Plant Physiol. (1976) 57, 430-436

Studies of Sulfate Utilization by Algae

15. OF ASSIMILATORY SULFATE REDUCTION IN EUGLENA AND THEIR CELLULAR LOCALIZATION'

Received for publication June 10, 1975 and in revised form November 18, 1975

CHRISTIAN BRUNOLD2 AND JEROME A. SCHIFF3 Institute for Photobiology of Cells and , Brandeis University, Waltham, Massachusetts 02154

ABSTRACT been found in non-photosynthetic organisms such as Escherichia coli (35, 37) and yeast (20, 40) where the nucleotide sulfonyl Crude extracts of wild-type Euglena grown in the light (WTL) or in donor is PAPS.4 the dark (WTD) and a mutant lacking detectable DNA (W3BUL) Sulfite reductases which reduce free sulfite to free sulfide have contain adenosine 5'-phosphosulfate (APS) sulfottansferase. Isotope also been found in many organisms (23) where they have been dilution experiments indicate that adenosine 3-phosphate 5'-phospho- suggested as participants in a pathway of sulfate reduction in- sulfate (PAPS) sulfotransferase is absent. volving free inorganic intermediates. In Chlorella, however, Thiosulfonate reductase, requiring addition of NADH or NADPH studies with mutants suggest that the bound pathway is the but not ferredoxin, and O-acetyl-L-serine sulfhydrylse, the two other physiological route of sulfate reduction in vivo with sulfite re- enzymes of the bound intermediate pathway of assimilatory sulfate ductase participating only when free sulfite is supplied exoge- reduction, are also present. Increasing levels of all three enzymes were nously. In E. coli the separation of the two pathways is less clear found in WTL, WTD, and W3BUL during logarithmic growth but the since thiosulfonate reductase and sulfite reductase activities may various activities were similar at comparable stages of growth in all three be contained in the same molecule (37), unlike Chlorella where types of . two separate enzymes appear to be present (11, 26). These results show that the three enzymes are not coded in the Since we wished to study the formation and cellular localiza- DNA and are not restricted to Euglena cells having fully tion of the enzymes of assimilatory sulfate reduction, we turned developed . Consistent with this, they do not increase during to Euglena where separations can be performed (5), light-induced chloroplast development in resting cells and are found to chloroplast development is under the experimenter's control and be enriched in the mitochondrial fraction. Further resolution of this mutants are available which lack chloroplast DNA (21, 22, 31). fraction on sucrose gradients shows that the APS sulfotransferase is In this paper we show that APSTase, TSRase, OASSase, and associated with both the (glyoxysomal) and mitochondrial sulfite reductase activities are present in Euglena cells, correlate fractions while the thiosulfonate reductase and O-acetyl-L-serine sulfhy- these activities with the presence or absence of functional plas- drylase are associated only with the mitochondria. Thus the three known tids and the presence or absence of plastid DNA, and show that enzymes of the bound pathway of assimilatory sulfate reduction are the three enzymes are localized in the mitochondrial fraction. present in Eugkna mitochondrina. A brief abstract of this work has appeared previously (4). Although the activity of the entire bound pathway (APS to cysteine) is low in extracts, addition of dithlothreitol which releases free sulfite from MATERIALS AND METHODS the product of the APS sulfotransferase reaction, causes an increase in reduction activity indicating that a sulfite reductase is also present. It Euglena gracilis Klebs var. bacillaris Pringsheim and a mutant remains to be shown which reducing system is the significant one in vivo (W3BUL) lacking detectable chloroplast DNA (21) were culti- in Eugkna. vated aseptically in Hutner's (10) pH 3.5 medium with shaking at 26 C, as previously described (18). Dark-grown resting organisms were obtained, as described previously (32), using pH 6.8 resting medium (3). Conditions for normal light-induced chloroplast development have also been given before (33). Cell densities were determined with a model A pathway of assimilatory sulfate reduction containing car- A Coulter counter with an aperture of 100 ,um as before (41). rier-bound intermediates has been described for Chlorella where For the preparation of cell extracts, the cells were centrifuged it appears to be the major pathway of reduction in vivo (26). at 1500g for 5 min and were washed twice in 10 mm tris-HCl, pH This reduction pathway begins with adenosine 5'-phosphosul- 9.25, containing 10 mm EDTA. For routine work the washed fate, the sulfonyl group of which is transferred via APS sulfo- cells were suspended in 200 mm tris-HCl, pH 9.25, to a final transferase to a carrier to form Car-S-SO3- which is then re- density of 15 to 20 x 106 cells/ml. For assay of TSRase with G- duced to Car-S-S- by a ferredoxin-dependent thiosulfonate re- S-S03- and for measurements of reduction of APS to cysteine, ductase. The thiol group is then transferred via 0-acetyl-serine the washed cells were resuspended in 2.3 ml of 200 mm tris-HCl sulfhydrylase to OAS to form cysteine. A similar pathway has (pH 8), BSA was added to a final concentration of 0.1% (w/v),

' Research was supported by Grant BMS73-00987 AOI from the 4Abbreviations: PAPS: adenosine 3'-phosphate 5'-phosphosulfate; National Science Foundation. APS: adenosine 5'-phosphosulfate; APSTase: adenosine 5'-phospho- 2 Supported by Schweizerischer Nationalfonds. Present address: sulfate sulfotransferase; OASSase: O-acetyl-L-serine sulfhydrylase; Pflanzenphysiologisches Institut der Universitaet Bern, Altenbergrain TSRase: thiosulfonate reductase; WTL: Euglena wild type grown in the 21, 3013 Bern, Switzerland. light; WTD: Euglena wild type grown in the dark; W3BUL: Euglena 3 Abraham and Etta Goodman Professor of Biology. To whom reprint mutant lacking detectable chloroplast DNA; OAS: O-acetyl-L-serine; requests should be sent. G-S-SO3-: glutathione-S-SO3-; DTT: dithiothreitol. 430 Physiol. Vol. 57, 1976 SULFATE REDUCTION IN EUGLENA ORGANELLES 431 and 50 ,lA of silicone defoamer (antifoam C emulsion, Dow Mitochondria were prepared as described by Buetow (5) from Corning diluted 1:50 with H2O) was also included, and the mutant W3BUL. For determination of activities, the suspension was gassed with N2 for 5 min. The resuspended cells pelleted mitochondria were resuspended in 200 Mm tris-HCI, pH cooled with ice water were broken in a Branson sonifier model S 9.25, to provide a final concentration of 1.5 to 3 mg/ml protein 75 at 2 amp with two bursts of 30 sec separated by an interval of and were sonicated as described above. The homogenate was 30 sec. The homogenate was centrifuged at 12,000g for 5 min, used without further centrifugation for the usual enzyme assays. and the supernatant fluid was used immediately for the enzyme For the separation of AP35S and PAP3S after incubation with assays. APSTase was measured as the production of sulfite-3S, Euglena extracts, the assay mixtures were inactivated in boiling assayed as acid volatile radioactivity from AP35S in the presence H20 for 1 min, 40 Mul of 1 N HCl were added to bring the pH to of DTT as previously described (14, 24). TSRase was measured 6.1, and after centrifugation, 50 ,l of the supernatant were as the production of H2S from dithionite or the formation of subjected to paper electrophoresis (12) in pH 5.8 citrate buffer cysteine-35S from G-S-35SO3- (25). In the dithionite assay, N2 on Whatman 3MM paper at 1800 v (30 v/cm) for 50 min at 4 C. was bubbled through the assay mixture during incubation. The AP35S and PAP35S were localized on the paper by UV, the areas gas stream was passed through 8.8 ml of zinc acetate solution containing the nucleotides were cut out, and the radioactivity (15) to remove continuously the H2S formed by the enzyme was measured with a Beckman liquid scintillation spectrometer, reaction, and sulfide was determined according to Johnson and The counting fluid was toluene-methanol (1:1) with 4 g/l PPO Nishita (15), using a molar extinction coefficient for the methyl- (Interex Corp.) and 50 mg/l POPOP (New England Nuclear ene blue formed of 35 x 103 (22). In the assay with G-S-35SO3-, Corp.). All the samples from each experiment were counted the reaction was stopped with 0.5 ml of 0.75 M formic acid-I M under identical quenching conditions. Proteins were determined acetic acid, pH 2 (8), containing 200 jig of cysteine. After according to Lowry et al. (17) using BSA as a standard. To removing the precipitated proteins by centrifugation 50 ,l of the determine Chl, a pinch of MgCO3 was added to cells washed by supernatant were subjected to paper electrophoresis on What- centrifugation in the tris-EDTA buffer used to prepare enzyme man 3 MM paper with formic-acetic buffer pH 2 (8) for 50 min extracts, and absolute acetone was used to extract the pigments. at 1800 v and 4 C. The cysteine was visualized by spraying with After adjustment of the extracts to 80% acetone with addition of 0.3% ninhydrin in 1-butanol-glacial acetic acid (100:3) and H20, absorbance was determined at 649 and 665 nm, and the heating at 100 C for 5 min (16). The spot was cut out, and its sum of Chl a and b was calculated (38). radioactivity was determined by scintillation counting. 3S-labeled sulfite and sulfate were purchased from the New OASSase was measured by the method of Becker et al (2). England Nuclear Corp. PAP35S was prepared by a modification Due to the high backgrounds in the cysteine assay caused by of the method of Hodson and Schiff (13). AP3S was obtained by substances in the crude enzyme extracts, the rates were routinely treating PAP35S with 3'-nucleotidase from ryegrass (29) and was measured as the difference in absorbance between two assays purified by column chromatography on DEAE (unpublished incubated for 2 and 4 min, respectively. For the determination of procedure). Ferredoxin and ferredoxin-NADP reductase pre- the specific radioactivity of the SO2 produced in the APSTase pared from Chlorella according to Schmidt (25) were a gift from reaction, 50 ,l of diluted silicone defoamer solution was added A. Vaisberg. G-S-35SO3- was prepared according to Schmidt to the reaction mixture after incubation. The reaction mixture (25). O-Acetyl-L-serine was obtained from Calbiochem; was acidified with 2 ml of 5 N H3PO4 and gassed with N2 at 50 C NADPH, NADP, NADH, dithiothreitol, 3'-nucleotidase from for 30 min. The gas stream was passed through 2 ml of 0.1 M ryegrass, and glucose 6-dehydrogenase (yeast) were purchased tetrachloromercurate (39), and the SO2 was determined accord- from Sigma. ing to West and Gaeke (39). Of this solution 200 Al were used to determine the radioactivity by scintillation counting. RESULTS AND DISCUSSION For separation on sucrose gradients, the cells from 1 liter of medium were collected from cultures in late log phase of growth Adenosine 5'-Phosphosulfate Sulfotransferase Activity. Table by centrifugation at 15OOg for 5 min; all manipulations here and I shows the requirements for optimal APSTase activity in crude following were performed at 4 C. The cells were washed in a extracts of Euglena WTL. The enzyme is unstable, losing up to buffer containing: 0.5 M sucrose, 0.01 M EDTA, 0.01 M MgC12, 50% of the initial activity in 2 hr at 4 C. Bovine serum albumin 0.01 M KCI, and 0.1% (w/v) BSA in 0.05 M sodium cacodylate, (0.1%, w/v) and glycerol up to 30% (v/v) do not stabilize the pH 7.2 (9). Previously published methods originally devised for enzyme activity completely, but their addition serves to slow zonal rotors were employed (9) using centrifuge tubes instead. A down the loss of activity. Using crude extracts without these homogenate of Euglena was prepared (9), centrifuged twice for 10 min at 10OOg and then at 3000g. Seven ml of supernatant Table I. Assay fluid were applied to 30.5-ml continuous linear sucrose gradients ofAdenosine 5'-Phosphosulfate (APS) Sulfotransferase in 40-mI Spinco centrifuge tubes. The gradients extended from Activity in Extracts of Light-grown Wild-type Euglena 25 to 48% (w/w) sucrose in 0.05 M sodium cacodylate, pH 7.2, The complete mixture contained in a final volume of 0.15 ml: AP3S containing 1 mM EDTA. The tubes were centrifuged in the SW- (18,484 cpm/nmole), or PAP35S (16,208 cpm/nmole), 150 nmoles; tris- 27 head of the Spinco ultracentrifuge at 25,000 rpm for 4.5 hr at HCI (pH 9.25), 40 ,umoles; DTT, 12 jtmoles; MgSO4, 100 jtmoles; and 2 to 5 C. The tubes were pierced at the bottom and fractions Euglena extract containing 54 lAg of protein. The incubation was for 30 were collected. The sucrose concentration of each fraction was min at 30 C under N2- determined by measuring the refractive a index with Bausch and AP2S Converted to Acid-volatile Radioactivity Lomb refractometer at 27 C. The enzymes used as markers were assayed as given in the following references: succinate dehydro- nmoleslhr-mg protein 10-3 pmolal genase (6), glyoxylate reductase with glyoxylate as substrate hr-cell (34), and malate dehydrogenase (19). These assays include the Complete system 97.8 12.04 addition of Triton X-100 to lyse the organelles; for the assay of Minus AP35S 0.16 0.02 APSTase, TSRase and OASSase, Triton was added to the ap- Minus DTF 0.24 0.03 propriate buffers to a final concentration of 0.1%. In the case of Minus MgSO4 13.41 1.65 the APSTase it was necessary to double the buffer concentration Minus AP3S + PAp3S 111.51 13.73 Heated used in the to the incubation mixture to the enzyme (3 0.24 0.03 assay bring proper min, 100 pH of 9.25. C) 432 BRUNOLD AND SCHIFF Plant Physiol. Vol. 57,1976 additives, the activity is linearly dependent on the incubation release of phosphate from PAPS (M. L.-S. Tsang, unpublished time up to 30 min, on the amount of protein added up to at least results). 70 ,ug, and is enhanced by high MgSO4 concentrations. An Thiosulfonate Reductase Activity. TSRase activity in crude enhancement was also obtained by of Na2HPO4 or extracts of Euglena WTL is shown in Table III. Activity can be Na2SO4. KCI, which is used at an ionic strength of 0.4 in Chlo- demonstrated with either dithionite or G-S-35SO3- as substrate. rella extracts for optimal activity (26) has an inhibitory effect in In the G-S-35SO3- assay the cooperative action of TSRase and of Euglena extracts. The requirement for high pH is characteristic OASSase is measured since cysteine is formed. NADPH, for APSTases and may be due to the need for an ionized -SH NADH, or NADP and an NADP-reducing system will serve as group for enzyme activity (26). Temperature and AP3S concen- reductants, but addition of ferredoxin is not required. The com- tration optima are the same as in the Chlorella system. Table I parable enzyme from Chlorella requires the addition of ferre- shows that PAP35S also serves as a nucleotide sulfonyl donor and doxin (26) for activity while the same enzyme from E. coli (35, that the reaction rate with PAP35S is higher than with AP35S. 37) does not. It is possible that the crude extracts used here Due to the instability of the present Euglena enzyme prepara- contain sufficient ferredoxin and ferredoxin-NADP reductase tions, it was not possible to determine the nucleotide specificity for maximal activity. of the sulfotransferase directly after purification of the enzyme O-Acetyl-L-serine Sulfhydrylase Activity. Euglena extracts and for this reason we turned to isotope dilution experiments. have appreciable OASSase activity which is heat-labile and re- Since there might be differences in the nucleotide specificity of quires OAS and sulfide for the formation of cysteine (2). Euglena WTL, WTD, and W3BUL, we included all three types Enzymes of Bound Intermediate Pathway of Assimilatory of cells in these experiments. Sulfate Reduction during Cell Growth of Euglena WTL, WTD, Figure 1 shows that over a wide range of AP3S concentra- and W3BUL. Figure 2 shows that APSTase, TSRase, and OAS- tions, nonradioactive PAPS does not dilute the label appearing Sase are present in Euglena WTL, WTD, and W3BUL. The in acid-volatile products indicating that APS is not converted to activity per cell of these enzymes changes during cell growth, PAPS prior to the transferase reaction. However, over a wide rising during logarithmic growth but falling as the cells enter range of PAP35S concentrations, nonradioactive APS effectively stationary phase. It is evident that optimum activities are ob- dilutes the label appearing in acid-volatile radioactivity. The tained in late log phase. The data also indicate that cell age must same results are obtained with all three types of cells and they be taken into account if enzyme activities from various strains suggest that PAPS is converted to APS prior to the transferase and culture conditions are to be compared. Using data collected reaction. from four to six experiments (Table IV), we conclude that the About 16 nmoles of AP35S are formed from PAP3S in 30 min three activities are not very different in WTL, WTD, and which would lead to a dilution of the specific radioactivity of W3BUL at comparable stages of growth, suggesting that these PAP3S in the transferase reaction by a factor of about 10. Table enzymes are not coded in chloroplast DNA and that their levels II shows that this magnitude of dilution is observed when the do not change during chloroplast development. specific radioactivity of the 3S02 recovered from the APSTase It was found in other experiments (data not shown) that, reaction is determined under similar conditions. Table II also although the cells grow well on cysteine or methionine, the levels shows that comparable amounts of SO2 are recovered in all of the three enzymes are comparable to those found for cells cases, which rules out the possibility that inhibition of product grown on sulfate, suggesting that these forms of sulfur do not formation occurs. These results verify that the predominant repress the formation of these enzymes of the sulfate-reducing enzyme activity present is an APSTase and that PAPS is utilized pathway. only by conversion to APS in all three types of cells. Euglena Levels of Sulfate-reducing Enzymes during Light-induced extracts contain 3 '(2'),5 '-diphosphonucleoside 3 '(2')-phospho- Chiloroplast Development in Nondividing Cells of Wild-type hydrolase activity which converts PAPS to APS as measured by Euglena. When dark-grown resting cells are placed in the light,

10 10 - 8- WILD TYPE (LIGHT) WILD TYPE (LIGHT) -8 > NO PAPS NO APS i 6- x6 4- xx/ ~~~~~4O o ~~2~~~~~ 0.5mM PAPS ~~~~~z0 0.5mM APS 0 M J_j 0. //_ o_0-o-O * o '° { WILD TYPE (DARK) WILD TYPE (DARK) 8 3 0 0 0.5mM PAPS NO APS 6 4 -NO u) ox x PAPS x X x ~~4 < E 2- CLJ, 0.5mM APS 2 ~0 /BL x--x-- mNOAPS 8 8- WBL 0.5mM PAPS x/o6- oz 6- 6 0 X---X-/ -4 U 4 -~~~4 0.~~~~~ i0..mm aps r2 -<

0 60 90 12( 30 60 90 12 AP35S ADDED (n moles) PAP35S ADDED (n moles) FIG. 1. Influence of nonradioactive PAPS and APS on the formation of acid-volatile radioactivity from AP"S and PAP"S by extracts of Euglena. The complete system contained in a final volume of 150 ,u: AP3S (20,380 cpm/nmole), or PAP35S (12,260 cpm/nmole), 150 nmoles; APS, or PAPS, as indicated, 75 nmoles; tris-HCI (pH 9.25), 40 ,umoles; DTT, 12 Amoles; MgSO4, 100 ,umoles; and Euglena extract from 304 x 106 cells. Plant Physiol. Vol. 57, 1976 SULFATE REDUCTION IN EUGLENA ORGANELLES 433

Table II. Influence ofAPS on PAP35S and PAPS on AP35S Conversion to Acid-volatile Radioactivity The complete mixture contained in a final volume of 1.5 ml: APS, AP`S, PAPS, PAP35S as indicated; tris-HCI (pH 9.25), 400 Amoles; DTT, 120 ,moles; MgSO4, 1 ,tmole; extracts of Euglena containing 400 to 500 jAg of protein. The incubation was for 30 min at 30 C under N2. Specific Radioac-SpcfcRdo- Source of Enzyme APS AP"S PAPS PAP"S tivity of AP"S or 'SO, Recovered SO, Recovered tivity of 3'SOa PAP2MS tvt f~ nmolesl cpm/nmok cpm nmoles cpmlnmok l.S ml Wild type grown in light 1500 11,350 27,599 3.12 8,846 750 1500 11,192 3,764 3.64 1,034 1500 16,430 70,650 4.53 15,596 1500 750 16,430 65,090 4.44 14,660 Wild type grown in dark 1500 11,305 20,989 2.03 10,339 750 1500 11,192 2,081 2.50 832 1500 16,600 66,802 4.25 15,718 1500 750 16,600 57,756 4.68 12,341 W3BUL grown in light 1500 11,305 28,589 2.81 10,174 750 1500 11,192 4,680 4.84 967 1500 16,430 92,406 5.36 17,240 1500 750 16,430 64,012 4.57 14,007

Table III. Thiosulfonate Reductase Activity in Extracts of Wild-type -150 6 Light-grown Euglena A. mixture contained in a final volume of 3.0 0 The complete reaction -100 4 D 0 ml: tris-HCl (pH 8.0), 141 j.moles; methyl viologen, 1.5 ,umoles; 0.7 ml r) of a solution of 250 mg of sodium dithionite and 250 mg of sodium (j) . in ml extract C r bicarbonate 10 of H20, and 100 ,lA of Euglena containing 50 -2 rL 0 280 ;Lg of protein. The incubation was for 30 min at 40 C. z -14 01m H2S Formed from Dithi- m m onite z 150 ;m -6 r a en nmoleslmg protein hr 0 r Complete system 863.5 m .100 c -1A 4 Minus methyl viologen 4.4 m 0 Minus sodium dithionite 1.1 .m Extract heated (3 min, 100 C) 6.5 -50 toU r- In B. The complete reaction mixture contained in a total volume of 200 0 .ul: tris-HCI (pH 8.0), 30 Amoles; MgCl2, 1 ,umole; G-S-35SO3-, 0.06 (A0 -6 o p.mole (21,214 cpm/nmole); OAS, 20 umoles, a reducing system as -150 0

indicated, and 50 p.l of Euglena extract from 0.96 x 106 cells. 0 - 0 Cysteine Formed from 00t oo -4 X G-S--SO,- 10 _ 10-' pmoles cell-' h-' o Complete system lacking reductant 0.11 51l - +NADPH, 0.4 p.mole 1.40 +NADH, 0.4 pAmole 1.01 +NADP, 0.02 p.mole + glucose-6-P, 0.4 ,umole 1.32 O L . . . 4.k 105 LO LO 10 30 50 70 + glucose-6-P dehydrogenase, 0.05 unit TIME (HOURS) +NADP, 0.2 ,umole + glucose-6-p, 0.4 + 1.33 pAmole FIG. 2. Changes in activity in extracts of Euglena cells at various glucose-6-P dehydrogenase, 0.05 unit + ferre- stages of growth. The incubation mixtures were composed as given in doxin, 0.005 pAmole + ferredoxin NADP-re- Tables I and III (dithionite assay). One ml of extract was prepared from ductase, 0.04 mg 15 to 20 x 106 cells, and for APSTase assay, 20 A±l, TSRase assay, 100 A.l, and OASSase assay, 10 Al, of extract was added. the proplastids develop into chloroplasts with a concomitant increase in many plastid constituents including plastid-localized events involved in plastid development and may be attributed to enzymes which are synthesized inside and outside the developing protein turnover which occurs in these starving cells (3). The chloroplast (21). Three enzymes of sulfate reduction decrease in result with W3BUL is consistent with this interpretation and activity during plastid development in wild type in the dark or suggests that during incubation on resting medium, turnover light and in mutant W3BUL (Fig. 3). This pattern is different sacrifices certain proteins which are not immediately indispensa- from that of normal chloroplast constituents and more closely ble for survival and, in wild type in the light, the amino acids resembles the behavior of enzymes which are not chloroplast released are probably used to form the constituents of the photo- localized and which are found in other cellular compartments synthetic apparatus. Since the resting cells are suspended in a (21, 22, 31). Since the loss of enzyme activity takes place in medium of diluted growth medium, there is ample sulfate pres- complete darkness, it is not part of the light-induced sequence of ent (about 1 mM) but this does not prevent the loss of the 434 BRUNOLD AND SCHIFF Plant Physiol. Vol. 57, 1976 Table IV. Activity of Sulfate-reducing Enzymes in Extracts of Euglena Each value is presented as the mean of data from several (usually six) different cultures in the mid-log phase of growth + the 95% confidence interval of the mean. Assay conditions are the same as those given in Tables I, III (dithionite assay), and IV; extracts were prepared and added as given in Fig. 3.

Ce Typeype APS-Sulfo-transferase Thiosulfonateductase Re- O-Acetyl-L-serineSulfhydrylase 10-3 pmoles hr-' cell-' Wild type, 7.8 ± 1.44 44.2 ± 19.61 2874 ± 894.9 light-grown Wild type, 7.2 ± 1.85 42.2 ± 18.76 3403 + 1341.3 dark-grown W3BUL, light- 7.7 ± 1.54 43.8 ± 16.71 5514 ± 2879.9 grown W3BUL, dark- 4.5 ± 0.92 31.0 + 9.77 4678 + 2049.8 grown enzymes of sulfate reduction during prolonged incubation. Other experiments showed that the loss of these enzyme activities during incubation of cells of wild type or W3BUL on resting medium in dark or light is not prevented by concentrations of cycloheximide or streptomycin (3) which effectively inhibit chlo- roplast development, indicating that loss of enzyme activity is not dependent on the synthesis of proteins which are translated 0 24 48 72 TIME (HOURS) on plastid or cytoplasmic . Cellular Localization of Sulfate-reducing Enzymes in Euglena. FIG. 3. Activity of sulfate-reducing enzymes and chlorophyll concen- Since the data already presented indicated that the three enzyme tration in extracts of dark-green resting Euglena cells in the light and in darkness. One ml of extract was prepared from 15 to 20 x 106 cells, and activities measured did not behave as though they were localized for the APSTase assay, 20 ,.l, for TSRase assay, 200 ,ul, and for exclusively or even predominantly in the chloroplasts, we turned' OASSase assay, 10 .ld, of extract was added to the incubation mixtures to studies with cell homogenates to localize further these activi- given in Tables I and III (dithionite assay). ties in the cells. Table V shows that when mitochondria are isolated from W3BUL by techniques yielding organelles which still possess oxidative phosphorylating activity (5), there is an these "proplastids" were contaminating the mitochondrial or enrichment of the sulfate-reducing enzymes in the mitochondrial microbody fractions. In the wild-type cells all proplastids de- fraction compared to the whole cell homogenate. velop into chloroplasts which can be removed in a preliminary This finding is confirmed and extended in experiments using centrifugation. Figure 5 shows that the gradients obtained are sucrose gradients (Fig. 4) which show that the thiosulfonate the same as those obtained with W3BUL homogenates suggest- reductase and 0-acetyl serine sulfhydrylase activities sediment ing that proplastids containing the sulfate-reducing enzymes are with mitochondrial marker enzymes. The APS sulfotransferase not contaminating these fractions. activity, like malate dehydrogenase, sediments with both the These results suggest that the thiosulfonate reductase and 0- mitochondrial and microbody enzyme markers. Since the possi- acetyl serine sulfhydrylase activities are localized to a large bility exists that the nuclear-coded plastid-localized components extent in the mitochondria of Euglena cells while the APS in W3BUL are still organized into lacking plastid DNA sulfotransferase is found both in the mitochondria and the mi- and components coded by this DNA, identical experiments were crobodies. The mitochondria of Euglena contain all of the en- performed with wild-type cells to rule out the possibility that zymes presently known to be associated with the bound pathway

Table V. Activity of Sulfate-reducing Enzymes in Unfractionated Cell Extracts and in Mitochondria of Euglena mutant W3BUL Assay conditions were as given in Tables I and III (dithionite assay).

APS Sulfotransferase Thiosulfonate Reductase O-Acetyl-L-Serine Sulfhydr lase Protein nmoles x h Protein nmoles x h Protein nmoles x h added(per -1 added(per -1 added(per 1 assay) x mg protein assay) x mg protein assay) x mg protein jig ~~~~~~,ugug Unfractionated Cell-

Extract 57 55.9 285 556 28.5 60,800 Isolated Mitochondria 48 163.5 240 1,025 24 215,000 Plant Physiol. Vol. 57, 1976 SULFATE REDUCTION IN EUGLENA ORGANELLES 435

20 25 30 35 40 45 50 20 25 30 35 40 45 50 SUCROSE CONCENTRATION t% W/W) SUCROSE CONCENTRATION (% W/W) FIG. 4. Enzyme and protein distribution after centrifugation of the 3000g supernatant fluid from homogenates of W3BUL Euglena cells on a linear sucrose gradient (15-50%, w/w). (One unit of succinate dehydrogenase, malate dehydrogenase and glyoxylate reductase = 1 j.mole x min- x ml-,.)

pletely but not from the cells of green algae or higher . Probably, the green algae and higher plants have certain indis- -I pensable cellular functions (perhaps including sulfate reduction) =0 which are carried out by plastid-localized enzymes, in addition to , which cannot be deleted without causing lethal- 3cnZrtl ity. Many of these same enzymes may not be plastid-localized in i, D I. ,v, Euglena (or at least, not exclusively); hence the plastids in this to,00Z organism can be deleted without lethal or debilitating conse- 3 IiD quences. The cellular localization of these enzymes may be ,^0 related to their biochemical requirements. The Euglena TSR 1n which is mitochondrial does not appear to require the addition of ferredoxin for activity (like the same enzyme from E. coli [35, 37]) while the enzymes from Chlorella and higher plants do require addition of ferredoxin (26). Presumably, the chloroplast- localized TSR enzyme would enjoy a ready source of reduced ferredoxin. At present, we have no explanation for the presence of the APS sulfotransferase in the microbody (or glyoxysome) fraction. The appreciable residual activity of all three enzymes in the supernatant of the homogenate (top of gradient in Fig. 4) indicates either that they are partially released from damaged organelles during extraction and separation or that they repre- sent enzyme molecules which are nuclear-coded and cytoplasmi- cally synthesized which are en route to their appropriate organ- elle. For a further discussion of organelle localization of sulfate- 41 45 37 41 45 reducing enzymes and its SUCROSE CONCENTRATION (% W/W) evolutionary implications see (23, 36). FIG. 5. Enzyme and protein distribution after centrifugation of the Sulfite Reductase Activity. Table VI shows that APS is con- 3000g supematant fluid from homogenates of light-grown wild-type cells verted to cysteine at low rates in the enzyme extracts. The low on a linear sucrose gradient (15-50%, w/w sucrose) in the fractions rates are perhaps due to the different conditions required for the between 37 and 47% (w/w) sucrose. (Units as in Fig. 4.) optimal activity of the APSTase and TSRase enzymes (26); conditions will have to be found which permit both enzymes to function more effectively together if higher rates are to be of assimilatory sulfate reduction. This situation is different from obtained. When an active thiol such as DTT is omitted from higher plants where these activities have been shown to be these incubation mixtures there is an appreciable decrease in the localized in the plastids of spinach leaves (22, 27) perhaps in the rate of cysteine formation from APS which can be ascribed to the lamellae (30). Since the green algae are very close to the higher release of sulfite in the APSTase reaction (26) by DTT and the plants in many characteristics, it is likely that the system in reduction of this sulfite by sulfite reductase to sulfide which can Chlorella is also chloroplastic. This would explain why it is be used by the OASSase reaction to form cysteine. Because possible to eliminate plastids from Euglena cells almost com- inorganic products are easily produced as side reactions of APS- 436 BRUNOLD AND SCHIFF Plant Physiol. Vol. 57, 1976 Table VI. Conversion ofAPS to Cysteine by Extracts from Light-grown 13. HODSON, R. C. AND J. A. SCHIFF. 1969. Preparation of adenosine 3'-phosphate 5'-phospho- Wild-type Euglena sulfate (PAPS): an improved enzymatic method using Chlorella pyrenoidosa. Arch. Bio- chem. Biophys. 132: 151-156. Complete system contained in a final volume of 150 Ml: tris-HCl (pH 14. HODSON, R. C. AND J. A. SCHIFF. 1971. Studies of sulfate utilization by algae. 9. Fractiona- 8.0), 20 MAmoles; MgSO4, 1 ,Mmole; NADPH, 0.6 Mmole; DTT, 12 tion of a cell-free system from Chlorella into two activities necessary for the reduction of iLmoles; OAS, 20 Mmoles; AP35S (7600 cpm/nmole), 150 nmoles; Eu- adenosine 3'-phosphate 5'-phosphosulfate to acid-volatile radioactivity. Plant Physiol. 47: 300-305. extract of 1.05-10 cells. Incubation was glena for 1 hr at 36 C. 15. JOHNSON, C. M. AND H. NISHITA. 1952. Microestimation of sulfur in plant material, soils, and Cysteine Formed irrigation waters. Anal. Chem. 24: 736-742. 16. KREBS, K. G., D. HEUSSER, AND H. WIMMER. 1969. Spray reagents. In: E. Stahl, ed., Thin- 10-3 pmoles/cell-hr layer chromatography. Springer, New York. pp. 854-909. Complete system 0.212 17. LoWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. I. RANDALL. 1951. Protein measure- Minus DTT 0.065 ment with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 18. LYMAN. H., H. T. EPSTEIN, AND J. A. SCHIFF. 1961. 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