Subcellular localization of phosphoenolpyruvate carboxykinase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata

Molecular and Bio chemical Parasitology, 6 ( 1982) 151 - 160 151

Elsevier Biomedical Press

SUBCELLULAR LOCALIZATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE TRYPANOSOMATIDS TR YPANOSOMA CR UZI AND

CRITHIDIA FASCICULA TA

JOAQUIN J.B. CANNATA 1 , ESTELA VALLE 2 , ROBERTO DOCAMPO 1 and JUAN JOSI~ CAZZULO 3

Cdtedra de Bioqufmica, Facultad de Medicina, Universidad de Buenos Aires. Paraguay 2155, 1121 Buenos Aires, Argentina; ~ Centro de Estudios Fotosint~ticos y Bioquimicos (CONICET- Fundaci6n Miguel Lillo - Universidad Nacional de Rosario), Suipacha 531, 2000 Rosario, Argentina; 3 lnsti tuto NacionM de Diagn6stico e Investigaci6n de la Enfermedad de Chagas 'Dr. Mario Fatala Chab~n, Ministerio de Salud l~blica y Medio Ambiente, Av. Paseo Col6n 568, 1063 Buenos Aires, Argentina.

(Received 15 February 1982; accepted 27 April 1982)

Particulate fractions obtained from Trypanosoma cruzi and Crithidia fasciculata by different procedures were subjected to isopycnic centrifugation in sucrose gradients, in order to determine the subceUular localization of phosphoenolpyruvate carboxykinase (PEPCK) in both organisms, and of malic enzyme (ME) I in T. cruzi. The more clear-cut results were obtained with T. cruzi by breaking the cells by grinding in a mortar with silicon carbide and using a gradient from 0.4 to 2.0 M sucrose, whereas with C. fasciculata, the best procedure was disruption of the cells by digitonin treatment and potter homogenization and use of a gradient from 1.1 to 2.0 M sucrose. PEPCK banded together with the glycosomal marker hexokinase in both organisms; there was a clear separation from the mito- chondrial markers, oligomycin-sensitive Mg2÷-APTase and citrate synthase. PEPCK showed a latency of 24% in the enriched 'glycosoma' fraction of T. cruzi. ME I from T. cruzi, on the other hand, banded together with the mitochondrial markers. These results indicate that PEPCK and ME are present in different subcellular compartments, a fact significant for the prevention of a futile cycle between C, - dicarboxylic acids and C 3 -monocarboxylic acids, which might take place ff both enzymes functioned in the same compartment.

Key words: Trypanosoma cruzL Crithidia faseiculata, Phosphoenolpyruvate carboxykinase, Malic enzyme, Subcellular fractionation, Glycosome, Aerobic fermentation of glucose.

INTRODUCTION

Trypanosoma cruzi, the causative agent of the American trypanosomiasis, Chagas' disease, and the insect trypanosomatid Crithidia fasciculata, catabolize glucose only partially to CO2, even under aerobic conditions, with a substantial amount of glucose

Abbreviations: MOPS, morpholinopropanesulfonic acid; PEPCK, phosphoenolpyruvate carboxykinase; ME, malic enzyme; PC, pyruvate carboxylase; CS, citrate synthase; HK, hexokinase.

0166-6851/82/0000-0000/$02.75 © 1982 Elsevier BiomedicalPress

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carbon being excreted into the medium as organic acids, mostly succinate [1 ,2] . Syn- thesis of succinate, which maintains the redox balance of the nicotinamide nucleotide coenzymes [1], requires fixation of CO2 on a C3 acid [3]. Both organisms contain an ADP-linked phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) [4-6] , which seems to be the main CO2-fLxing enzyme, presumably responsible for the CO2. fixation observed in vivo. In addition, C fasciculata, but not T. cruzi, contains a low activity of pyruvate carboxylase (PC; EC 6.4.1.1) [5-7]. However, the fact that this PC activity,

when measured under optimal conditions, is 60-fold lower than that of PEPCK [7], makes unlikely that it could be, as in other organisms, the main CO2-fixing enzyme. T. cruzi [8, 9] and C fasciculata [2, 10] also contain a NADP-linked malic enzyme (ME; EC 1.1.1.40). T. cruzi contains two isoenzymes of ME with different kinetic and re- gulatory properties [11]; ME II is activated by L-aspartate, whereas ME I is inhibited by the amino acid [11]. C. fasciculata, on the other hand, contains only one ME, activa- ted by L-aspartate [10].

From a physiological point of view, a strict regulation of the activities of PEPCK and ME is required to avoid a wasteful recycling of the C4-dicarboxylic acid, synthesized by PEPCK, back to the level of C3-monocarboxylic acid by the decarboxylation reaction catalysed by ME. Such a regulation seems to be accomplished both through a system of allosteric controls and also by subcellular compartmentation, the latter being perhaps the most important. In fact, whereas PEPCK is particulate in both trypanosomatids [6, 7, 12], the L-aspartate-activated ME, the most likely to be involved in C4-acid de- carboxylation [11], is located in the cytosol in both organisms [12]. The precise location of PEPCK and of the particulate ME I of T. cruzi has not been determined; the chief candidate organelles were the mitochondrion and a microbody-like structure, the glyco- some [13, 14]. The results presented in this paper suggest that PEPCK is present in the glycosome in both organisms studied, whereas ME I from T. cruzi is present in the mito- chondrion.

MATERIALS AND METHODS

Maintenance and growth o f the organisms. T. cruzi, Tulahu6n strain, was grown at 28°C without shaking in liquid medium described by Warren [15], except that bovine serum

was used at 4% (v/v) instead of 10%. C. fasciculata, anopheles strain (ATCC 11745), kindly supplied by Dr. S.H. Hutner of the Haskins Laboratories, Pace University, New York, U.S.A., was maintained and grown as previously described [16].

When the cultures of T. eruzi reached a cell density of about 1 × 108 organisms per ml (early stationary phase; usually after 6 days), the epimastigotes were harvested by cen- trifugation (4000 × g, 10 min) and then washed twice in 25 mM Tris-HC1 buffer (pH 7.6) containing 0.32 M sucrose, 1 mM EDTA and 4 mM KC1 (buffer 1). C fasciculata was harvested and washed in 0.25 M sucrose, 5 mM KC1 solution as previously described [16]. All harvesting, washing, disruption and fractionation procedures were carried out at 4°C except where stated otherwise.

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Cell disruption. The washed cells were disrupted by three different procedures, namely: (1) grinding with glass powder in a mortar, performed as previously described [12]; (2) grinding with silicon carbide (Crystalon) in a mortar, essentially as described by Toner and Weber [17]. The cell paste was mixed with silicon carbide (1.5 g per g cells, wet weight) and ground in an ice-refrigerated mortar for 1.5-2.0 min. The mixture was suspended in buffer 1 (12 ml per g cells, wet weight); (3) digitonin treatment, adapted from the procedure of Zuurendonk and Tager [18]. The cells were suspended in 20 mM MOPS buffer (pH 7.0), containing 0.25 M sucrose and 3 mM EDTA (buffer 2) (4 ml per g cells, wet weight) and digitonin was added (as a 40 mg/ml solution in dimethylforma- mide) to a final concentration of lmg/ml. The suspension was incubated at 20°C for 5 min, with 15 s shaking in a Vortex mixer every min. The suspension was then centri- fuged (27 000 × g, 5 min); the supematant, containing the cytosol, was discarded, and the pellet was resuspended in buffer 2 (0.8 ml per g cells, wet weight) with a potter homogenizer.

Subcellular fractionation. Differential centrifugation was carried out in a Sorvall RC-2 refrigerated centrifuge, equipped with an SS-34 rotor. The resuspended ground paste from procedures 1 and 2 was centrifuged at 100 X gfor 3 min to get rid of the abrasive. The sediment was washed once with buffer 1. The recombined supematants were cen- trifuged at 3000 rev./min (1000 × g) for 10 min. After washing once with buffer 1, the pellet containing nuclei and cell debris was discarded and the combined supernatants were centrifuged again at 11 000 rev./min (14 500 X g) for 10 min. The supernatant was discarded, the pellet was washed once with buffer 1 and then resuspended in the same buffer (0.7 ml per g cells, wet weight).

The suspension of digitonin-treated ceils (procedure 3) was centrifuged at 2500 rev./ min (600 × g) for 20 min; the pellet was discarded, and the supematant was centrifuged again at 16 500 rev./min (32 000 × g) for 30 min. The supematant was discarded and

the pellet was resuspended in buffer 2 and centrifuged again (16 500 rev./min, 30 min). The washed pellet was resuspended in buffer 2 (0.8 ml per g cells, wet weight).

lsopycnic centrifugation. Linear density gradients from 0.4 to 2.0 M sucrose or from 1.1 to 2.0 M sucrose, containing 1 mM sodium EDTA and 25 mM Tris-HC1 buffer (pH 7.4) [19] were prepared with a Gilson Minipulse peristaltic pump and layered on top of a 2.5 M sucrose cushion (0.86 ml). Samples (0.5 ml, containing 3 -10 mg of protein) of the resuspended particulate fractions obtained from T. cruzi and C. fasciculata by any of the cell disruption procedures described ('large granule' fractions [13] ) were applied to the top of the pre-cooled gradients (11.2 ml) with a 0.5 ml overlay of 0.32 M sucrose, 25 mM Tris-HC1 buffer (pH 7.8) and 1 mM EDTA. Gradients were centrifuged in a SW- 40 rotor in a Beckman L2-65B ultracentrifuge at 23 000 rev./min for 2 h (C. fasciculata) or in a 6 × 14 Titanium Swing-Out rotor in a MSE Prepsin 50 ultracentrifuge at 30 000 rev./min for 13 h (T. cruzi), at 6°C in both cases. The gradients were pumped out through a free bore metal tube from the bottom of the centrifuge tube. 15-20 fraction~ per

154

gradient were collected. The activity of total and oligomycin-sensitive ATPase was deter- mined immediately after centrifugation. All other enzyme activities were determined after dilution with an equal volume of 50 mM Tris-HCl buffer (pH 7.2), containing 0.2% Triton X-100 and freezing at -20°C (C fasciculata) or dilution with two volumes of 20 mM Tris-HC1 buffer (pH 7.6), 1 mM EDTA, and disruption by sonic disintegration in a Branson Sonifier (three treatments, 5 s each, at 50 W, with the microtip) (T. cruzi).

Sucrose concentration was determined by refractometry after dilution with water of aliquots of the fractions. The activities of PEPCK [4], ME [9], citrate synthase [20]; oligomycin-sensitive Mg2÷-ATPase [21]; succinate dehydrogenase [22]; hexokinase [23]; catalase [24]; malate synthase [25] and isocitrate lyase [26] were determined as de- scribed in the respective references. Protein was determined either by the original pro- cedure of Lowry et al. [27] or by its modification by Bensadoun and Weinstein [28], using bovine serum albumin as standard.

For the calculation and presentation of the results of isopycnic centrifugation ex- periments, the methods described by De Duve and co-workers [29, 30] were used. For comparison of density distributions, the density range covered by each fraction was standardized [30].

Latency of PEPCK in the 'glycosomal" fraction of T. cruzi. The 'glycosomal' fraction obtained from isopycnic sucrose gradients was preincubated in the reaction mixture for PEPCK, lacking oxaloacetate, or for hexokinase, lacking ATP, in the presence of 0.25 M sucrose, for 5 min at 30°C, and then the reaction was started by addition of oxaloacetate or ATP, respectively. Free activity and maximal activity were measured in the absence and presence of 0.1% Triton X-100, respectively. Latency is expressed as

total activity - free activity) tot--0~ac~-y / × 100 [31].

Chemicals. All coenzymes and substrates, and digitonin, were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. All other reagents were analytical reagents of the highest purity available.

RESULTS

Fig. 1 shows that in both T. cruzi (Fig. 1A) and C. fasciculata (Fig. 1B) PEPCK banded at the same density as the glycosomal marker hexokinase (about 1.20 g/cm 3 in the case of T. cruzi and 1.26 g/cm a in the case of C fasciculata). This peak was clearly separated from the mitochondrial peak, as defined by the activities of citrate synthase and the oligomycin-sensitive Mg2+-ATPase. In the case of T. cruzi, ME I banded at exactly the same density (1.15 g/cm 3) as the latter enzymes, thus suggesting a mitochondrial locali- zation. In the case of C fasciculata there was some discrepancy between the peak bands for citrate synthase (1.22 g/cm s) and ATPase (main peak at 1.19 g/cm3), but both of them were clearly separated from hexokinase and PEPCK (1.26 g/cm s).

155

>b 0

0

O"

i1

A

0 I

5 A T P a s e

o I - 1 , 1 ~J , , ~ , I

lO ~-

o

lO C S

O =

HK 5

0 ~ i

1.05 1.10 1.15 1.20 1.25

E q u i l i b r i u m d e n s i t y

B

,•__•, ] lO

' | ! o

i ! ! i o

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5 0

4 0 HK

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2O

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1.15 1.20 1,25 1.30

( , /om ) Fig. 1. Isopycnic centrifugation in sucrose of 'large granule' fractions from Trypanosoma cruzi and

Crithidia fasciculata. A. T. cruzi, disrupted by grinding with silicon carbide, linear gradient from 0.4 to 2.0 M sucrose. B. C. fasciculata, disrupted by digitonin treatment, linear gradient from 1.1 to 2.0 M sucrose. Density frequency distribution histograms were calculated for density increments of

0.014 g/cm 3 (T. cruzi) or 0.010 g/cm 3 (C. fasciculata). One representative experiment is shown for each flagellate. The percentages of recovery (sum of the activity of all fractions with respect to the activity of the layered samples) was 107% for protein, 118% for oligomycin-sensitive Mg2+-ATPase, 130% for citrate synthase, 81% for ME I, 103% for hexokinase and 321% for PEPCK in the case of T. cruzi; the corresponding values for C. fasciculata were 80% for protein, 173% for oligomycin- sensitive Mg2*-ATPase, 160% for hexokinase, 143% for citrate synthase and 205% for PEPCK. In the case of T. cruzi there was a pellet at the bottom of the tube, which was rich in flagella as judged by electron microscopy, and accounted for 26% of protein, and 28%; 11%; 4%; 20% and 11% of the re- covered activities of oligomycin-sensitive ATPase, ME I, citrate synthase, hexokinase and PEPCK, respectively. These values were taken into account for the calculation of frequencies. In the case of digitonin treatment of both organisms, no pellet was observed. Frequency, on the ordinate, is defined as the percentage of recovered activity in a fraction divided by the density range covered by the same fraction. The upper frame in both A and B corresponds to protein.

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The PEPCK activity present in the 'glycosomal' fraction of T. cruzi presented some

latency (24%), as shown in Table I, since full activity was not expressed unless 0.1%

Triton X-100 was present. Under the same conditions, hexokinase showed a slightly

higher latency (35.8%). Sonication was as effective as Triton in unmasking latent activ- ities (not shown).

TABLE I

Latency of phosphoenolpyruvate carboxykinase and hexokinase in the 'glycosomal' fraction of Trypanosoma cruzi

Enzyme Experimental condition Activity Latency (%) (nmol/min/ml)

PEPCK - Triton X-100 13.9 24 + 0.1% Triton X-100 18.3

HK - Triton X-100 353 35.8

+ 0.1% Triton X-100 550

PEPCK and HK were assayed in the 'glycosomal' fraction of T. cruzi as described under Materials and Methods, with the additions stated in the table.

DISCUSSION

SubceUular fractionation of trypanosomatids is a difficult problem, due to the pre-

sence o f the single mitochondrion, present as a kinetoplast-mitochondrion complex, which is disrupted even with mild breakage procedures, and can become smeared all

over the gradient when isopycnic sucrose gradient centrifugation is performed, probably due to the formation of different types of mitochondrial vesicles. For this reason, dif- ferent disruption procedures were tested in the present work. In the case of T. cruzi,

three different methods were tried. Although digitonin treatment afforded the better-

looking subcellular fractions when observed in the electron microscope (Dr. W. de Souza, personal communication), the enzyme distribution after isopycnic centrifugation was

very unsatisfactory, with most of the enzyme markers concentrating in the same fractions. The glass-grinding procedure afforded better results, but mitochondrial vesicles seemed to be distributed at least in two different bands. The pattern obtained was quite reproduc-

ible, with no significant differences in five different experiments. The silicon carbide-

grinding procedure proved to be the most satisfactory, and therefore it was used for the

latter experiments with T. cruzi, such as the representative one presented in Fig. 1A. It

is worth noting that the equilibrium densities of the peaks o f enzyme activities were the

same with both grinding procedures, but the peaks were sharper with silicon carbide. In the case of C. fasciculata, on the other hand, digitonin treatment gave much better enzyme prof'fles than glass-grinding, and therefore a representative experiment obtained

by the former procedure is shown in Fig. 1 B. In this case, silicon carbide was not tried.

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Another difficulty was the choice of appropriate marker enzymes, as previously discussed by Opperdoes et al. [32] in the case of T. brucei. Citrate synthase was chosen as a marker of the mitochondrial matrix, and succinate dehydrogenase and the oligo- mycin-sensitive Mg2+-ATPase as markers of the mitochondrial inner membrane. Suc- cinate dehydrogenase was discarded after a few experiments, since the activities were low and appeared to be scattered throughout the gradients. This might be due to inhibi- tion of the enzyme by treatments which affect the structure of the membrane. Citrate synthase showed in many cases a bimodal distribution, which suggested the possibility of the presence in these trypanosomatids of a second, glyoxysomal, location for this enzyme, as reported in the case of Tetrahymena pyriformis [33]. However, no activity of the characteristic enzymes of the glyoxylate cycle, malate synthase and isocitrate lyase, could be detected in either crude subcellular fractions or gradient-purified sub- fractions of C. fasciculata. Since about two-thirds of the citrate synthase leaks to the cytosol after grinding [7, 12], the discrepancies found in some experiments between citrate synthase and the oligomycin-sensitive Mg2*-ATPase might be explained by resealing of membranes after disruption, trapping some of the citrate synthase which had leaked out of the mitochondrion. Such an effect would not be observed, of course, with the membrane-bound ATPase, which would be a much better marker ofmitochondrial vesicles.

Catalase was tried as a microbody marker in the case of C. fasciculata (T. cruzi does not contain this enzyme; ref. 34), but most of the enzyme was solubilized upon cell disruption, and the remaining particulate catalase was scattered all over the gradient, also noted by Opperdoes et al. [19] in the case ofC. luciliae. This enzyme was discarded as a possible marker, and hexokinase, which is present in the glycosome in both T. brucei [13] and T. cruzi [14], was chosen instead.

The results presented in Fig. 1 show that in both T. cruzi and C. fasciculata PEPCK banded at the same density as the glycosomal marker hexokinase, and clearly separated from the mitochondrial peak, as defined by the activities of citrate synthase and the oligomycin-sensitive Mg2÷-ATPase. In the case of T. cruzi, ME I banded at exactly the same density as the latter enzymes, thus suggesting a mitochondrial localization.

The differences found in the equilibrium densities of glycosomal and mitochondrial particles in T. cruzi and C. fasciculata can be accounted for both by differences inherent to the different organisms and to the differences in the disruption procedures used in the representative experiments shown in Fig. 1. In fact, in experiments performed with particles obtained after grinding of C. fasciculata with glass powder, the equilibrium density of the peak of PEPCK activity ranged from 1.21 to 1.24 g/cm 3 , thus closer to the value (1.20 g/cm a) obtained for T. cruzi with the same procedure. The equilibrium density for the PEPCK activity in T. cruzi disrupted by digitonin treatment ranged from 1.21 to 1.23 g/cm 3 , thus closer to the value (1.26 g/cm 3) obtained for C. fasciculata with the same procedure. Oduro et al. [35] have reported differences in the enzyme profdes obtained after isopycnic centrifugation of subcellular fractions from T. brucei obtained by grinding or digltonin treatment.

The differences found, using essentially identical disruption procedures, between T.

158

cruzi, C. luciliae and T. brucei (this paper and refs. 17 and 36) can probably be account- ed for by differences inherent to the different organisms.

The PEPCK activity present in the 'glycosomal' fraction of T. cruzi presented a low latency (24% in the experiment shown in Table I), whereas hexokinase showed a higher degree of latency (35.8%). These values are low as compared with those for hexokinase and other glycosomal enzymes in T. brucei [31 ]. This sugge4ts that part of the.T, cruzi glycosomal population was damaged, probably due to the high hydrostatic pressures encountered during isopycnic sucrose centrifugation. It is noteworthy that in other

experiments PEPCK presented a different degree of latency, ranging from 10 to 49%. In all cases the latency of hexokinase was higher than that of PEPCK, up to 73% (not

shown). It is tempting to speculate that the difference between the degree of latency of PEPCK and hexokinase might be due to a different suborganellar localization inside

the glycosome, as recently suggested for other glycosomal enzymes in T. brucei by Opperdoes and Nwagu [37].

The results presented in this paper clearly indicate that PEPCK and ME I in T. cruzi

are placed in different sub cellular compartments. The coincidence between PEPCK and hexokinase in both T. cruzi and C. fasciculata suggests that PEPCK is placed in the glycosome in both organisms. ME I from T. cruzi, on the other hand, behaved exactly

like citrate synthase and the oligomycin-sensitive Mg2+-ATPase, thus suggesting a mito- chondrial localization. The possibility of a futile cycle between C4-dicarboxylic acids and

C3-monocarboxylic acids in trypanosomatids would be prevented, therefore, through a strict compartmentation on the enzymes involved, together with a 'fine' allosteric control, which would prevent or considerably decrease the misuse of metabolites diffused from the respective compartments.

ACKNOWLEDGEMENTS

This research was supported by funds from the Consejo Nacional de Investigaciones Cientfficas y T6cnicas de la Repfiblica Argentina (CONICET), the Subsecretarfa de Estado de Ciencia y Tecnologfa, the Ministerio de Salud Pfiblica y Medio Ambiente, and the Fundaci6n Lucio Cherny. J.J.B.C., R.D. and J.J.C. are members of the Carrera.del Investigador Cientffico of CONICET, and E.V. holds a research scholarship of the same institution.

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