compartmented metabolite pools in protoplasts from the green alga chlamydomonas reinhardtii: changes...

410 BJochtmica et B~ophysica Act~ 1073 (1991) 410-415 © 1991 Elsevier Science Publishers B.V. 0304-416s/91/$03.50

ADONIS 030441659100110U

Compartmented metabolite pools in protoplasts from the green alga Chlamydomonas reinhardtii: changes after transition

from aerobiosis to anaerobiosis in the dark

G e r d Kl~ck a n d K a r l h e i n z K r e u z b e r g

Boranisches Instirut, Universltiit Bonn, Bonn (F R.G,)

Key words: Protoplast fractionation; Subcallular mctabolite distribution; Chloroplast; Starch catabolism; (C, mi~hardrii)

A rapid fractionation meflJed for determination of metabolite levels in the chloroplast and the extraehloroplast compartment of Chlamydomonas reinlmrdtii has been developed, Protoplasts containing one large chloroplast were fractionated by passing them tlmmgh a multtlayer gradient containing dlgitonln, polyasrylamide, and a mixture of silicone oll and bromodecane. Lysis of the plasma membrane and the separation of the chloroplasts from most ol' the exiradfloroplast material was achieved within less than 5 s. The chloroplast enriched fraction was contaminated with 3% fumarase (mitochondria) and 13% phosphoenolpyruvate earboxylase (cytosul). Metabolites of the upper glycolytic chain were detected mainly in the dfloroplasts, whereas 2-phosphoglycerate was found only in the extracldomplast compart- men1. Analysis of changes in metabalite concentrations after transition to anaerobic conditions in the dark pointed to a regulation of carbohydrate catabolism by chloroplast phospbofructoklnaso aml by cytesolie pymvatekinase.

Green algae from the genus Chlamydomoaas can survive many days under anaerobic conditions in the dark [1,2]. Cellular starch, which is completely localized within the single chloroplast, is the main carbohydrate reserve for dark fermentation in C. reinhardtii. The products and the pathways of anaerobic fermentation in C. reinhardtii are well known [1,3]. Peavey et al. [4] described a Pasteur-effect during starch catabolism in Chiamydomoaas and Kreuzberg and Martin [2] found an oscillatory starch degradation under anaerobic conditions in C. reiahardtiL Nevertheless there is only limited information on the regulation of carbohydrate metabolism during starch degradation in unicellular algae available.

Abbre,~ations: Fr~-6-P, fructose 6-phosphate; Fm-I,6-P2, [tuc~s¢ 1,6-bisphosphate; GIu-I-P, glucose 1-phosphate; GIu-6-P, glucose 6-phosphate; glycerol-3-P, sn.glycerol.3.phosphate; Hepes, 4-(2-hy- droxyethyl)-l-plperazineethanesulf0nic acid; MTT, 3-(4,5.dimethyl- thlazoyl-2)-2,5-diphenyltetrazo[iurn bromide; Mops, 3.(N-nlolpho- lino)propanesulfordc acid; PES, phenanzine elhosulfate; 2-PGA, 2- phosphoglyceric acid; 3-PGA, 3-phosphoglycefic acid; p. inorganic phosphate; TP, glyeeraldehyde 3-phosphate+ dihydrosyacetone phosphate; Tris, 2- amin~2-hydroxyrnet hylptopane*1,3-di0l.

Correspondence: K. Kxeuzber~ Deuteche Forschungsaustoli ftir Luft- und Raumfahrt e.V. (DLR), Linder Hehc, D-5000. Keln 90, F.R.G.

Recently, the cornpartmentation of enzymes ~nvolved in glycolysis and the oxidative pentose phosphate cycle has been reported for this alga [5,6[ The enzymes for degradation of starch to trios*, phosphates (TP) were found solely in the stroma, whereas TP and 3-phosphoglyceric acid (3-PGA) seemed to be metabolized outside of the chloroplast as drawn from the results of the enzyme localization study. To understand the regulation of starch degradation, detailed knowledge of metabolite concentrations in the different sl:~ellular compartments is necessary. But up to now, no data on subcellular metabolite pool,~ in unicellular green algae are available, Attempts to use the published methods for rapid subcellular fractionation of hisber plant protoplasts failed for C. reinhardtii and other unicellular green algae because of the similar size of algal cells and chloroplasts. Thus we developed a method for the rapid fractionation of Chlamydom~.as protoplasts and report the first study on subc~llalar dis;.~;bufion of metabolites in thb alga.

Materials and Methods

Pratoplast preparation Protoplasts from synchronously grown C. reinhardtii

(11/32-b from the Sammlung fftr Algenkuhmen, G~fingen, F.R.G.) were prepared as described earlier [6]. Protoplasts were washed in 15 mM Mops/KOH

(pH 6.8, 3 rain 350 × g) and stored on ice until use ~2-5 mg Chl/ml, in 15 mM Mops/KOH (pH 6.8), 0.5 mM ethylenediaminetetraaeetate, 0.5% w / v bovine serum albumin).

Fractionation of protoplasts 3.3% polyacrylamide gel was prepared from l0 ml

50% (w/v) poiyaerylamide, added to 140 ml 20 mM Hepes/Tris (pH 7.0). The solution was degassed under reduced pressure, and then 0,1 ml N.N.N,N-tetrameth- ylethylenediamine and 0.1 ml 10% (w/v) ammonium- peroxydisulfate were added. The gel was polymerizcd 2 h at 35aC. Subsequently, it wa~, dialyzed three limes for 12 h against 0.1% (w/v) Dowex 1 × 8 (CI-) in distilled water and stored for a ma:dmum of 2 weeks.

The separation gels were prepared as follows: P-gel: 2.5 mi 3.3~ (w/v) polyacrylamide, 2 ml of 250

mM Mops/NaOH (pH 6.8), 25 mM MgLI z and 5.5 ml distilled water.

C-geh2.5 ml 3.3% (w/v) polyaerylamide, 2 ml of 250 mM Mops /NaOH (pH 6.8), 2 ml 600 mM man- nitol, 1 ml 5 mM MgCI 2 and 2.5 ml distill,zd water.

The silicone oil-bromodecane mixture was prepared from 20 ml AR 200/AR 20 (17.9 v/v , Wacker Chemie, Miinchen, F.ILG.) and 20 ml bromodecane.

Separation procedure 50 pl of the protoplast suspension were pipetted into

the cap of a conical microcentrifuge tube (1.5 ml Ep- pcodorf, Hamburg, F .R.G, cf. Fig. 1), prepared in one of six different ways. Tubes for protoplast analysis without fractionation contained 1 ml P-gel, followed by 0.15 rnl silicone oil-hromodecane mixture and either: (a) 50 /~1 3 M HCIO4 in 105 (w/w) sucrose (tubes for metabolite assays); or (b) 50 /tl 3 M NaOH, 0.02% (w/w) Triton X-100 in 105 (w/w) sucrose [NAIMP)H analysis]; or (c) 50 / l l 20 mM Tris /aseta te (pH 7.4) in 305 (w/w) glycerol (marker enzymes); or (d- f ) tubes for chloroplast enrichment contained 1 nd C-gel, followed by the same solutions as described for the corre- sponding protoplast tubes.

Then, 50 ,¢1 of a freshly prepared digitonin solution (55, w / v recrystulllzed digitonin [6]) were mixed into the C-gel layer. The tubes were carefully closed and incubated at 3 7 ° C in the dark. The protoplasts in the cap of the tubes must not drop into the gel during this procedure. After 5 rain, the tubes wci'¢ transfered to a centrifuge (Mod. 5114, Eppendorf, Hamburg, F.R.G.) and immediately centrifuged for 15 s. Darkness was mai:Rnined during the whole procedure.

During centrifugafion the protoplusts drop into the separation gel. in the C-gel layer, protoplasts are immediately lyzed by digltouin. The chloroplasts pass the silicone-oil layer and form a pellet at the ~;~om of the

Fig. 1. Photo~aph of a mlc~'adient aft~ ccntfifu~tion showing from t~e top to I~ bottom d~ layers or C-get and sil~one c~t- bromodecane mixture, and the pelleted ddoroplasts in the Tris/

acclatv/glycerol mixture,

tubes (Fig. 1). Protoplasts dropping in the P-gel (without digitonin) are not lyzed and are pelleted intact.

Anaerobic conditions were achieved by working in an Atmosbag ITM> (Sigma, Miinchen, F.R.G.) under a pure argon atmosphere. All solutions were flushed with argon before use.

Extract proce~ing After centrifugation, the tubes were frozen in liquid

nitrogen. Shortly before analysis, the frozen tubes were cut through the silicone oil layer and the oil was re- moved. The pellets were thawed in 450 /xl of 1 mM ethyienediamine tetraaeetatc and tt=.'entrifuged for 5 min.

The supcrnatants from HCIO 4 extracts were neutralized with 3 M KOH/0.5 M Mops to pH 7.1. For analysis of sugar phosphates and P., 25 / i l of a freshly prepared suspension of acid washed activated charcoal (205, w/v) in double distilled water were added. After 1 -2 h on ice, t i c tubes were centrifuged (5 rain) and the

412

clear supernatants used for metabolite analysis, The supernatants from NaOH extracts were heated (5 min, 100°C). Then, 50 ~1 1 M Mops (free acid) were added and, after centrifugation (5 rain), aliquots (5-25 ~tl) were used for NAD(P)H determination by enzymatic cycling. Bec:~use of the limited stability of NAD(P)H, this cycling procedure had to be performed as soon as possible after protoplast fraetionation,

Analysis of metabolites Metabolites were quantified in a two-step procedure:

first, specific assays, coupled to reduction or oxidation of pyridine nucleotides were used [7]. Subsequently, the resulting NAD(H) was amplified using the enzymic-cycling technique (for details see Ref. 7).

Glycerol-3-P was estimated according to Ref. 7. De- termination of 2-PGA, and of the ATP. ADP and AMP followed the methods previously given [8]. The other metabolites were measured as described in [9]. Usually, 50 p.l of the neutralized extracts were assayed in a total volume of 0.1 ml. In general, all assays were run in triplicate from the pooled supematants of six neutralized HCIO4-extracts.

Enzymatic reactions were stopped after 30 rain by addition of 50 #1 2 M HCI (NAD-coupled assays) or 50 /tl 2 N NaOl-I OOADH coupled assays). After heating (10 min, 95°C) and eentrifugation (3 min), 5-25 #1 of the superuatants were used for enzymatic cycling of NAD(H).

Each determination contained a standard series (5-25 /LM) of the authentic compounds. Reaction blanks were obtained by adding the extract to the reaction mixture after including 2 M HCI or 2 M NaOH. Recovery of metabolites was routinely assayed and usually 90-105~ except for TP (72 + 10~) and NADPH (75 4- 7~).

Enzymic cycling The cycling method used for NAD(H) determina-

tions resembled that described by [10]. NADP(H) was quantified by the same method with following modifica- tions: 100 mM Tris-l-ICl (pH 7.6) containing 1 mM MnSO4; substrate: 5 mM isocitrate. Alcohol dehydrogenase was replaced by 1 U isocitrate dehydrogenase.

Oxidized pyridhie nueleotides were determined from HCIO4 extracts before neutralization. Aliquo~s (5-20 ~1) were heated (5 min. 95°C) and used for the cycling procedure.

NAD(P)H was determined from NaOH extracts (20- 30/d , heated for 10 rain 95°C).

It should be mentioned that only highly purified chemicals were used. The coupling enzymes for metabolite analysis were dialyzed against 5 mM Hopes/ NaOH (pH 7.4). Buffers were prepared weekly from double-distilled, charcoal-treated water and filtered through 0.2 #M polycarbonate membranes (Sehleieher & $ehgll, BA 83, Diiren, F.R.G.) pri~r to use.

Determination of inorganic phosphate Inorganic phosphate was determined using the

malachite-green method [11]. Usually, 50 #1 of the neutralized HCIO 4 extracts were measured in a total volume of 200 ~1 at 660 nm, using a Zeiss PMQ I11 conuected to a recorder.

Assay of marker enzymes Marker enzymes were measured spectrophotometri-

cally using a Zeiss PMQ IlI (Zeiss, Oberkochem, F.R,G.) connected to a recorder, at 25°C in a total volume of 1.2 ml. Reactions were initiated by the addition of substrate.

The fumarase assay cocktail contained 100 ul extract, 100 mM Tris /aceta te (pH 7.4) and 20 mM L-malate. Enzyme activity was determined following the increase in absorption at 240 nm [12]. Chloroplast fructose-l,6- bisphosphatase was determined as in [6].

PEP-carboxylase was measured spectrophotometri- caily as previously described [13] except that 5 mM oxamate was included to inhibit interfering pyruvate consuming enzyme activities. Up to 10 mM oxamate did not inhibit PEP-carboxylase from C. reinhardtii as tested by the 14CO2 fixation assay (Ref. 13, results not shown).

Usually, marker enzyme activities had been determined within 6 h after protoplast fractionatian.

Chlorophyll was measured as described earlier [6]. Crosscontaminatian in chloroplast-enriched fractions

was corrected using the formula of Klein 15]. The ex- tsachloroplast concentration of metabolites was determined by subtraction of corrected chloroplast concentrations from proloplast values.

All enzymes and substrates were from Boehringer (Mannheim, F.R.G.). MTT, PES and malachite green were from Sigma (Miinchen, F.R.G.). All othex chemicals were at least analytical grade (Marek or Aldrich, Darmstadt and Dreieich, F.R.G., respectively).

Results and Discussion

The fra~ionation procedure Rapid lysis of the plasmalemma was achieved by

centrifugation of protoplasts through a digitonin solution follov,ed by a hydrophobic layer. Our procedure is similar to the methods of Zuurendonk and Tager [14] and Janski and Cornell [15:1 for rapid subeelhilar fraetionation of isolated liver cells.

To illustrate the principle of the fractionation proce- dut'e, the distribution of organdie markers in a typical experiment is shown in Table I. Fraction P represents the intact protoplasts. Fraction C is chloroplast enriched and contains 98% of the chlorophyll from "roto- plasts, bu! only 13% of PEP-carboxylase and 3~, of fumarase activity, respectively. Neither digitonin, nor

TABLE 1

Df~tribution of marker enzymes in protoplasts follo~ing the fractwnatwn

The dala represent the mean of six experiments and wet,,, nol corrected for crosscontamination.

Fraction PEP-earboxylase Fnmarase Fmcto~1.6-bisphosphatase Chlorophyll ~mol fraction" i h ~ t (%) ~"

Pr(( p)plasts 4.51 (100) 0.59 (1(30) 1.8g (100) 93.3 (100)

Chloropl~ts (C) 0.6 031 0.02 (3) 193(103) 97.6 (98)

• pg fraction -I.

the gels inhibited the measared marker enzyme activities.

A critical factor in cellular fractionatioa for metabolic analysis is the time between the beginning of cell rupture and the complete quenching of metabolic activities. As sho:~,'a in Fig. 2A more than 80% of the chloroplasts are quenched in less than three sec after starting the centrifuge. The time required for the method de-

'°t/ ,01: . . . . U /°1

~° % - ° - / l 0 l ' , , , O0 o . . . . 0 3 5 15 0 100 200 300 z.O0

hme {set:) Chlorophyll (Mg)

t ~ ,00 oco.o,o,o D

TomI~retufo (°CI D,g,tcn,n lmglmt)

Fig. 2. C~ract~atlon of the separati~ procedure. Separation was performed as described under Matslials and Methods, only the indi- cated parameters were varied. The F i l e t represents the chloroplast enriched fraction. (A) Effect of centrifugation time on the amount of chlorophyll (o) found in the pellet. I00~[ are 102 PB chlorophyll. (B) Effect of incubation te~aperalme oa (£3) chlorophyll and (o) PEP- ¢.athoxyl~ is the pellet f~cti~. (C) Sep~tinu ~paclt r of the microgradient shown by the effect of increa.qn 8 the load of prolo- plasts (expressed in lib ¢mc¢ophyn) on the ~.para,~on of the chlo~- plastic ~ chlorophyll) aad exttachloroplastic (o, PEP-carboxylase) fraetio~ (D) Effect of dil~toinn on ([:l) ddorophyll and (o) PEP-

car boxyl~e in the pelleL

scribed here is of the same order as for silicone-oil procedures published for the fractionation of higher plant protoplasts [16,17]. Rapid digitonin fractionation of isolated hepatooytes has been performed within 1-3 s [15]. Another critical step in a fractionation procedure is the exact calculation of extraplastidic metabelite pools as the difference between hhc a,mo'ants measured fo[ protoplasts and chloroplasts. Such a calculation is valid, if no significant change~ in metabolite levels occur during the cellular fractionation. To ensure the validity of the method, intact isola,.ed chloroplasts from C. rcinhardtii [6] were centrifuged through the C-gel i n t o

HCIO 4 in the dark. The hereby estimated levels of metabolites corresponded directly to those measured in these chloroplasts before centrifugation.

AS can be seen from the experiment without manni- tol in the C-gel (Table I1), only intact eh!oroplasts passed the silicone-oil layer.

The experimental details of the fractionation procedure, e.g., temperature, digitonin concentratim~, and the amount of protoplasm which may be ftactionated, were optimized (Fig. 2). Temperature (Fig. 2B) was the most critical factor in the separation procedure. Only above 3 0 " C was the contamination of the chloroplast-enriched fraction with PEP-c, arboxylase satisfactory small,

TABLE II

Recovery of ,awabol#~ a l ~ ¢ ~ / ~ ol ~ntact ~ , , l a * ~ e, to~-

Chloroplasts (94% intaotl were isolated as desclihed 161. Allqnots (50 BI) were eenmfuged through the C-gel and smcone oll into 3 M HCIO 4 as deseribod for protoplast~ ~ t that digitollin was omitted and 0.5% bovine serum albumin wen: added. Control measu~a,,e~ts were made with chloroplast& centrifuged through slticDne ml into 3 M HCIO4. The pellets were anal~:d as described under Matsrials and Methods.

Metabolim Corer ): c=&el C~gel (manintol omitted)

(nm~l m8 Chl - I )

p, 60.1 M.4 n.d. ATP 15.0 14.4 n.d. Fruc-6-P 8.9 8.0 n.d.

n.d. not dc0eclable.

414

but at 37 4 0 ° C many chloroplasts were damaged toe,. As can be seen from Fig. 2C, protoplasts with up to 240 /~g chlorophyll could be fractionated on one micrograoi- ent. The amount of digitonin necessary for rapid lysis of the plasma membrane was highly affected by temperature: the higher ilia temperature, the more critical was the digitonin concentration (not shown). At 3 5 ° C and with more than 50 #g Chl/gradient , 2.5 mg d ig i tonin /ml was found optimal. At lower concentrations, lysis of the plasma membrane was not quantitative, but at higher concentrations, chloroplasts were also lyzed (Fig. 2D).

Several media of similar viscosity to the 3.3% polyacrylamide gel (e.g., dextran T40, TS00, FICOLL, sucrose, bovine serum albumin, and several crosslinked polyacrylamide gels) were tested but did not give a recovery of intact chloroplasts, or else the chloroplasts s~e, reown.~ighly contaminated with cytoplasm (results not

The introduction of the low-viscosity bromodecane- silicone oil mixture as hydrophobic layer permitted fractionation with shorter centrifugation time than was possible with silicone oil alone [15].

Compartmented metabolite pools The results of the analysis of compartmented

metabolite levels during starch degradation in protoplasts of C. reinhardtii are shown in Table 11I. Most of

TABLE Ill

Metabolde levels m chloroplasts and the extrachloroplast compartment of protvplast~ ,from C. remhardtii m the dark

The protoplasts were incubated 10 rain under air or under anaerobic conditions in the dark and subsequently fractlonated as described under Materials and Methods. Melabolile levels are corrected for crosscomamination of the chloroplasts. The values are means of four independent experiments; the S.E. was between 6-24%.

Chloroplast Extrachloroplast

(nmol mg Chl - i )

Pi 55.4 46.5 11,5 280 Olu-l-P 1.4 2,3 0.l 0,2 GIu~-P 18.7 15.1 3.6 7,7 Fm-6-P 7.0 1.4 0.1 0.9 Fm-l,6-~ 4.0 9,0 1.2 G,6 TP 33 7.2 5.3 5.3 Glycerol-3-P 0.7 2.6 0,7 11.3 3-PGA 12.0 3.8 O.l t2.7 2-PGA 0.2 0.l 2.8 5.8 ATP 9.7 10.3 9.6 6,5 ADP 7.S 7.9 6.2 7.5 AMP 2.5 2.7 2.3 6.4 NAD 12.9 11.9 4.2 5.7 NADH 2.9 2.9 0,9 2.4 NADP 1.9 1.9 1.3 1,8 NADPH 0.5 0.6 1.3 1.5 R~uction state * 0,23 0.25 0.40 0,52

* Reduction slate: (NADH ÷ NADPH)/(NAD + HA[)P).

,900 ~ CYTOSOL

60°- / \ ? ' ° E

i o - -loo , - , , 300 I I

r-I CHLOROPLAST

/\ 16o- o f -o \

o/\o/ \ . z Fig. 3. Crossover plot of the change~ in subcclhSar melabolite concentrations after transition to anaerobic conditions. The data are from

Table Ill.

the sugar phosphates show significant differences in their distribution between stroma and tile extrachloroplast compartment.

Under anaerobic conditions, most of the P, (85~) was found in the stroma of C. reinhardtii chloroplasls. This result is contrary to that from higher plants, where only a small port ion of cellular Pl was localized within the chloroplasts [8]. The subcellular distribution of glycolytic intermediates coincided well with the compartmentation of the glycolytic enzyme activities in this alga [5,6]. Thus, most of Gin- l -P , Gin-6-P, Fru-6-P and Fru-l,6-P2 were detected in the stroma, whereas TP, 3-PGA and glycerol-3-P were distributed within both the chlo~Dla:;t and the extrachioroplast compartments. However, 2-PGA was measurable only in the latter. After transition to anaerobiosis, significant changes of metabolite levels and in metabolile compartmentation were observed (Fig. 3). After 5 mill under argon, levels of sugar phosphates in the stroma showed changes typical of a Pasteur-effect: metabolites produced in the glycolytic chain after phosphofructo~nase were lowered, whereas those before were increased. Pl decreased i,nder argon by 16~b. It should be mentioned that no significant changes in the energy charge or the redox state of the chloroplast could be detected. However, the ¢x- trachloroplast energy charge decreased under anaerobiosis. The reduction state outside the chloroplast rose from 0.4 (air) to 0.52 (argon), whereas in the chloroplast

it was not affected. Significant changes were observed in the distribution of glycerol-3-P, TP and 3-PGA. Thus, glycerol-3-P and 3 -PGA increased dur ing anaerobiosis outside the chloroplast. The ratios of P G A to T P were reversed ( O z / N 2 chloroplast 4.00 : 0.50, extrachloroplast 0 .92 :2 .30 , respectively). These results may be explained by the concomitant rise of the reduction st~-te and the decrease of ATP level in the extrachloroplast compartment . The regeneration of reduc- ing equivalents may be l imited without 02 .

A 3-PGA-TP-sbut t le is known for the indirect trans- por t of A T P from the cytosol into the chloroplast in the dark [18]. A T P resulting from mitochondrial respiration is required for the conversion of 3 -PGA to TP. In the chloroplast, TP can be rcoxidized to 1,3-PGA, which is the substrate for ATP-generat ion via plastidic phosphoglycerate kinase.

Unde r anaerobic conditions, eyto~olie ATP-level is too low to sustain phosphorylat ion of 3 - P G A [19] and the A T P necessary for plastidic phosphofruetokinase react ion has to be generated completely within the chloroplast. Thus, the product ion of T P as substrate for plast idic glycereldehyde-3-phosphate dehydrogenase is strictly coupled to a glycolytie flux through the plios- phofructokinase reaction and the rate o f plastidie T P oxidat ion is lowered under anaerobic conditions.

As can be seen from Fig. 3, the plastidie TP-level increased after t ransi t ion to anaerobic condit ions. whereas the level of 3 - P G A was lowered. A lower con- centrat ion of 3 -PGA may increase glycolytic flux via act ivation o f the phospofructokinase react ion [20].

415

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compartmented metabolite pools in protoplasts from the green alga chlamydomonas reinhardtii: changes...

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