SYMBIOSIS (2009) 47,99-107

©2009 Balaban, PhiladelphiaiRehovot

ISSN 0334-5114

Amino acid transport systems of Japanese Paramecium symbiont F36-ZK Yutaka Kato and Nobutaka Imamura* Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu City, Shiga 525-8577, Japan, Tel. +81-77-561-2825, Fax. +81-77-561-2659, [email protected]@se.ritsumei.ac.jp

(Received June 5, 2007; Accepted July 28, 2008)

Abstract Analyses of amino acid transport systems in Japanese Paramecium symbiont F36-ZK were performed using 14C- amino acids. Kinetic analyses of amino acid uptake and competitive experiments revealed three transport systems; a basic amino acid transport system, which catalyzed transport of L-Arg and L-Lys, a general amino acid transport system, which had broad specificity for 19 amino acids (but not L-Arg), and an alanine transport system. These three systems were considered to be capable of active transport. Amino acid-proton symport was indicated by the following data: decreases in pH of the medium observed during L-Ser and L-Ala uptake, and uptake of L-Arg, L-Ser and L-Ala being inhibited by carbonyl cyanide m-chlorophenylhydrazone, sodium azide and vanadate. The optimal pH for uptake of neutral amino acids and L-Arg was around 5 and 5 to 6.5, respectively. Uptake of L-Asp and L-Glu was very sensitive to pH and little uptake of L-Asp was measured above pH 6.0. Amino acid uptake was not inhibited by nitrate or ammonium, and cultured cells with ammonium also possessed constitutive uptake systems. Keywords:

Paramecium bursaria, Chlorella, amino acid transport

1. Introduction The green color ciliate Paramecium bursaria is a common inhabitant of fresh water all over the world. The green is derived from its endosymbiotic algae, which were classified into the genus Chlorella by morphological (Nakahara et al., 2003) and phylogenetic studies (Hoshina et al., 2004, 2005; Nakahara et al., 2004). The ciliate can grow as an autotroph in inorganic medium due to its endosymbiotic algae. The host and symbionts must exchange substances and information for homeostasis as a "green paramecium" (Reisser, 1986). Studies on the transfer of nutrients between host and symbionts were performed using European and American paramecia in the past. The transfer of maltose from the symbiont to the host is the well known result in these studies (Brown and Nielsen, 1974; Kessler et aI., 1991; Reisser and Widowsk i, 1992; Schilling et al., 1991; Ziesenisz et al., 1981). Few studies exist describing transport of nitrogenous compounds between P. bursaria and Chlorella spp.

"tt« author to whom correspondence should be sent.

While nitrogen in the form of nitrate is a common nutrient for photosynthetic organisms, such as Chiarella spp., no reports exist that show provisioning of nitrate to the algae by their hosts. Albers et al. presumed that ammonium was the key form of supplied nitrogen because the aposymbiotic European paramecia released ammonium in contrast with symbiotic ones, which imported it (Albers et al., 1985). They also suggested the possibility of glutam ine as a minor supplied nitrogen compound. The symbiotic alga F36-ZK was recently isolated from Japanese P. bursaria F36 (Kamako et al., 2005). The symbiont released maltose the similar to other Paramecium symbionts; however, it could not utilize nitrate due to lack of nitrate reductase. These findings suggest that F36-ZK was more adapted to its host than hosts associated with European and American symbionts. Japanese symbiont F36-ZK multiplied in the presence of amino acids rather than ammonium (Kamako et al., 2005; Kato et a!., 2006). In addition, preliminary study indicated that F36-ZK could transport more kinds of amino acids than the American Paramecium symbiont, which could import a few amino acids (McAuley, 1986; 1989), and the free-living C. vulgaris, which was phylogenetically close to F36-ZK

100

Y. KATO AND N. IMAMURA

(Hoshina et al., 2004, 2005) but could transport only one amino acid, arginine (Kato et al., 2006). These differences between symbiotic Chlorella F36-ZK and free-living C. vulgaris on the utilization and the transport of amino acids suggest the existence of the developed amino acid transport system in symbiotic Chlorella F36-ZK and the importance of amino acids in this symbiotic relationship. In this paper, we describe the analysis and characterization of the amino acid transport systems of F36-ZK, and discuss amino acids supplied from host to symbionts in the Paramecium endosymbiosis.

2. Materials and Methods Material and chemicals The Paramecium endosymbiont F36-ZK isolated from Japanese P bursaria F36 was cultured in C medium (Ichimura, 1971) plus L-Ser (200 ug mr l ) and provided a 16:8 light and dark cycle with a light intensity of 30 umol photons m- 2 S-I at 25°C (Kamako et al., 2005; Kato et al., 2006). When the alga was cultured with ammonium as the sole nitrogen source, nitrate was exchanged with the corresponding sulfate and NH 4CI (0.12 mg mr 1) as described previously (Kato et al., 2006). L-[U- 14C]Arginine, 14C]lysine, 14C]aspartic L-[UL-[Uacid, L-[U- 14C]glutamic 14C]glutamine, acid, L-[UL-[U- 14C]serine, L-[U_ 14C] 14C]leucine, alanine, L-[UL-[U- 14C]proline and [U_l4C] glycine were obtained from Moravek Biochemicals (USA). L-[U- J4C]Asparagine, L-[U- 14C]threonine, L-[U- 14C]valine, L-[U_ 14C]isoleucine, L-[methyl-14C]methionine, L-[side chain 3 YC]tryptophan and L-[1- 14C]cysteine were obtained from American Radiolabeled Chemicals Inc (USA). L-[carboxyl-v'Cjhistidine, L-[U-14C]tyrosine and L_[U_ l4C] phenylalanine were obtained from Amersham International pic (United Kingdom). Tris(hydroxymethyl)aminomethane, L-arginine, L-asparagine, L-glutamine, L-cysteine, nigericin sodium salt, N, N'-dicyclohexyl carbodiimide (DCCD), and ouabain were purchased from Wako Pure Chemical Industries Ltd (Japan). Sodium orthovanadate was purchased from Mitsuwa's Pure Chemicals (Japan). Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was obtained from ICN Biomedicals Inc (USA). 2-Morpholinoethanesulfonic acid monohydrate (MES) was purchased from Dojindo (Japan). Valinomycin was purchased from Sigma (USA). Ammonium chloride, sodium azide and scintillation cocktail (clear-sol I) were purchased from Nacalai tesque (Japan).

Uptake experiments Amino acid uptake was measured using 14C- labeled amino acids as described previously (Kato et al., 2006). Algal cells in log phase were washed with 25 mM sodium

phosphate buffer (pH 6.0) three times by centrifugation (1, 200 g, 5 min, room temperature) and resuspended in 8 l buffer at a cell density of approximately 2.0x I 0 cells mr • Each 360 ul of cell suspension was poured into a well of a 48-well plate (Sumilon) and incubated with 90 umol photons m- 2 S-I at 25°C. Forty microliters of labeled amino acid solution (0.038 to 120 mCi mrnof ') were added to start the experiment. A hundred microliters of the aliquot were filtered with a membrane filter (Advantec, pore size 0.2 urn, 25 mm diameter, cellulose acetate) at intervals of 0.5, 1, or 1.5 min and washed with 10 ml of ice-cold sodium phosphate buffer. The membrane filter was transferred into a vial. After the addition of 5 ml of cocktail (Clear-sol I), algal radioactivity was measured using a liquid scintillation counter (Beckman LS6000TA). In the case of L-Asp, the pH of the experimental buffer was adjusted to 5.0 because little uptake of L-Asp was observed at pH 6.0 (Kato et al., 2006).

Determination ofkinetic parameters Amino acid uptake was measured at a range of final amino acid concentrations between O. I u.M and 30 mM (0.038 to 120 mCi mmol"). Data obtained were plotted on a Lineweaver-Burk plot from which Km and Vmax were calculated. The experiments were independently performed twice.

Competitive experiments Competitive experiments were carried out according to Cho and Komor (1985). Forty microliters of unlabeled amino acid solution were added to 320 u] of algal suspension. After 30 seconds, labeled amino acid solution (0.23 to 34.7 mCi mmol'") were added. One hundred microliters of the suspension were taken at intervals of I or 1.5 min to measure algal radioactivity. The ratio of tracer to competitor ranged from 4 to 48. The experiment was performed once.

Measurement of intracellular and extracellular free amino acid Algal cells in 25 mM sodium phosphate buffer (pH 5.0) at a cell density of approximately 1.5x I 0 8 cells m ,-I were incubated with labeled amino acid for I or 30 min. After incubation, 100 f.ll of the suspension were transferred to a tube equipped with a cup holding a membrane filter (Ultra-free MC, pore size 0.45 urn, Millipore), and centrifuged for 3 min at 18,000 g at 4°C to separate algae from filtrate. Extracellular free amino acid concentration was calculated from radioactivity of 50 u.l offiltrate. The method for determination of intracellular free amino acid radioactivity depended on the incubation time (Sauer et aI., 1983). Algal cells on the membrane were washed with

AMINO ACID TRANSPORT SYSTEMS OF JAPANESE PARAMECIUM SYMBIONT 200 I-ll of 25 mM sodium phosphate buffer. The membrane filter cup was subsequently transferred into a vial containing 10 ml cocktail to measure total intracellular radioactivity. The radioactivity measured was assumed to be equal to intracellular free amino acid radioactivity for a 1 min incubation according to Sauer et al., 1983. Labeled algal cells were prepared in the same manner and incubated for 30 min, after which time the reaction was stopped using 100 I-ll of 10% (w/v) trichloroacetate (TCA) for 1 h at 4°C. The suspension was filtered by centrifugation as above, and the membrane filter cup was transferred into a vial with 10 ml cocktail. Radioactive proteins containing the labeled amino acids were measured. Radioactivity of intracellular free amino acid was defined as the value of the protein radioactivity subtracted from the total intracellular radioactivity. The experiments were performed three times. Cell volume was calculated using the formula V = 4pr 3/3, with the assumption that a cell was a complete sphere (Reisser, 1986) and the mean radius obtained by the measurement of 100 cells with a microscope could be used. Intracellular free amino acid concentration was calculated from radioactivity of free amino acid and cell volume.

101

Table I. Apparent kinetic parameters of amino acid uptake in F36-ZK. System 2

System 1

Km

(JlM)

Arg Lys His Asp Glu Asn Gin Ser Thr Tyr Ala Val Leu IIe Pro Phe Met Trp Gly Cys

12.3 36.5 1232 2589 1315 1002 1992 113 73.8 11.2 50.3 66.2 37.8 36.4 96.9 27.3 22.8 27.5 145 91.6

Vmax

(nmol/min/ 5x107cells) 4.2 0.51 14.4 13.3 7.5 9.0 125 8.2 7.8 2.0 9.3 7.4 5.6 6.0 10.1 4.6 3.9 2.6 11.8 14.5

Km

(JlM)

Vmax

(nmol/minl 5 x l 0 7cells)

602

2103

2.46

15.7

Measurement ofproton flow during uptake

Each value is the mean of two independent experiments.

Cells in log phase (approximately 2.5xl0 7 cells mr l ) were washed three times with 5 mM NaCI solution and resuspended at a cell density of approximately LOx 109 cells l mr . Eighteen milliliters of algal suspension were poured into a vial with a magnetic stirrer and pH electrode (Horiba H-7 SO), and then incubated at room temperature (23°C). Two milliliters of 10 mM amino acid solution adjusted to the pH of the cell suspension were added with stirring. The external pH was monitored by a recorder (056-1001 Hitachi, Ltd). The experiments were performed twice.

experiments were performed three times.

Determination ofpH dependence on uptake The pH dependence was determined according to Sauer (1984). Amino acid uptake at pH 4.0 to 5.0 was measured in 25 mM sodium citrate buffer, and uptake at pH 5.0 to 8.0 was measured in 25 mM sodium phosphate buffer. Uptake in citrate buffer was corrected to that in phosphate buffer using a correction factor calculated from rates at pH 5 in each buffer. The experiments were performed three times.

Effect ofuncoupler and inhibitor on amino acid uptake Effect ofinorganic nitrogen on amino acid uptake Initial amino acid uptake rates were measured using cells preincubated with an uncoupler or an inhibitor for 10 min before the addition of labeled amino acid solution. The following uncouplers were used in the experiments: valinomycin as a K+ ionophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a protonophore, and nigericin as a K+/H+ exchanger. The following inhibitors were used: vanadate, which is a structural analogue of phosphate, as an inhibitor of phosphorylation, N, N'-dicyclohexyl carbodiimide (DCCD) and sodium azide as an inhibitor of ATP synthesis, and ouabain as an inhibitor of Na+/K+-ATPase. In the case of vanadate, 25 mM 2-morpholinoethanesulfonic acid (MES) -NaOH buffer (pH 6.0) was used as experimental buffer instead of sodium phosphate. Valinomycin, CCCP, nigericin, and DCCD were dissolved in 100% ethanol. The final concentration of ethanol in the experiments was less than 0.2%. The

Uptake of L-Arg and L-Ser was measured in the presence of 2.3 mM inorganic nitrogen as NH 4CI, NH 4N0 3 or NaN0 3 . Amino acid solution (final concentration of 0.5 mM) and inorganic nitrogen solution were added to algal cells suspended in 25 mM sodium phosphate buffer (pH 6.0) at a cell density of approximately 1.5 x10 8 cells mr l to start the experiments. One hundred microliters of the sample were taken at 10 or 15 min intervals and algal radioactivity was measured. The amino acid uptake of F36-ZK grown with ammonium as sole nitrogen source was measured as described above. The experiments were performed three times.

Statistical analysis Statistic analyses were performed using a statistical soft-

Y. KATO AND N. IMAMURA

102

Table 2. Competitive study of amino acid uptake in the presence of unlabeled amino acid. Competitive AA Target A.A. Argl Arg2 Arg 4 15 Lys 92 105 His 80 97 Asp 106 112 132 122 Glu Asn 102 108 Gin 120 112 91 90 Ser Thr 97 102 Tyr* 90 102 91 94 Ala Val 97 105 Leu 97 97 lie 93 93 90 93 Pro Phe 84 85 III Met 95 Trp 71 89 Gly 99 89 Cys 108 94

Uptake of tracers (% control) Lysl Lys2 40 24 54 22 85 65 107 99 108 103 102 101 108 88 49 71 65 87 48 69 40 66 69 90 32 55 60 90 67 86 35 64 42 70 24 59 65 84 49 85

Lys3 His 32 76 35 81 84 55 96 99 102 77 92 76 88 51 81 30 89 19 85 12 84 14 19 90 69 8 104 12 89 23 72 7 87 7 70 8 84 33 84 12

Asp' 95 96 86 89 20 51 65 20 14 20 9 15 14 16 10 14 10 10 14 10

Glu 88 74 60 117 69 74 60 59 61 II

18 17 10 10 26 7 5 10 37 15

Asn 74 87 55 94 78 53 66 20 19 26 57 61 28 31 23 19 21 15 22 27

Glnl 86 100 80 101 84 83 60 41 27 15 23 26 17 20 31 15 9 12 41 20

Gln2 Serl 83 84 99 107 64 99 121 119 71 123 77 105 94 56 24 70 15 58 13 20 48 66 52 15 9 33 8 39 28 55 9 30 24 5 6 22 26 61 II 45

Concentration oftracers(mM) 0.026 0.014 0.026 0.026 0.12 0.036 0.012 0.012 0.009 0.006 0.006 Concentration of competitors (mM) 0.1 0.5 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.25 0.5 'In the case of L-Asp, the pH of experimental buffer was 5.0.

Ser2 96 III

71 121 101 90 66 31 17 15 13 21 12 12 20 10 6 6 28 15

0.025 0.027 0.1

0.5

Alai 84 104 93 106 107 109 84 64 45 14 35 54 34 37 59 30 17 19 61 40

Ala2 84 98 63 112 91 75 70 24 15 13 13

15 9 13 17 9 6 7 23 12

Ala3 81 87 29 35 12 9 9 4 14 15 8 8 13 7

Pro 79 83 65 112 80 75 58 31 21

Val 75 99 75 106 90 100 68 33 30

17

II

12 25 17 16 24 16 13 15 34 16

13 16 8 12 24 9 4 6 36 26

0.027 0.027 4.670 0.026 0.025 0.1

0.5

20

Table 3. Amino acid transport systems in F36-ZK. Systems

Amino acids transported (K", value)

Basic amino acid transport system L-Arg (12.3 !-lM), L-Lys (602 !-lM) General amino acid transport system L-Lys (36.5 !-lM), L-His (1232 !-lM), L-Asp (2589 !-lM). L-Glu (1315 !-lM), L-Asn (1002 !-lM), L-Gln (1992 !-lM), L-Ser (113.4 !-lM). L-Thr (73.8 !-lM), L-Tyr (11.7 !-lM), L-Ala (2103 !-lM), L-Yal (66.2 !-lM), L-Leu (37.8 !-lM), L-Ile (36.4 !-lM). L-Pro (96.9 !-lM), L-Phe. (27.3 !-lM), L-Met (22.8 !-lM), L-Trp (27.5 !-lM), Gly (145 !-lM), L-Cys (91.6 !-lM) Alanine transport system

L-Ala (50.3 !-lM)

ware SPSS 12.01 for windows (SPSS, Chicago, IL, USA). The detail of statistical methods was indicated in figure and table legends.

3. Results

Determination a/kinetic parameters a/uptake Kinetic analyses of uptake of all amino acids were performed, and the results are given in Table I. The K",

values for L-Arg and L-Tyr uptake were lower than the others, indicating higher affinity to their transporters. In the case of L-Gln, L-His, L-Asn and acidic amino acids, higher K", values were measured, indicating low affinity to their transporters. For all amino acids except L-Gln, the range of 15xl07cells- l . Although the V",ax was 0.51 to 15.7 nmolminapparent affinity of L-Gln for its transporter was very low, L-Gln showed the highest V",w; 125 nmol min-I 5xI0 7cells- l . Kinetic data for L-Lys and L-Ala uptake on a Lineweaver-Burk plot were biphasic in nature (Fig. 1). Uptake of these amino acids had two K", values, indicating that they were transported by at least two systems. High and low affinity phases of L-Lys uptake were in the range of 0.0167 to 0.1 mM and 0.1 to 0.5 mM, respectively. In the case of L-Ala, high and low affinity phases were assigned to a range of 0.03 to 2 mM and 2 to 33 mM, respectively. The other 18 amino acids had single K", values, indicating transport by one system.

Comparative experiments To sort amino acids into their respective transport systems, competitive experiments were carried out. Uptake rates of several amino acids in the presence of unlabeled amino acids are indicated in Table 2. It is expected that L-Arg acts as a good competitor due to its high affinity (Table I). When unlabeled L-Arg was added, uptake of labeled L-Arg and L-Lys was inhibited strongly, indicating

AMINO ACID TRANSPORT SYSTEMS OF JAPANESE PARAMECIUM SYMBIONT

A

20

40

liS (mMr

60 l

B 0.3

-?

0.2 0.1 0 10

0

20

30

40

liS (mlvl)"

Figure I. Lineweaver-Burk plot of L-Lys and L-Ala uptake by F36-ZK. Uptake rates of L-Lys (A) and L-Ala (B) in the range of 0.016 to 27 mM were ploned on Lineweaver-Burk plot. The insert is a close-up of L-Ala uptake at high concentration. Arg !

120

nmol H+

Ser !

~---------!

~---Time

103

that these amino acids were transported by the same system; a basic amino acid transport system. Uptake of most amino acids, including L-Lys, L-His, L-Asp and L-Glu, was inhibited by the addition of neutral amino acids. These results indicated the existence of a common broad transport system, a general amino acid transport system, which catalyses the transport of 19 amino acids but not L-Arg. In addition, the existence of an alanine transport system was suggested because Ala was transported by at least two systems as mentioned above and one of them was identified as a general amino acid transport system but another was unidentified. L-Lys and L-Ala seemed to be transported via two systems from the results of kinetic studies, as shown in Fig. 1. Uptake of 0.026 mM (38.5 mM- 1 in Fig. lA) and 1 0.12 mM (8.3 mM- in Fig. lA) L-Lys was performed by a high and low affinity system, respectively. Transport of L-Lys via the high affinity system (Lys 1 in Table 2) was inhibited more strongly than that of the low affinity system (Lys 3 in Table 2) by the addition of neutral amino acids, which were transported via the general amino acid transport system. These results indicated that the high affinity system of L-Lys transport is identical with the general amino acid transport system, and also that the low affinity system was a basic amino acid transport system. Similarly, uptake of 1 0.027 mM (37.0 mM- in Fig. 1B) L-Ala (Ala I in Table 2), which was carried out by the high affinity system, was affected less than that of 4.67 mM (0.21 mM- 1 in Fig. 1B) L-Ala (Ala 3 in Table 2) by the addition of neutral amino acids. These results also indicated that high and low affinity systems of L-Ala transport were assigned respectively to a third system (the alanine transport system) and to general amino acid transport system. The transport systems of F36-ZK are summarized in Table 3.

Measurement of intracellular and extracellular free amino acids

-

30 s

Figure 2. Change in external pH during amino acid uptake. The external pH was monitored by a pH electrode during amino acid uptake by F36-ZK. Bar on X axis represents 30 s. The column bar represents the scale 01'20 nmol proton.

To classify whether these systems are capable of active or passive transport, intracellular and extracellular concentrations of L-Arg (for the basic amino acid transport system), L-Ser (for the general amino acid transport system)

Table 4. Accumulation of amino acid by F36-ZK. I min after addition of tracer Intracellular AA (rnlvl ) Extracellular AA (mM) Arg Ser Ala

3.66 ± 127 13.0 ± 2.22 0.76±013

0.99 ± 0.01 0.94 ± 0.03 0.10 ± 0.0002

Each value is the mean of three experiments ± S.D.

Ratio

30 min after addition of tracer Intracellular AA (mM) Extracellular AA (mM)

Ratio

3.7 13.8 7.6

11.6 ± 1.66 51.6 ± 4.35 5.90 ± 0.28

12.7 103 101

0.92 ± 0.01 0.50 ± 0.01 0.06±0.OOI

Y. KATO AND N. IMAMURA

104

Table 5. Effect of uncouplers and inhibitors on amino acid uptake. Uncouplers and inhibitors

Uptake rate (% control)" Arg Ser

Ala

lonophores CCCP (100 mM) Valinomycin (10 mM) Nigericin (10 mM) ValinomycinlNigericin (each 10 mM)

5.11 ± 1.56" 98.0 ± 5.90 133 ± 7.82** 127 ± 8.36**

1.26 ± 0.48" 100±14.2 123±11.6* III ± 6.69*

3.20 ± O. \7" 100 ± 12.2 125 ± 9.09* 109 ± 1.99**

Inhibitors of ATP synthesis NaN] (I mM) DCCD (100 mM)

17.6 ± 0.82** 87.9 ± 3.29**

4.87 ± 0.87** 100 ± 3.12

4.70 ± 0.74** 90.2 ± 4.96*

Structural analogue of phosphate Vanadate (I mM)

52.4 ± 4.93**

74.3 ± 7.26**

75.7 ± 2.57**

Inhibitor ofNa+/K+-ATPase Ouabain (I mM)

100 ± 3.52

92.4 ± 2.75**

103 ± 4.87

'Each value is the mean of three experiments ± S.D. Asterisks indicate a significant difference from control (non-treated cells) based on t-test (**. p
L-Arg Group I

120 1fXI

xo 40

l

1~1

IlKI

60

100

41l

20

t ,/

•

t·t

60

+.1,

-.

4U

.~.

20

4

..

120

Xil

t\f

i

20

4

i.-Ala Group 3

120 1f~1

~

Xll

41l

20

60

L-Ser Group 2

120

L-Gln Group 2

J(XI

1\

f

L-Glu Group 4

120

·1

J(XI

so

f

60

+'.

40

10

L-Asp Group 4

120

so 60

~

t

40

."

20

'.

pH Figure 3. Relative amino acid uptake of F36-ZK at several pH values. Amino acid uptake was measured using cells in 25 mM sodium citrate buffer at pH 4.0 to 5.0 and in 25 mM sodium phosphate buffer at pH 5.0 to 8.0. Each point is the mean of three experiments. Bars represent means ± S.D. of three replicates. Significantly different groups were distinguished by two-way ANOVA followed by Tukey test (p<0.05) based on the data between pH 4.5 to 8.

and L-Ala (for the alanine transport system) were measured (Table 4). At I min after the addition of tracers, intracellular concentrations of all amino acids were higher than extracellular ones. The differences between intracellular and extracellu lar am ino acid concentrations became larger after 30 min of incubation, indicating that the transport systems were active. L-Ser and L-Ala accumulated more in the cells than L-Arg at 30 min.

Proton flow during uptake and effect of uncoupler and inhibitor The pH of the medium was monitored during amino acid uptake (Fig. 2). The only one data was shown because the same data were obtained. No change in external pH was observed during L-Arg uptake. Conversely, a decrease of external proton concentration was observed for approximately I min after the addition of L-Ser and L-Ala,

AMINO ACID TRANSPORT SYSTEMS OF JAPANESE PARAMECIUM SYMBIONT Table 6. Amino acid uptake of F36-ZK grown with L-Ser or ammonium as sole nitrogen source.

pH Dependence ofamino acid uptake

Amino acid uptake (nmoll min 15 x 107 cells)" Ser(n=3) NH/(n=2) Arg 0.05 mM Lys I mM Lys His Asp (pH 5) Glu Asn Gin Ser Thr Tyr 0.1 mMAla 3 mM Ala Val Leu

1.95 ± 0.15 0.24 ± 0.06 1.27 ± 0.15 1.03±0.16 1.27 ± 0.37 0.54 ± 0.08 0.50 ± 0.08 10.8 ± 1.43 5.05 ± 0.83 5.12±0.85 203 ± 0.39 5.77 ± 0.83 7.85 ± 0.92 4.34 ± 0.67 4.35 ± 0.57 4.31±0.53 5.49±0.48 3.84 ± 0.50 2.87 ± 0.44 3.15±0.41 4.80 ± 0.69 10.8±0.71

lie

Pro Phe Met Trp

Gly Cys

To clarify the features of these amino acid transport systems, uptake of several amino acids was measured in the pH range 4.0 to 8.0. The pH dependence of uptake is indicated in Fig. 3. The maximum uptake rates of neutral amino acids, such as L-Ser, L-Ala and L-Gln, were observed at around pH 5.0. Similar data was obtained during L-Pro uptake (data not shown). A broad optimal pH for L-Arg uptake was observed in the range of 5.0 to 6.5. In the case of L-Asp and L-Glu, characteristic curves were obtained; uptake of L-Asp was inversely related to pH over the range of pH 4 to pH 5.5 with little uptake occurring at pH 6 and above.

3.85 ± 0.22 0.35 ± 0.13 1.62 ± 0.40 0.45±0.07 0.15 ± 0.001 0.28 ± 0.07 0.35 ± 0.04 3.60 ± 0.22 1.60 ± 0.11 1.63±0.08 0.72 ± 0.05 2.61 ± 0.37 8.42 ± 1.19 1.28 ± 0.06 2.26 ± 0.10 1.61±0.17 1.87±0.16 1.90 ± 0.33 1.72 ± 0.03 2.12±0.91 2.03 ± 0.22 5.94±0.19

Effect ofinorganic nitrogen on amino acid uptake Uptake of several amino acids was measured in the presence of nitrate and ammonium. The data are given in Fig. 4. These forms of inorganic nitrogen did not affect L-Arg and L-Ser uptake. Uptake of L-Gln was not affected by these inorganic nitrogen compounds (data not shown). To examine the effect of cultivation with ammonium, amino acid uptake of F36-ZK was measured using cells grown with ammonium as sole nitrogen source. The results are shown in Table 6. The alga also transported all 20 amino acids, which clearly indicated that all of the transport systems are expressed under these culturing conditions, although the uptake rate of the general amino acid transport system decreased slightly.

Uptake of 0.1 mM amino acid was measured. "Each value is the mean of three experiments ± S.D. (for n = 2, mean ± difference from the mean). and plateaus were observed after approximately I min. To study the energy of these transporters in detail, amino acid uptake was measured in the presence of several kinds of uncoupler and inhibitor. The results presented are demonstrated in Table 5. A protonophore (CCCP) and an inhibitor of ATP synthesis (sodium azide) inhibited amino acid uptake strongly approximately 90% inhibition. An inhibitor of phosphorylation (vanadate) also inhibited uptake of these amino acids, especially L-Arg.

A v

..:.: es

c..::l -0 'w ~

0

'" '6 -<

"i:) (,)

r-.

120

,0

100

:!5 20

X

15


'0 E 5-

4. Discussion Kinetic analyses and competitive experiments revealed three amino acid transport systems of F36-ZK (Table 3), a

B

,5

0

105

80

...-NH4+

-u- N0 3'

10

60 40

- . NH4+, N0

0

0

10

20

Time (min)

30

' 20 3 0 40 0

20

40

Time (min)

60

80

Figure 4. Amino acid uptake by F36-ZK in the presence of inorganic nitrogen. Uptake of 0.5 mM L-Arg (A) and L-Ser (B) was measured in the presence of 2.3 mM inorganic nitrogen at 90 umol photons m'2 S'I at 25°C. Values were means of three experiments ± S.D.

106

Y. KATO ANON. IMAMURA

basic amino acid transport system for L-Arg and L-Lys, a general amino acid transport system that showed very broad specificity for 19 amino acids (but not L-Arg), and an L-Ala transport system. These three systems were shown to be active transporters carrying out amino acid-proton symport based on the following results: 1) an observed decrease of proton concentration in the medium during L-Ser and L-Ala uptake, 2) uptake inhibition by a protonophore, sodium azide (an inhibitor of ATP synthesis), and vanadate (an inhibitor of phosphorylation). Amino acid uptake of F36-ZK was not inhibited by ammonium. Furthermore, the expression of the amino acid transport systems was also independent of nitrogen source. The constitutive systems characterized above were one of the specific features of F36-ZK. Uptake of several amino acids was measured at various pH levels. Optimal uptake of all tested neutral amino acids was observed near pH 5.0 and a broad optimal pH range existed for L-Arg uptake was at pH 5.0 to 6.5. Amino acids with a slight positive charge seemed to be suited for the transportation because the optimal pH for uptake was slightly more acidic than the isoelectric points. These results also supported the amino acid-proton symport mechanism which is found in many organisms (Bush, 1993; Cho and Komor, 1983; Young et aI., 2003). In the case of L-Asp and L-Glu, uptake was very sensitive to external pH; i.e., uptake increased when the pH became acidic and little uptake was measured above pH 6. It was reported that Paramecium endosymbionts could not utilize L-Glu in the previous studies (Albers et aI., 1982; McAuley, 1986; Kato et aI., 2006), but the reported experiments were carried out in a medium with a pH above 6.3, a value where little uptake of L-Glu was measurable for F36-ZK. Therefore, we speculate that nonutilization of L-Glu by Paramecium symbionts is due to the lack of uptake at the studied pH. In fact, F36-ZK could utilize L-Glu slowly at pH 5.5, doubling time was 4.4 days. Many microalgae grown photoautotrophically can import few amino acids (Cho et aI., 1981; Kato et aI., 2006; Kirk and Kirk, 1978). However, amino acid transport systems in free-living C kessieri can be induced by glucose (Cho et aI., 1981). In the case of C vulgaris, sugars or glycine induce an amino acid transport system for 4 neutral amino acids (Plakunov et aI., 1995; Seifullina et aI., 1995). Thus, free-living Chiarella possesses the potential to build various systems, but these are usually repressed in photoautotrophic conditions (Cho et aI., 1981; Plakunov et aI., 1995; Sauer, 1984; Seifullina et aI., 1995). Three constitutive amino acid transport systems of F36-ZK were sufficient for transport of all amino acids and the general amino acid transport system showed extremely broad specificity. The induced general system ofC kessleri also exhibited broad specificity for 10 amino acids (Sauer, 1984); however, even broader specificity for 19 amino acids in F36-ZK was observed.

Similar broad specificity of amino acid permease was reported for GAPI in Saccharomyces cerevisiae, which was induced on poor nitrogen sources (Jauniaux and Grenson, 1990), and AAP3, which had a role in uptake and distribution of amino acids in roots of higher plants (Fischer et aI., 1995; Okumoto et aI., 2004). Therefore, am ino acid transport system, which had broad specificity, could be induced as a result of an amino acid requirement. The characteristics of the general amino acid transport system of F36-ZK perhaps suggest that the algae require amino acids that the host supplies to their symbionts. Sugar contributes to the development of amino acid transport systems in free-living C kessleri and C vulgaris (Cho et aI., 1981; Plakunov et aI., 1995; Sauer 1984; Seifullina et aI., 1995). In addition, symbionts in Paramecium cells are considered to live in the presence of sugar, because the algae enclosed by the perialgal vacuole membrane release maltose during the entire day (Ziesenisz et aI., 1981). Therefore, amino acid transport systems of symbiotic Chiarella should develop under these conditions. Furthermore, since the optimal pH value for amino acid transport by F36-ZK was almost identical to that for maltose release by symbiotic Chiarella (Kessler et al., 1991; Reisser and Widowski, 1992), it is possible that the type of transport of maltose and amino acids in F36-ZK antiport that occurs at the same time. The characteristic feature of symbiotic Chiarella F36-ZK implies the possibility of the exchange of maltose and amino acids between host and symbionts in Paramecium symbiosis.

Acknowledgments We would like to express our sincere thanks to Prof. Bun-ichiro Ono for his great advice on this study.

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