growth conditions The cell-wall deficient strain Chlamydomonas reinhardtii cw-15 (Davies and Plaskitt 1971) was obtained from the Sammlung von Algenkulturen at the University of Göttingen. Cells were grown at 24 ˚C under a photon fluence rate of 40 µmol m –1 s –1 in a high-salt medium supplemented with 0.2 % (w/v) sodium acetate as previously described (Voigt and Münzner 1987). Cell concentrations were determined by duplicate hemocytometer counting.

Preparation of cell lysates Cells were harvested by centrifugation at 6,000 g for 10 min at 4 ˚C: All subsequent steps were performed at 0 – 4 ˚C. The cells were washed with and resuspended to a final cell density of 0.5–1.0 × 109 cells mL –1 in ice-cold homogenization buffer A consisting of 25 mmol/L Tris-HCl, pH 7.2, 2 mmol/L dithiothreitol, and 0.1mmol/L EDTA. After addition of the protease inhibitors phenylmethylsulfonyl fluoride (final concentration: 0.1mmol/L) and chymostatin (final concentration: 5 µg mL –1), cell lysis was performed by addition of Triton X-100 to a final concentration of 0.5–1.0 % (v/v). After 5 min, efficiency of lysis was checked microscopically and the particulate constituents removed by centrifugation for 15 min at 400,000 g in a Beckman (München, Germany) ultracentrifuge rotor TLA 100.2. The supernatants were stored at –75 ˚C.

Ornithine decarboxylase assay ODC activity was determined by measuring the release of 14CO2 from L-[1-14C]ornithine (Schulz et al. 1985). No release of 14CO2 from L-[114 C]ornithine was detected when Chlamydomonas lysates were preincubated in the presence of the ODC inhibitor DFMO at a concentration of 0.1 mmol/L for 10 min at 4 ˚C before incubation with L-[114 C]ornithine. One unit of ODC activity catalyzed the decarboxylation of 1 µmol of ornithine per min at 37˚C.

Determination of protein Quantitation of protein was performed by the method of Minamide and Bamburg (1990) using bovine serum albumine as standard.

Polyamine uptake and ODC activity

Determination of protein biosynthesis Biosynthesis of proteins was analyzed in vivo by pulse-labelling with [3H]arginine and measuring the radioactivity incorporated into protein. Aliquots (1mL) were taken from cultures and incubated for 30 min in the presence of 370 KBq of [3H]arginine (spec. radioactivity 1.7TBq/ mmol; Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany). After pulse-labelling, the cells were rapidly cooled to 0 ˚C and centrifuged through a 0.4 mL cushion of 40 % (w/v) Percoll in PBS. The cells were washed twice with 1mL PBS and finally dissolved in 100 µL urea-SDS buffer containing 8 mol/L urea, 2 % (w/v) SDS, 10 mmol/L EDTA, 200 mmol/L 2-mercaptoethanol, and 20 mmol/L Tris-HCl, pH 7.5. Aliquots (50 µL) were plated onto glas microfiber filters (GF/C, Whatman Ltd, Maidstone, England) and boiled for 15 min in 10 % (w/v) trichloroacetic acid to hydrolyse the aminoacyl-tRNAs. The filters were then washed twice for 1 min with 5 % (w/v) trichloroacetic acid, twice for 1 min with 95 % (v/v) ethanol and finally with diethylether. After addition of 4 mL UltimaGold (Packard Instruments, Groningen, The Netherlands), the dried filters were measured for radioactivity using a liquid scintillation counter.

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rified chloroplasts and nuclei were analyzed for radioactivity and protein.

Thin layer chromatography of [3H]putrescine and its metabolites [3H]Putrescine and its metabolites were analyzed by thin layer chromatography of the dansylated products (Seiler and Schmidt-Glenewinkel 1975). The dansylated polyamines were detected and identified by comparison with dansylated standards run in parallel, and marked under uv-light (365 nm). The spots were scraped out and the silica gel extracted by shaking with 1mL of methanol – 25 % ammonia (99 : 1) for 30 min at room temperature. After centrifugation for 10 min at 6,000 g, the supernatants were transferred to 5-mL scintillation vials and the solvent removed by a gentle stream of air. After addition of 4 mL UltimaGold (Packard Instruments, Groningen, The Netherlands), radioactivity was measured using a scintillation counter.

HPLC analysis of polyamines Uptake and subcellular distribution of [3H]putrescine To determine the kinetics of [3H]putrescine uptake and its dependence on polyamine concentration, 37 kBq of [3H]putrescine (spec. radioactivity 2960 GBq/mmol; Hartmann analytics, Braunschweig, Germany) were placed into each 1.5-mL reaction tube with or without addition of unlabelled polyamines. Aliquots (1mL) of a mixotrophically grown C. reinhardtii culture (adjusted to a cell density of 1 × 106 cells mL –1) were added, mixed, and incubated at room temperature. After the indicated incubation time, the uptake was stopped by placing the tubes in ice-water. Each cell suspension was then immediately layered on top of a 0.4 mL cushion containing 40 % (w/v) Percoll and centrifuged at 14,000 g for 10 min. The cell pellet was washed twice with 1 mL PBS: Finally, the cells were lysed in 100 µL urea-SDS buffer and measured for radioactivity after addition of 4 mL UltimaGold using a liquid scintillation counter. To investigate the subcellular distribution of [3H]putrescine and its metabolites, 3.7 MBq of [3H]putrescine and unlabelled putrescine (final concentration: 10 µmol/L) were added to 1-litre cultures of mixotrophically grown C. reinhardtii (cell density 1× 106 mL –1). After 2 h in the light, the cells were harvested by centrifugation at 6,000 g for 10 min, washed twice and resuspended in ice-cold homogenization buffer B containing 250 mmol/L sorbitol, 1 mmol/L MnCl2, 1 mmol/L MgCl2, 2 mmol/L EDTA, 20 mmol/L KCl, and 50 mmol/L HEPES-NaOH (pH 7.5) to a final cell density of 0.5–1.0 × 109 cells mL –1. The cells were disrupted using a Potter-Elvejhem homogeniser. The homogenates were layered on top of three-step Percoll gradients (4 mL of 40 % Percoll, 4 mL of 60 % Percoll, and 4 mL of 80 % Percoll in 15-mL Pyrex tubes) and centrifuged at 10,000 g for 30 min at 4 ˚C in a swinging bucket rotor (Sorvall HB4; Kendro Laboratory Products, Hanau, Germany). Microsomes banded at the 0 %/40 % Percoll interface. Intact chloroplasts banding at the 40 %/60 % Percoll interface were harvested, diluted with homogenization buffer B, collected by centrifugation at 8,000 g and washed twice with homogenization buffer B. This fraction also contained the mitochondria as shown by determination of cytochrome c oxidase activity. Crude nuclei banding at the 60 %/ 80 % Percoll interface were diluted, collected by centrifugation at 1,000 g for 10 min and twice washed with homogenization buffer B contining 0.2 % (v/v) Triton X-100. Crude subcellular fractions and pu-

Frozen cells were extracted with 5 % (w/v) trichloroacetic acid for 1 h in an ice bath and centrifuged for 30 min at 4 ˚C and 20,000 g in a Sorvall SS34 rotor. The supernatants were evaporated at 70 – 80 ˚C. Dried samples were redissolved in 200 µL of 5 % (w/v) perchloric acid and the polyamines benzoylated according to Flores and Galston (1982). Aliquots were analyzed by reversed-phase HPLC according to Kotzabasis et al. (1993) using the HPLC system Gold (Beckman Instruments, San Ramon, CA, USA) equipped with an Ultrasphere C18 column, 4.6 × 250 mm, 5 µm particle size (Phenomenex, Aschaffenburg, Germany). Elution of the benzoyl-polyamines was performed at 25 ˚C and a flow-rate of 1 mL/min with 58 % (v/v) aqueous methanol and monitored at 254 nm.

Results Effects of exogenous polyamines on the intracellular polyamine level In mammalian cells, extracellular polyamines affect the intracellular polyamine level both by their uptake and their effects on polyamine biosynthesis (Tabor and Tabor 1984, Hayashi and Murakami 1995, Marton and Pegg 1995, Seiler et al. 1996). Therefore, we have investigated the effects of exogenous polyamines on the intracellular polyamine level of C. reinhardtii (Fig. 1). Cytotoxic effects were observed when putrescine concentrations were higher than 1.5 mmol/L or when spermidine or spermine was applied at a concentration above 0.3 mmol/L (data not shown). Therefore, the uptake of putrescine was studied at an exogenous concentration of 1.0 mmol/L, the uptake of spermidine and spermine at concentrations of 0.1 mmol/L. Since C. reinhardtii does not produce spermine (Theiss et al. 2002), the results with exogenous spermine can be interpreted most easily because any spermine found in the cells must have been taken up. A slow, linear increase in the spermine level occurred during the first 7 h and no saturation was observed even after 10 h (Fig. 1 C).

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Figure 1. Effects of extracellular polyamines on the intracellular polyamine level of C. reinhardtii. Cultures of the walldeficient C. reinhardtii strain cw-15 were grown under continuous light to a cell density of 1 × 106 cell mL –1. At zero time, putrescine, spermidine or spermine were added to 5-litre cultures of C. reinhardtii. At the indicated time intervals, aliquots of 800 mL were harvested and the cells washed and frozen at –75 ˚C until use. Polyamines were extracted from frozen cells, benzoylated and analysed by HPLC as described in the ‹Materials and Methods› section. Values are means ± SD of 4 separate experiments. (A) Addition of putrescine (final concentration: 1 mmol/L), (B) addition of spermidine (final concentration: 100 µmol/L), (C) addition of spermine (final concentration: 100 µmol/L). (䉭) Putrescine, (䉲) spermidine, (䊊) spermine.

During this time period, the intracellular putrescine and spermidine levels declined continuously (Fig. 1 C). Extracellular addition of putrescine (Fig. 1A) caused a slow increase in the intracellular levels of both putrescine and spermidine indicating that part of the accumulated putrescine was transformed to spermidine (Fig. 1 A). Assuming that increased putrescine levels cause a diminished putrescine synthesis as observed in the experiments with exogenous spermine (Fig. 1 C), the amounts of putrescine taken up by the cells should be higher than the measured increase in the cellular putrescine level (Fig. 1 A). An increase in the intracellular levels of both spermidine and putrescine was also observed after addition of exogenous spermidine (Fig. 1B). In this case, the increase in the putrescine level might be due to a diminished transfomation to spermidine and/or an increased degradation of spermidine to putrescine (Slocum 1991). No spermine was detected in these experiments (Fig. 1A, B).

Uptake, subcellular distribution and metabolization of [3H]putrescine When C. reinhardtii cells were incubated in the presence of 10 µmol/L [3H]putrescine, a rather slow uptake of radioactivity was measured (Fig. 2 A) as in the experiments on the uptake of unlabelled putrescine determined by HPLC analysis of intracellular polyamines (Fig. 1 A). Furthermore, no decrease in the extracellular [3H]putrescine level was detected (Fig. 2 B). These data indicate that C. reinhardtii cells may lack high- affinity putrescine and polyamine transporters. The rate of [3H]putrescine uptake was considerably diminished by addition of spermidine or spermine (Fig. 2 A). The latter finding was corroborated when [3H]putrescine uptake was measured at increasing putrescine concentrations (Fig. 3). Again, uptake of putrescine was decreased in the presence of either spermidine or spermine (Fig. 3). A KM-value of about

Polyamine uptake and ODC activity

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Figure 2. Kinetics of [3H]putrescine uptake in the absence and presence of spermidine or spermine. Aliquots (1mL) were taken from C. reinhardtii cultures adjusted to a cell density of 1 × 106 cell mL –1, added to 1.5-mL vials containing 37 KBq of [3H]putrescine and 10 nmoles of unlabelled putrescine. The samples were incubated at room temperature in the absence and presence of spermidine (0.1mmol/L) or spermine (0.1mmol/L). After the time periods indicated in the figure, radioactivities of the cells were analyzed as described in the ‹Materials and Methods› section. (A) Radioactivity taken up by the cells. (B) Radioactivity remaining in the culture medium. Values are means ± SD of 6 separate experiments. (䉭) in the absence of spermidine and spermine, (䉲) in the presence of spermidine, (䊊) in the presence of spermine.

360 µmol/L was calculated for the uptake of [3H]putrescine in the absence of spermidine and spermine. Since no saturation of [3H]putrescine uptake was observed in the presence of spermidine or spermine (Fig. 3), it was not possible to find out whether or not the [3H]putrescine uptake was inhibited by spermidine and spermine in a competitive manor. To study the subcellular distribution of [3H]putrescine and its metabolites, C. reinhardtii cells incubated for 2 h in the presence of 10 µmol/L [3H]putrescine were disrupted and the homogenates fractionated by centrifugation using three-step Percoll gradients. The predominant proportions of the radioactivity were found in the cytosol (on top of the gradients) and in the microsomal fraction banding at the 0 %/40 % (v/v) Per-

coll interface. Increased radioactivities were also measured in the chloroplast fraction and in the crude nuclear fraction banding at the 40 %/60 % and at the 60 %/80 % (v/v) Percoll interfaces, respectively. The crude subcellular fractions as well as purified nuclei and chloroplasts (which also contained the mitochondria as revealed by determination of cytochrom c oxidase activities) were analyzed for their specific radioactivities and their relative contents of [3H]putrescine, [3H]spermidine, and [3H]spermine (Table 1). The specific radioactivities in the nuclei and in the chloroplasts were lower than in the cytosol and in the microsomal fraction. To analyse the proportions of [3H]putrescine and its metabolites, the HClO4-soluble fractions were dansylated and the products separated

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Figure 3. Effect of the exogenous putrescine concentration on [3H]putrescine uptake in the absence and presence of spermidine and spermine. Cultures of the wall-deficient C. reinhardtii strain cw-15 were grown under continuous light to a cell density of 1× 106 cell mL –1. Aliquots (1mL) were added to 1.5-mL reaction tubes containing 186 KBq of [3H]putrescine and increasing amounts of cold putrescine in the absence or presence of spermidine (final concentration: 0.1 mmol/L) or spermine (final concentration: 0.1 mmol/L). After 1 h at room temperature, uptake of radioactivity by the cells was analyzed as described in the ‹Materials and Methods› section. Values are means ± SD of 7 separate experiments. (䉭) Absence of spermidine and spermine, (䉲) in the presence of spermidine, (䊊) in the presence of spermine.

Table 1. Subcellular distribution of polyamines. Cultures of the wall-deficient strain C. reinhardtii cw-15 (1-litre) grown under continuous light were incubated for 2 h in the presence of 3.7 MBq [3H]putrescine and unlabelled putrescine (total concentration: 10 µmol/L). Cells were harvested, washed, homogenized and the homogenates subjected to subcellular fractionation as described under ‹Materials and methods›. The subcellular fractions were analyzed for protein and radioactivity. Furthermore, the proportions of [3H]putrescine and its metabolites were determined after separation by thin-layer chromatography. Mean values ± SD of 4 separate experiments are given. Subcellular fraction

Total radioactivity (dpm)

Specific radioactivity (dpm/mg protein)

[3H]Putrescine (%)

[3H]Spermidine (%)

[3H]Spermine (%)

Homogenate Cytosol Microsomes Chloroplasts (crude) Chloroplasts (purified) Nuclei (crude) Nuclei (purified)

3,250,000 ± 127,000 1,890,000 ± 210,000 1,010,000 ± 90,000 163,000 ± 17,000 91,000 ± 8,500 95,000 ± 15,000 27,000 ± 8,200

68,000 ± 6,500 47,500 ± 5,100 82,800 ± 7,500 79,000 ± 8,700 28,500 ± 3,100 62,500 ± 8,300 7,800 ± 1,600

92.5 ± 2.4 93.2 ± 2.1 93.5 ± 1.7 91.3 ± 2.8 91.6 ± 2.6 92.7 ± 2.8 90.4 ± 1.8

7.5 ± 2.4 6.8 ± 2.1 6.5 ± 1.3 8.1 ± 2.8 8.4 ± 2.4 7.7 ± 2.7 9.6 ± 2.3

0.0 0.0 0.0 0.0 0.0 0.0 0.0

by thin-layer chromatography. About 90 % of the radioactivity comigrated with the dansylated putrescine and 5–10 % with dansylated spermidine (Table 1). The same ratio of [3H]putrescine and [3H]spermine was found in all the subcellular fractions (Table 1). [3H]Spermine could not be detected. HPLCanalyses of benzoylated polyamines also revealed that in the absence of exogenous polyamines, C. reinhardtii cells contain putrescine and spermidine in a 15 : 1 ratio and do not contain spermine (Theiss et al. 2002).

Effects of cycloheximide and polyamines on the uptake of [3H]putrescine Specific polyamine transporters of mammalian cells were shown to be proteins with a short half-life whose synthesis is regulated by the intracellular polyamine level (Hayashi and Murakami 1995, Seiler et al. 1996). Therefore, the uptake of polyamines by mammalian cells was found to be down-regulated after inhibition of protein biosynthesis or after preincu-

Polyamine uptake and ODC activity

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Figure 4. Effects of cycloheximide and pretreatment with spermidine or spermine on the uptake of [3H]putrescine by C. reinhardtii cells. Cultures of the wall-deficient C. reinhardtii strain cw-15 grown under continuous light were divided and diluted with fresh culture medium to a density of 0.3 × 106 cells mL –1 at the day before the experiment. (A) Effects of cycloheximide on the uptake of [3H]putrescine. Cycloheximide was added to one of the 6-mL subcultures to a final concentration of 10 µg mL –1 (indicated by arrow). After 30 min, 222 KBq of [3H]putrescine and 60 nmoles of unlabelled putrescine were added to each culture. After the indicated time intervals, 1-mL aliquots were analyzed for radioactivity taken up by the cells as described in the ‹Materials and Methods› section. Values are means ± SD of 4 separate experiments. (䉭) Without cycloheximide, (䊏) with cycloheximide. (B) Effect of pretreatment with spermidine or spermine on the uptake of [3H]putrescine. Spermidine or spermine (final concentrations: 0.1 mmol/L) were added to 2 of the 6 subcultures (10 mL) 30 min before the beginning of the experiment (arrow). At zero time and after 3.5 h, respectively, 222 KBq of [3H]putrescine and 60 nmoles of unlabelled putrescine were added to 3 of the subcultures (one with spermidine, one with spermine, and one without spermidine and spermine). After the indicated time intervals, 1-mL aliquots were analyzed for radioactivity taken up by the cells as described in the ‹Materials and Methods› section. Values are means ± SD of 5 separate experiments. (䉭, 䉱) Absence of spermidine and spermine, (䉮, 䉲) pretreatment with spermidine, (䊊, 䊉) pretreatment with spermine. Open symbols: [3H]putrescine added 30 min after application of spermidine or spermine; closed symbols: [3H]putrescine added 210 min after application of spermidine or spermine.

bation in the presence of unlabelled polyamines (for references see: Hayashi and Murakami 1995, Seiler et al. 1996). In the case of C. reinhardtii cells, however, the uptake of [3H]putrescine was neither influenced by addition of cycloheximide, an inhibitor of protein biosynthesis (Fig. 4 A), nor by preincubation with spermidine or spermine (Fig. 4 B).

Down-regulation by exogenous polyamines of ODC activity in Chlamydomonas cells To study the effects of polyamines on ODC activity, the timecourse of the specific ODC activities was determined after addition of exogenous putrescine, spermidine, and spermine,

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Figure 5. Time-course of ODC activity in C. reinhardtii cells after addition of exogenous polyamines. Cultures of the wall-deficient C. reinhardtii strain cw-15 were grown under continuous light to a cell density of 1× 106 cells mL –1. At zero time, putrescine (final concentration 1mmol/L), spermidine (final concentration 0.1 mmol/L), and spermine (final concentration 0.1 mmol/L), respectively, were added. Aliquots (400 mL) of the cultures were harvested after the indicated time intervals and cell lysates prepared and analyzed for ODC activity and protein as described under ‹Materials and Methods›. ODC activities are expressed in percent of maximal specific activity (between 350 and 580 µunits [mg protein] –1). Values are means ± SD of 4 separate experiments. (䉭) Putrescine, (䉲) spermidine, (䊊) spermine, (䉫) control.

Figure 6. Effects of polyamines on ODC inactivation. At the day before the experiment, cultures of the wall-deficient C. reinhardtii strain cw-15 grown under continuous light were divided into four 5-litre Erlenmeyer flasks, diluted with fresh culture medium to a final cell density of 0.5 × 106 cells mL –1 and further incubated in the light. On the day of the experiment, putrescine (final concentration 1 mmol/L), spermidine, and spermine (final concentrations 0.1 mmol/L), respectively, were added to 3 of the 4 subcultures. After 3 h, cycloheximide (final concentration 10 µg mL –1) was added. Aliquots (400 mL) were taken at the time periods after addition of cycloheximide indicated in the figure. Cell lysates were prepared and analyzed for ODC activity and protein as described in the ‹Materials and Methods› section. ODC activities are expressed in percent of maximal specific activity (between 380 and 620 µunits [mg protein] –1). Values are means ± SD of 4 separate experiments. The half-logarithmic plots of the data used for determination of t1/2 are shown in the inserted figure. (䉭) Putrescine, (䉲) spermidine, (䉱) spermine, (䉫) control.

respectively, to asynchronously growing cultures of C. reinhardtii. All these commonly occurring polyamines caused a decrease in ODC activity after a lag-phase of 2 h (Fig. 5). After 5 h, this effect was more pronounced for spermidine and spermine than in the case of putrescine. After longer time periods, a further decrease in ODC activity was observed in the cultures treated with putrescine whereas in the case of sper-

midine and spermine, recovery of ODC activity was observed (Fig. 5). The latter effect might be due to the rather different polyamine levels necessary to cause a decrease in ODC activity. Concentrations of 50–100 µmol/L were sufficient to cause a down-regulation of ODC activity by spermidine and spermine, respectively, whereas concentrations of > 0.5 mmol/ L were required in the case of putrescine.

Polyamine uptake and ODC activity

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Figure 7. Effects of polyamines on the light-induced increase in ODC activity in dark-adapted C. reinhardtii cells. Cultures of the wall-deficient C. reinhardtii strain cw-15 grown under continuous light were divided at the day before the experiment and the subcultures diluted with fresh culture medium to a final volume of 4 L (in 5-L Erlenmeyer flasks) and a cell density of 0.5 × 106 cells mL –1 and wrapped with two layers of aluminium foil. After 16 h in the dark (= zero time), the first aliquots (400 mL) were harvested prior to the addition of putrescine (final concentration 1 mmol/L), spermidine (final concentration 0.1 mmol/L), and spermine (final concentration 0.1 mmol/L), respectively. The aluminium foils were removed 3 h after addition of polyamines (marked by arrow) and the cultures transferred to the light. Aliquots (400 mL) were harvested at the time intervals indicated in the figure. The 400,000 g supernatants of the cell lysates were analyzed for ODC activity and protein as described in the ‹Materials and Methods› section. ODC activities are expressed in percent of maximal specific activity (between 310 and 660 µunits [mg protein] –1). Values are means ± SD of 5 separate experiments. (䉭) Putrescine, (䉲) spermidine, (䊊) spermine, (䉫) control.

Polyamines affect biosynthesis of ODC but not ODC half-life in Chlamydomonas cells A decrease in ODC activity by exogenous polyamines (Fig. 5) has also been reported for mammalian cells, which was caused in part by an increased rate of degradation of ODC protein via polyamine-induced antizyme (Murakami and Hayashi 1985, Hayashi and Murakami 1995, Hayashi et al. 1996). Therefore, we have comparatively studied the decrease of ODC activity after inhibition of protein biosynthesis by addition of cycloheximide in polyamine-treated and untreated cultures of C. reinhardtii (Fig. 6) and calculated the ODC half-lifes. The inactivation rate of ODC in Chlamydomonas cells was not affected by polyamine treatment (Fig. 6). ODC half-lifes between 1.2 and 1.5 h were calculated both in the absence and presence of polyamines. Therefore, in the case of C. reinhardtii, the polyamine-induced down-regulation of ODC activity is not due to an increased rate of degradation or inactivation. As previously reported, a rapid 10- to 20-fold increase in ODC activity was measured upon illumination of darkadapted (starved) C. reinhardtii cultures (Voigt et al. 2000). This light-induced increase in ODC activity could be prevented by inhibition of either photosynthesis or protein biosynthesis, but not by inhibition of RNA synthesis and is due to an ATP-dependent increase in translation of pre-existing ODC mRNA (Voigt et al. 2000). Therefore, we have investigated the effects of exogenous polyamines on up-regulation of ODC ac-

tivity by comparative studies of the light-induced increase in ODC activity in untreated and polyamine-pretreated, darkadapted C. reinhardtii cells. As shown in Figure 7, the lightinduced increase in ODC activity was considerably reduced by preincubation of the dark-adapted C. reinhardtii cultures in the presence of putrescine, spermidine or spermine. Both in the absence and presence of polyamines, this light-induced increase in ODC activity was slightly diminished by actinomycin D and completely abolished by cycloheximide (Table 2). These data indicate that polyamines essentially do not affect the level of ODC mRNA, but cause a decreased up-regulation by affecting the translation of ODC mRNA. As shown by pulse-labelling experiments with [3H]arginine, protein biosynthesis in dark-adapted C. reinhardtii cells rapidly increased after transfer to the light (Fig. 8). Prior to illumination, polyamines caused an increase in the incorporation of [3H]arginine into protein (Fig. 8). No effects of polyamines on the incorporation of [3H]arginine, however, was observed during the light period. Therefore, the decrease in ODC activity by exogenous polyamines is not caused by a diminished rate of overall protein biosynthesis but must be due to a specific effect on the translation of ODC mRNA.

Discussion In C. reinhardtii, ODC activity is down-regulated by exogenous polyamines (Fig. 5) as in other organisms (Murakami

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Christine Theiss et al.

Table 2. Effects of actinomycin D and cycloheximide on the light-induced increase in ODC activity in the absence and presence of exogenous polyamines. Cultures of the wall-deficient C. reinhardtii strain cw-15 grown under continuous light were divided at the day before the experiment and the subcultures diluted with fresh culture medium to a density of 0.5 × 106 cells mL –1 and wrapped with two layers of aluminium foil. After incubation for 16 h in the dark, polyamines were added (final concentrations: 1 mmol/L putrescine, 0.1 mmol/L spermidine, and 0.1 mmol/L spermine, respectively). After 2 h, actinomycin D (final concentration: 5 µg mL –1) and cycloheximide (final concentration: 10 µg mL –1), respectively, were added as indicated. After further incubation for 1 h in the dark, the cultures were transferred to the light. Aliquots (500 mL) were harvested prior to illumination (zero time) and 30 min and 60 min after onset of illumination, respectively. The cell lysates and 400,000 g supernatants were prepared and analyzed for ODC activity and protein as described under ‹Materials and methods›. ODC activities are expressed in % of maximal specific activity measured in the individual experiments in the absence of polyamines and inhibitors (between 195 and 450 µ units ODC [mg protein] –1). Mean values ± SD of 5 separate experiments are shown. Polyamine

– – – Putrescine Putrescine Putrescine Spermidine Spermidine Spermidine Spermine Spermine Spermine

Actinomycin D

– + – – + – – + – – + –

Cycloheximide

– – + – – + – – + – – +

and Hayashi 1985, Fonzi 1989, Hayashi and Murakami 1995, Hayashi et al. 1996, Hoyt et al. 2000). The polyamine-induced decrease in ODC activity occurs after a lag-phase of about 2 h (Fig. 5). This delay in the response of ODC activity to the addition of polyamines is obviously due to the slow uptake of polyamines by C. reinhardtii cells (Figs. 1 and 2). The latter finding clearly shows that C. reinhardtii cells do not contain high-affinity polyamine transporters in contrast to other organisms (Hayashi and Murakami 1995, Seiler et al. 1996, Huber et al. 1996, Le Quesne and Fairlamb 1996, Sakata et al. 2000). This conclusion is corroborated by the observation that rather high exogenous putrescine concentrations are required for maximal uptake of [3H]putrescine (Fig. 3). [3H]Putrescine uptake was found to be decreased in the presence of spermidine or spermine (Fig. 3). In contrast to the polyamine uptake via high-affinity polyamine transporters found in mammalian cells (Hayashi and Murakami 1995, Seiler et al. 1996, Huber et al. 1996, Sakata et al. 2000), the rate of [3H]putrescine uptake by C. reinhardtii was not influenced by inhibition of protein biosynthesis throughout a period of 8 h (Fig. 4 A) and was not affected by pre-treatment of the cells with spermidine or spermine (Fig. 4 B). The predominant proportion of [3H]putrescine taken up by Chlamydomonas was found in the cytosol and in the microsomal fraction, 5–10 % being metabolized to [3H]spermidine after 2 h (Table 1). No [3H]spermine could be detected (Table 1). These findings are in agreement with HPLC anal-

ODC activity [%] 0 min

30 min

60 min

8.3 ± 1.2 7.1 ± 1.4 6.9 ± 1.7 6.1 ± 0.9 5.2 ± 1.1 5.0 ± 0.9 3.4 ± 0.5 2.9 ± 0.7 2.8 ± 0.7 3.2 ± 0.3 2.7 ± 0.6 2.5 ± 0.5

64.1 ± 12.5 52.8 ± 9.3 6.7 ± 1.5 42.8 ± 8.1 35.1 ± 6.4 4.8 ± 1.1 31.7 ± 8.2 27.2 ± 7.4 2.7 ± 0.8 24.8 ± 6.5 20.7 ± 3.9 2.4 ± 0.6

100.0 82.0 ± 10.4 6.4 ± 1.3 56.2 ± 8.9 48.8 ± 7.4 4.4 ± 1.2 40.9 ± 7.5 33.7 ± 6.9 2.5 ± 0.9 31.4 ± 5.9 26.3 ± 4.2 2.2 ± 0.5

yses of polyamines present in Chlamydomonas cells (Theiss et al. 2002). A putrescine/spermidine ratio of 15 : 1 was observed and no spermine was detected in these studies. Furthermore, no spermine was found when the intracellular polyamines were analyzed by HPLC at different time intervalls after application of exogenous putrescine or spermidine (Fig. 1 A, B). Spermine is also lacking in Scenedesmus obliquus cells (Kotzabasis and Senger 1994). Furthermore, the genome of Saccharomyces cerevisiae does not contain a spermine synthase gene. Exogenous spermine (0.1 mmol/L) not only resulted in an uptake of this polyamine, but also in a continuous decrease in the intracellular putrescine and spermidine levels (Fig. 1 C). ODC activity was down-regulated by application of exogenous polyamines after a lag-phase of 2 h (Fig. 5) when an increase of the intracellular polyamine level was measured (Fig. 1). In other organisms, ODC activity is regulated by polyamines both at the translational level (Tabor and Tabor 1984, Marton and Pegg 1995) and via controlled, ATP-dependent proteolysis by the 26S proteasome (Bercovich et al. 1989, Li and Coffino 1993). In mammalian cells, degradation of ODC is regulated by polyamines via the polyamine-dependent level of a small labile protein called antizyme (Murakami and Hayashi 1985, Hayashi and Murakami 1995). In Chlamydomonas cells, however, the ODC inactivation was not affected by polyamines (Fig. 6). Translation of ODC mRNA, but not overall protein biosynthesis is diminished by increased polyamine levels (Figs. 7 and 8, Table 2). A spe-

Polyamine uptake and ODC activity

Figure 8. Effects of exogenous polyamines on protein biosynthesis at the beginning of the light period. Cultures of the wall-deficient C. reinhardtii strain cw-15 grown under continuous light were divided at the day before the experiment and the subcultures diluted with fresh culture medium to a cell density of 0.5 × 106 cells mL –1 and wrapped with two layers of aluminium foil. Where indicated, polyamines were added after 16 h in the dark (final concentrations: 1 mmol/L putrescine, 0.1 mmol/L spermidine, and 0.1 mmol/L spermine, respectively). After incubation in the dark for another 3 h in the presence or absence of polyamines, the cultures were transferred to the light. Aliquots of 1mL were taken at the indicated time intervals before and after onset of illumination and pulse-labelled with 37 KBq of [3H]arginine for 30 min. After pulse-labelling, the cells were analyzed for radioactively labelled protein as described in the ‹Materials and Methods› section. Data are expressed in dpm [3H]arginine incorporated into protein per 106 cells. Mean values ± SD of 4 separate experiments are given. (䉭) Putrescine, (䉲) spermidine, (䊊) spermine, (䉬) control.

cific down-regulation of the translation of ODC mRNA was also observed in other organisms (Fonzi 1989, Stjernborg et al. 1991, Marton and Pegg 1995, Shantz and Pegg 1999, Hoyt et al. 2000). Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft (grant no. Vo 327/9).

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