Asymmetrical orientation of phospholipids and their interactions with marker enzymes in pig heart mitochondrial inner membrane

ARCHIVES OF E~OCHEMISTRY AND BIOPHYSICS Vol. 208, No. 1, April 15, pp. 305-318, 1981

Asymmetrical Orientation of Phospholipids and Their Interactions Marker Enzymes in Pig Heart Mitochondrial Inner Membrane JE:AN S. HARB, Laboratoire

a!e Biologie &’

JANE

COMTE,

AND

DANIELE

C. GAUTHERON

et Technologie des Membranes du CNRS, lJniversit6 Claude Bernard Bd du 11 Novembre 1918, 69622 Villeurbanne &dex, France Received

October

with

de LYON I,

16. 1980

The transverse distribution of phospholipids and their interactions with marker enzymes were investigated in pig heart mitoplasts and inverted vesicles, using phospholipase AZ from N. naja venom and chemical labeling with TNBS and FDNB. Morphological integrity was checked by freeze-fracturing. Fifty percent of phosphatidylcholine was hydr’olyzed in mitoplasts as well as in inverted vesicles, suggesting an even distribution of this phospholipid on the two halves of the inner membrane; however, the fatty acid distribution did not appear the same in the two membrane fractions. Cardiolipin is exclusively hydrolyzed in inverted vesicles proving its location on the inner face of the inner membrane. The results obtained from phospholipase hydrolysis and TNBS labeling suggest that three different pools of phosphatidylethanolamine occur in the membrane: a first pool-about 50-60% of the total membrane phosphatidylethanolamine-is quickly accessible from the two sides of the membrane, a second pool-about 20-30% is slowly available, and finally 20-30% are buried within the membrane and inaccessible to the phospholipase and the probe. The cytochrome c oxidase activity increased in mitoplasts with the phospholipase attack suggesting a better accessibility of added cytochrome c after the attack. The rotenone-sensitive NADH-cytochrome c reductase was activated in mitoplasts but completely inactivated in inverted vesicles by the attack; the addition of cardi’olipin liposomes restored the latter activity. The soluble matricial malate dehydrogenase was released, but the particulate form of this enzyme, strongly associated to the membrane, was detached only after attack of inverted vesicles.

This work represents a continuation of our studies on the functions of mitochondrial inner membrane isolated from pig heart. During recent years, investigations have established that enzyme activities, in membranes, are regulated by the interactions between lipid and protein components and by their uneven arrangement (l-7). We have shown in a recent work (8) that malate dehydrogenase (EC 1.1.1.37), reported to be a soluble matricial enzyme, exists in tw’o forms in pig heart mitochondria: a soluble form in the matrix fluid and a particulate form strongly associated with inner membrane. Besides, a detailed s’tudy of both lipid components and protein profiles of pig heart inner mitochondrial membrane was performed (9). It has also been shown in our laboratory

that although the outer face of inner membrane appeared rather insensitive to trypsin (lo), trypsin hydrolyzes a protein located near the outer surface of the inner membrane that is structurally involved in the oligomycin sensitivity of the ATPase complex. The present study proves on the one hand, that phospholipids are unevenly distributed in pig heart inner mitochondrial membrane, and on the other hand, that the particulate form of malate dehydrogenase as well as the rotenone-sensitive NADH-cytochrome c reductase (EC 1.6.99.3) are tightly associated with cardiolipin, and to a lesser extent, with phosphatidylcholine. It describes experiments on the release of enzymes and phospholipids from pig heart mitoplasts (inner membrane right side out) and inverted 305

0003-9861/81/050305-14$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form resewed.

306

HARB,

COMTE,

vesicles (inside out), as a function of mild attacks by phospholipase A2 from N. naja venm. In parallel the structural integrity of inner membrane was studied by freezefracturing. Besides, a chemical labeling of phosphatidylethanolamine, in mitoplasts and inverted vesicles, was performed with TNBS and FDNB.’ The use of phospholipase A2 from pig pancreas was finally avoided in this study as its attack appeared too drastic for the structural integrity of the particles and since it penetrated through the membrane. A preliminary account of this work has been presented at the 11th International Congress of Biochemistry, Toronto (11). MATERIALS

AND

METHODS

Phospholipase Az from N. naja wenom (EC 3.1.1.4) (specific activity 1000 units/mg protein), fatty acidfree bovine serum albumin, paminobenzamidine (PAB), c-amino-n-caproic acid (EACA), and 2,4,6trinitrobenzene sulfonic acid (TNBS) were purchased from Sigma Chemical Company. Phenylmethane sulfonyl fluoride (PMSF) was obtained from Merck. Butylbenzene and 1-fluoro-2,4-dinitrobenzene (FDNB) were purchased from Fluka. Membrane preparations. Pig hearts obtained from the slaughterhouse and brought to the laboratory in a chilled medium, consisting of 0.25 M sucrose, 10 mM KHzPO,, 1 mM EGTA, and 20 mM KCl, pH 7.6, were used within 60 min after the electrocution of the animals. Mitochondria were isolated in 0.25 M sucrose, 10 mM phosphate, 1 mM EGTA, 0.05% bovine serum albumin, pH 7.4, by a procedure derived from that of Crane et al. (12), washed twice, and tested for respiratory control ratios, protein concentration, and ADP/O, as previously described (13). Purified mitoplasts were obtained according to Mai’sterrena et al. (14): washed mitochondria (400 mg of protein) were homogenized in 400 ml of 10 mM potassium phosphate at pH 7.4 and O’C and allowed to swell for 20 min. Then the suspension was centrifuged at 105,OOOg for 60 min. The pellets were resuspended in 0.25 M sucrose, 10 mM Tris-HCl at pH 7.4 and centrifuged at 11,500g for 15 min in order to separate the outer membranes from the mitoplasts (inner membranes and matrix). ’ Abbreviations used: TNBS, 2,4,6-trinitrobenzene sulfonic acid; FDNB, 1-fluoro-2,4-dinitrobenzene; PE, phosphatidylethanolamine; BSA, bovine serum albumin; PAB, paminobenzamidine; EACA, c-aminon-caproic acid, PMSF, phenylmethane sulfonyl fluoride; BAEE, a-N-benzoyl arginine ethyl-ester.

AND

GAUTHERON Inverted vesicles were prepared according to Mdirouch and Godinot (10). Mitoplasts were homogenized at 0°C in 0.25 M sucrose, 10 mM Tris-HCl at pH 7.4 at 20 mg of protein/ml and sonicated three times for 10 s each time in 5-ml aliquots with a Branson Sonifier B12. Care was taken to prevent the temperature from raising above 8°C during sonication. Unbroken mitoplasts were spun down at 25,OOOg for 10 min and sonicated vesicles were collected after centrifugation at 78,OOOg for 90 min and the pellets suspended in 0.25 M sucrose, 10 mM Tris-HCl at pH 7.4 and 0°C; over 90% of these vesicles have an inverted polarity of inner membrane since they are not retained on a Sepharose-cytochrome c column (15). Marker enzymes. Cytochrome c oxidase (EC 1.9.3.1) was estimated by the disappearance of 0.05% reduced cytochrome c in 0.1 M phosphate buffer (pH 7.4) at 550 nm and 30°C. Rotenone-sensitive NADH-cytochrome c reductase was measured spectrophotometrically at 550 nm and 30°C by the reduction of 60 pM cytochrome c in the presence or not of 1.5 PM rotenone (16). Malate dehydrogenase was determined spectrophotometrically at 30°C (17) by the decrease in absorbance at 340 nm in the presence of added 0.11 mM NADH, 1 pM rotenone, and 80 FM NazS; to stimulate enzyme activity, a pretreatment with 0.02% Triton X-100 of membranes was performed for 5 min. ATPase activity was determined by a spectrophotometric method using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes (18) in a medium containing 40 mM Tris-SO,, 3 mM MgSO&, 4 mM phosphoenolpyruvate, 0.3 mM NADH, 2 mM NazS, 2 mM ATP. Protein. This was estimated by the Lowry procedure (19). Digestion of membranes by phospholipase A2 from N. naja venom. Incubations were carried out as described by Nilsson and Dallner (5) at 30°C in the presence of 50 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mg of membrane protein (final volume: 1 ml). In some cases, the incubation medium contained also 50 mg defatted bovine serum albumin/ml. The digestion was started by the addition of 7 units of phospholipase. At different time intervals, the reactions were stopped by the addition of 15 mM EDTA. After centrifugation at 100,OOOg for 45 min, lipid determination and marker enzyme analyses were performed on pellets and supernatants. The preparations of phospholipase A2 from N. naju venom were checked for the absence of contamination by proteolytic enzymes: first, no trypsic activity could be detected with a-N-benzoyl arginine ethylester (BAEE) as substrate; second, addition of protease inhibitors-2 mM PMSF, 10 mM PAB, and 10 mM EACA-in the incubation medium, did not influence the results of digestion. Rebinding of lipids to lipid-de&cient preparations. Dispersion of lipids was performed according to Feo

LIPID-PROTEIN

INTERACTIONS

IN HEART

et al. (6): 8 ml of a medium containing 20 mM TrisHCI (pH 8) and 1 mM EDTA were added either to 75 mg of lipids (Folch’s extract) or of cardiolipin or of phosphatidylet:hanolamine or of phosphatidylcholine. After vortexing for 15 min, the dispersions were sonicated for 10 min under nitrogen bubbling with a Branson Sonifier B12. Care was taken to prevent the temperature from raising above 8°C and to avoid foaming. The suspensions were centrifuged for 18 min at 114,OOOg. Clear supernatants were taken as the source of 1:ipid vesicles (about 10 pmol of phospholipid/ml). For the lipid-rebinding experiments, phospholipase AZ-treated mitoplasts (about 100 pg of protein), suspended in 0.88 M sucrose, 10 mM Tris-HCl (pH ‘7.4), were added directly into the spectrophotometric cuvettes containing the different reaction mixtures needed to the enzymatic determinations. After a lomin incubation at 3O”C, with various amounts of added lipid dispersions, enzymatic activities were initiated by additions of the respective substrates. Labeling with TNBS and FDNB. Mitoplasts or inverted vesicles (10 mg of protein) were incubated at 23°C in a medium containing 50 mM KCl, 0.1% albumin, 100 mM NaHC03, 0.25 M sucrose, pH 8.2. The reaction was initiated by TNBS or FDNB additions; final’volume was 10 ml. After the incubation period, the reaction was stopped by acidification of the medium to pH 6.8 and centrifugation for 20 min at 40,OOOg. The supernatant fluids were discarded and the pellets washed twice with 10 ml of the suspending medium without the probes. Lipid extra&on, phospholipid determination, and fatty acid analysis were conducted as previously described (9). Freeze-fracture electron microscopy. Membrane fractions were spray-frozen in liquid propane (20). After evaporation of propane, frozen particles were bound with n-butylbenzene at -85”C, transferred to gold-nickel holders, and rapidly cooled in liquid nitrogen. Fracturing was done at -150°C and l-2 X 10e6Torr in a Balzers BAF 300 apparatus equipped with an electron-beam ‘evaporation device and a quartz crystal thin-film monitor. After Pt/C shadowing, replicas were cleaned in chromic acid, washed in distilled water, and mounted on uncoated 400-mesh copper grids. Electron micrographs were performed, at the Centre de Microscopic Electronique Appliqube 1 la Biologie, de l’Universit.8 Claude Bernard de Lyon, using Philips E:M 300 (at 80 kV) and Hitachi HU 12 (at 75 kV) elec,tron microscopes. The freeze-etching nomenclature according to Branton et al. (21) was used to describe the different fracture faces. For quantitation of intramembranous particles, micrmographs (initial magnification 40,000 or 60,000) of different replicas were printed at a final magnification of 260,000. Particles were counted on

MITOCHONDRIAL

INNER

307

MEMBRANE

small surfaces in the central part of the various fracture faces, to minimize errors due to the membrane curvature. The mean value of the number of particles per unit of membrane surface (1 pm’) and the standard deviation of the mean were calculated.

RESULTS

The absence of outer membranes and the intactness of the mitoplasts were controlled as before (8) by the lack of monoamine oxidase activity, by the detergent stimulation of malate dehydrogenase and cytochrome c oxidase activities, by electron microscopy, and by freeze-fracture replicas (Fig. 2). Likewise, the opposite orientation of inverted vesicles was checked as previously by measuring the activities of marker enzymes in the presence or not of detergents (8, 10) and by freeze-fracture replicas (Fig. 3). Table I shows that the distribution of main phospholipids in inverted vesicles was the same as in mitoplasts. We will see later (Table II) that the distribution of fatty acid chains in phosphatidylcholine is also the same both in mitoplasts and inverted vesicles. Therefore sonication did not induce important alterations of membrane structure. TABLE PHOSPHOLIPID MITOPLASTS

I

DISTRIBUTION AND INVERTED

IN PIG HEART VESTICLES

Percentage of total phosphorus”

Phospholipid Phosphatidylcholine

Inverted vesicles

Mitoplasts 35.2

f 1.2

33.8 +- 2.1

(20) Phosphatidylethanolamine Cardiolipin

32 + 2.3

(20) 19.7 f

1.6

(20) Minor

components

10.5 f

1.5

(20) ‘Values parentheses,

are followed by standard number of determinations.

(6) 34.5

k 1.2

(6) 18.1 + 0.7

(‘3 10 f 1.2

(6) deviation.

In

308

HARB,

COMTE,

AND

GAUTHERON

TABLE FATTY

II

ACID DISTRIBUTION IN PHOSPHATIDYLCHOLINE AFTER A 30-min TREATMENT MITOPLASTS AND INVERTED VESICLES BY PHOSPHOLIPASE Az FROM N. Naja Mitoplasts’

Fatty

acids

Control

Inverted

Phospholipqse treated*

Control

OF PIG HEART venom vesicles” Phospholipase treated*

Cl40 c15:o C16br C16:O Cl&l c17:o ClSbr C18:O Cl&l C18:2 C18:3 C20:4

2.8 10.4 1.7 22.6 1.6 0.5 1.7 6.7 14.0 29.9 1.4 5.1

2.9 6.4 2.0 19.0 2.5 1.7 1.9 12.0 17.5 25.8 1.9 4.9

2.1 6.0 1.4 23.5 1.7 1.0 0.9 8.1 15.2 32.1 1.5 4.9

10.1 3.3 2.9 47.7 2.5 1.4 0.7 14.0 2.3 11.0 1.1 2.9

Z Unsaturated

52.0

52.6

55.4

19.8

a Fatty acid methyl esters were analyzed on a 10% diethylene glyeol succinate are expressed as percentages of recording area of the sum of fatty acids. *For experimental conditions, see legend of Fig. 1.

Effects of Treatment by Phospholipase A2 from N. naja Venom on the Phospholipid Distribution in Mitoplasts and Inverted Vesicles Figure la shows that the attack of phosphatidylethanolamine in mitoplasts was biphasic. In 2 min, about 50% of this phospholipid were hydrolyzed; then, during the following 10 min, the degradation was slower and after 15 min, a plateau was reached. In contrast, a rapid and maximal hydrolysis of phosphatidylcholine (4550%) was attained in about 5 min and after this time, no more degradation occurred. Cardiolipin was not altered at all, even after a 45-min attack; however, it was checked that cardiolipin in liposomes was hydrolyzed by this enzyme in the same conditions (not shown here). The presence of bovine serum albumin (BSA) which is known to bind fatty acids and lysophospholipids, slowed down the hydrolytic attack of phosphatidylethanol-

polyester

column.

Values

amine, but finally only slightly protected phosphatidylcholine and -ethanolamine against hydrolysis. Figure lb shows that the rate of hydrolysis of phosphatidylcholine in inverted vesicles was slower than it was in mitoplasts (20% at 5 min). It reached a maximum of 50% after 15 min incubation. The cardiolipin was progressively attacked and after 60 min about 50% were hydrolyzed, although this phospholipid is a rather poor substrate for the N. naja venom phospholipase; if inverted vesicles are incubated for a longer time with phospholipase (2 h), about 90% of cardiolipin can be hydrolyzed. The attack of phosphatidylethanolamine in inverted vesicles was markedly biphasic, about 50-60s being very rapidly hydrolyzed while the remaining phosphatidylethanolamine was slowly attacked. Apparently, two pools of phosphatidylcholine exist: one pool directly available for phospholipase AZ attack and a second

LIPIDPROTEIN

INTERACTIONS

15

IN HEART

30

MITOCHONDRIAL

INNER

MEMBRANE

309

45 TIME

FIG. 1. Effects of treatment by phospholipase A2 on phospholipid distribution in mitoplasts (a) and inverted vesicles (b). Incubations were performed in the absence (open symbols) or in the presence (solid symbols) of bovine serum albumin (50 mg/ml) in 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose, 10 mg of membrane protein; final volume: 1 ml. Reactions were started by the addition of 7 units of phospholipase Ae. The values in ordinate are given as percentage of the total phosphorus which is recovered in the pellet after treatment, relatively to the control and are means of two determinations. (0) Phosphatidylethanolamine; (A) phosphatidylcholine; (0) cardiolipin.

pool not easily degradable. Do these pools have a different composition in terms of molecular species of phosphatidylcholine? Table II shows that no noticeable changes occurred in the fatty acid distribution in mitoplast phosphatidylcholine after phospholipase attack; in contrast, the content of unsaturated Cl8 acids-mainly oleic acid-strongly decreased after phospholipase treatment in inverted vesicles, inducing a high increase in the relative distribution of Cl6 and Cl8 saturated acids.

Morphological

Studies

The appearance of pig heart mitoplasts (prepared b;y the hypotonic method (14)) in freeze-fracture replicas is shown in Figs. 2a-c. Besides, we have determined that the convex fracture or the protoplasmic (21)1 fracture faces (Fig. 2b) contain a high density of intramembranous particles (3516 + 259/pm2, 27 determinations) with ,a diameter of 70 A, while the concave fracture or exoplasmic (21) fracture faces (Fig. 2a) contain similarly sized particles but in much lower density (1898 + 9’7/pm2, 20 determinations). After a 15-min treatment with phospholipase (Fig. 2d), the spherical aspect of mito-

plasts was preserved; but their size was somewhat higher, which is in agreement with some unfolding of inner membrane. There was an increase in the relative occurrence of cross sections as compared to fracture faces, as it can be expected if the outer phospholipid layer is disorganized. In contrast to well-oriented mitoplasts, the concave fracture faces of inverted vesicles contain a high density of intramembranous particles (4665 + 387 particles/ pm2, 16 determinations), while the particle density in the convex fracture faces is twofold lower (Fig. 3a): 2191 + 170 particles/ /*m2, 30 determinations. It should be remembered that the concave fracture faces of inverted vesicles correspond to the convex fracture faces in normal mitoplasts, i.e., to the matrix side of the inner membrane. The analysis of the frequency of the occurrence of membrane faces shows slightly more concave than convex fracture faces. Cross sections (Fig. 3b) showed that the soluble matrix proteins of mitoplasts are largely removed during the preparation of inverted vesicles. The statistical study (40 fracture faces) revealed a vesicle diameter between 0.11 and 0.15 pm. Inverted vesicles appeared very sensitive to phospholipase A2.

310

HARB,

COMTE,

AND

GAUTHERON

FIG. 2. Freeze-fracture appearance of control pig heart lipase AZ treatment for 15 min (d). X80,000. (a) Concave convex fracture face or exoplasmic face; (c) cross section.

If the treatment did not induce important morphological changes after 5 min (Fig. 3c), however, after 15 min (Fig. 3d), the cardiolipin hydrolysis was accompanied by a disappearance of fracture faces, the formation of protein aggregates.

Labeling

with TNBS and FDNB

The second approach for studying the phospholipid arrangement in the mem-

mitoplasts fracture

(a-c) and effects of phosphoface or protoplasmic face; (b)

brane was the comparative labeling of phosphatidylethanolamine by the reputed slowly penetrating probe TNBS and the fast-penetrating probe FDNB (22-27). We see in Fig. 4a, that as the concentration of TNBS increased, the amount of phosphatidylethanolamine labeled after 3 h in mitoplasts also increased until it reached a plateau at approx 0.5-0.7 mM TNBS, for which about 82% of the to-

LIPID-PROTEIN

INTERACTIONS

FIG. 3. Freeze-fracture appearance effects of phospholipase A2 treatments. section. Treatments by phospholipase

IN HEART

MITOCHONDRIAL

INNER

of inverted vesicles prepared from pig heart X194,750. (a) Concave and convex fracture for 5 min (c), 15 min (d).

MEMBRANE

mitoplasts and faces; (b) cross

311

312

HARB,

;”

COMTE,

b

P

0.6 [ TNBS]

or [FDNB]

0.9

1 1.2

(mM)

FIG. 4. Labeling of phosphatidylethanolamine by TNBS and FDNB in pig heart mitoplasts (a) and inverted vesicles (b) in correlation with the effects of TNBS and FDNB on ATPase (c) and malate dehydrogenase (d) activities of pig heart mitoplasts. The values, in ordinate, represent the percentage of phosphatidylethanolamine reacted relative to the untreated control (lower part) or the percentage of enzymatic activities measured in mitoplast pellets relative to the unreacted mitoplasts (upper part). Membrane fractions were incubated for 3 h in the presence of increasing concentrations of TNBS (0) or FDNB (B) as described under Materials and Methods; means of two determinations.

tal phosphatidylethanolamine has reacted with the probe. At even higher TNBS concentrations, the labeling was increased again (about 95% of the total phosphatidylethanolamine being labeled at 1 mM TNBS). These results indicate that in intact mitoplasts, the high concentrations of TNBS facilitate the penetration of the probe. With inverted vesicles (Fig. 4b), the profile is different: at low TNBS concentrations (0.1-0.2 IIIM), a noticeable amount of phosphatidylethanolamine (about 55% ) was labeled after 3 h, then the labeling

AND

GAUTHERON

increased as a function of the concentration; but at 0.6 IIIM TNBS, maximal labeling of about 85% was reached. In contrast, the amount of phosphatidylethanolamine in mitoplasts reacting with FDNB (Fig. 4a) reached a maximum (about 95% of total phosphatidylethanolamine) at 0.6 mM FDNB. The extent of labeling above this concentration remained constant. In inverted vesicles (Fig. 4b), the phosphatidylethanolamine labeling by FDNB was low-even lower than with TNBS-and no plateau was reached as in mitoplasts. Control experiments concerning the penetration of both reagents into mitoplasts have been conducted. We see in Fig. 4, that the matricial soluble enzyme-like malate dehydrogenase (interior vesicular space) is affected only by high concentrations of TNBS after a 3-h reaction, while the penetrating FDNB immediately affected the major part of the activity (Fig. 4d). Even with the transmembrane ATPase-ATPsynthase, high concentrations of TNBS were necessary to affect the enzyme (Fig. 4~). In contrast the penetration of FDNB rapidly abolished all the activity even at low concentrations. In Fig. 5, we see the kinetics of phos-

TlYE(min)

FIG. 5. Kinetics of phosphatidylethanolamine labeling by TNBS in mitoplasts and inverted vesicles. The values, in ordinate, represent the percentage of phosphatidylethanolamine reacted relative to the untreated control. 10 mg protein of mitoplasts (0) or inverted vesicles (m) were incubated at 23°C in the presence of 0.6 mM TNBS. At different times, the reaction was stopped as described under Materials and Methods.

LIPID-PROTEIN

INTERACTIONS

IN HEART

phatidylethanolamine reaction with TNBS in the membranes. At 0.6 mM TNBS, 50% of phosphatidylethanolamine were labeled in mitoplast,s and 60% in inverted vesicles during the first 5 min. After this time, the labeling was slower, with both types of membrane preparations and reached a plateau, within 30 min. About 25% of phosphatidylethanolamine have not reacted with the probe in mitoplasts and somewhat less in inverted vesicles.

Effects of Phospholipase A2 Treatments on Inner Membrane Marker Enzymes Figure 6a shows that the preincubation of mitoplasts at 30°C in the absence of phospholipa.se induced a slight activation of cytochrome c oxidase in the first min. After 15 min, the activation decreased and reached the basis value at 30 min. In presence of phospholipase, the activation of the enzyme was twice higher than it was in the control. In inverted vesicles (Fig. 6b) a similar activation of the enzyme activity was observed during the preincubation but it was not affected by phospholipase treatments. Phospholipase induced, in mitoplasts, a high increase of the rotenone-sensitive NADH-cytochrome c reductase activity (sevenfold the control value in 10 min), but after 15 min, the activation rapidly decreased (Fig. 7a). A high increase of rotenone-sensitive NADH-cytochrome c re-

MITOCHONDRIAL

INNER

MEMBRANE

ductase activity was also observed in control inverted vesicles, while phospholipase treatments completely abolished the activity (Fig. 7b). The total NADHcytochrome c reductase activity (sum of rotenone-sensitive and insensitive activity) exhibited, in both cases, a similar profile (not shown). No enzyme activity was detected in supernatant fluids after phospholipase treatment (not shown), in normal mitoplasts as in inverted vesicles. We know that phospholipase treatment in inverted vesicles hydrolyzes cardiolipin as well as other phospholipids. After incubation of inverted vesicles with N. naja venom, the addition of total mitochondrial lipids (Folch’s extract) not only restored the rotenone-sensitive NADH-cytochrome c reductase activity, but also produced an extrastimulation (Fig. 7~); the addition of cardiolipin liposomes (0.2 to 1 pmol) increased by 1.5 to 2-fold the enzymatic activity; in contrast phosphatidylethanolamine liposomes were without effect. The effects of phospholipase AZ treatment on malate dehydrogenase are shown in Fig. 8. The preincubation of mitoplasts at 30°C in the absence of phospholipase induced an increase in malate dehydrogenase activity which can still be stimulated by Triton (Fig. 8a). A rapid release of the enzyme was observed after phospholipase treatment; about 10 to 20% of the enzymatic activity remained bound to the membrane, thus indicating that the

TIME (min)

FIG. 6. Elffects vesicles (b). For (A) phospholipase;

313

of phospholipase A2 on cytochrome c oxidase activity in mitoplasts (a) and inverted experimental conditions, see the legend of Fig. 1. Incubations without (0) or with means of two determinations.

314

HARB,

pmol

COMTE,

2 01 phosphohpid

AND

GAUTHERON

n

4 added

b

3

15

30

45

\ A

15

30

45

FIG. 7. Effects of phospholipase A2 on rotenone-sensitive NADH-cytochrome c reductase activity in mitoplasts (a) and inverted vesicles (b) and effects of lipid additions to phospholipase-treated inverted vesicles (c). For experimental conditions, see the legend of Fig. 1: incubations without (0) or with (A) phospholipase. In the experiments shown on curve (c), inverted vesicles were treated for 15 min with phospholipase; pellets were suspended in 0.88 M sucrose, 10 mM Tris-HCl (pH ‘7.4), and aliquots of lipid dispersions (Folch’s extract) were added (O), as described under Materials and Methods. Untreated vesicles were used as the source of native enzyme (+). Means of two determinations.

particulate form of malate dehydrogenase is resistant to phospholipase attack, in these conditions. The lost enzymatic ac-

tivities were almost completely recovered in supernatant fluids (not shown here) and correspond to matricial enzyme. In con-

15 _ a

b

TIME ( mid

FIG. 8. Effects inverted vesicles nations.

of phospholipase (b). Incubations

A2 on malate dehydrogenase activity without (0) or with (A) phospholipase;

in mitoplasts means of two

(a) and determi-

LIPID-PROTEIN

INTERACTIONS

IN HEART

trast, the preincubation of inverted vesicles at 30°C in the isotonic medium in the absence of phospholipase induced, after 5 to 10 min, some decrease of malate dehydrogenase activity from the membrane; after phospholipase treatment, a greater decrease of the activity was observed; only 3% of the activity was measured after 30 min indicating that an inactivation of the particulate enzyme occurred, as cardiolipin hydrol.ysis proceeded (Fig. 8b). As in mitoplasts, the major part of the released enzyme was recovered in supernatant fluids (not shown here). DISCUSSION

The purpose of this work was to study on the one hand the phospholipid orientation in the mitochondrial inner membrane and on the other hand, the interactions between the phospholipids and some marker enzymes using mitoplasts and inverted vesicles. Our pig heart mitoplasts show a good structural appearance, in freeze-fracturing. The particle density in the two fracture faces (protoplasmic and exoplasmic) are in good agreement with the data of Packer et al. (28) for beef heart mitochondrial membranes. In inverted vesicles, the matrix face of inner membrane appeared well oriented to the outside. Indeed, in mitoplasts, the convex fracture face which corresponds, to the half-membrane facing the matrix, has a high particulate density; in contrast, this high particulate density was seen on. the concave fracture faces in inverted vesicles. In contrast to Packer et a2. (28), more concave fracture faces were observed in inverted vesicles, but no special explanation can be given. The profiles of phospholipid hydrolysis in mitoplasts and inverted vesicles suggest that phosphatidylcholine is evenly distributed and well compartmented between the two halves of the membrane: the same percentages of 50% hydrolysis of this phospholipid are obtained from both types of membrane preparation, although the attack is somewhat slower in inverted vesicles. The fatty acid chains involved in the

MITOCHONDRIAL

INNER

MEMBRANE

315

fraction being on the inside of the membrane appear more unsaturated than those involved in the external phosphatidylcholine fraction. Cardiolipin is exclusively located on the inner half of the inner membrane; indeed, this phospholipid is only hydrolyzed in inverted vesicles and not in mitoplasts which proves that the phospholipase does not penetrate through the membrane. This last result differs from previous data (5,29), where the authors localize cardiolipin on the inner face of the inner membrane, although they observe some cardiolipin degradation in their “right side-out” oriented membranes attacked by phospholipase AZ; this might be due to the fact that they use higher concentrations of phospholipase (5,29) in the presence of CaC12 (29). As to phosphatidylethanolamine location, the results obtained with labeling experiments as well as with the biphasic phospholipase attacks are in favor of the presence of different pools of this phospholipid in the inner membrane. Indeed, about half of phosphatidylethanolamine (50-60%) is quickly hydrolyzed by phospholipase AZ and less rapidly labeled with the slowly penetrating probe TNBS (in 5 min) in mitoplasts as well as in inverted vesicles, suggesting that a pool of phosphatidylethanolamine is easily accessible in each half of inner membrane. Moreover, the results with marker enzymes seem to show that no enzymatic activity is dependent on this pool of phosphatidylethanolamine. A second pool (about 20-30%) is less available to reaction with TNBS and phospholipase from either side. It is slowly attacked in 10 to 15 min, but at this time, freeze-fracture replicas show that the membrane was ruptured in the case of inverted vesicles or that the outer monolayer of phospholipids was disorganized in the case of mitoplasts. Thus, the phospholipase or the reagents become accessible to the unmasked pool. A third pool (20-30%) is inaccessible to both types of treatments in “right side-out” as well as “inside-out” oriented membranes. The comparative studies between phospholipase hydrolysis and TNBS labeling

316

HARB,

COMTE,

AND

of phosphatidylethanolamine seem to exclude the probability of a rapid TNBS penetration through the membrane, since the phospholipase attack is more rapid than the reaction with TNBS, as shown also by Bishop et al. (30) with protoplast membranes of Bacillus subtilis. Control experiments on the effects of TNBS on marker enzymes (malate dehydrogenase, ATPase) are in good agreement with this conclusion. The incomplete labeling of phosphatidylethanolamine with TNBS and FDNB in inverted vesicles can be explained in view of the studies of Bishop et al. (31) with monolayers: this might be due, at least partly, to the presence of negatively charged phospholipids such as cardiolipin. This incomplete labeling indicates that the use of chemical labels in the study of lipid asymmetry of biological membranes must be approached with great caution (31). These conclusions are in agreement with the results of Crain and Marinetti (27) on rat liver mitoplasts labeled with TNBS. Previously, Nilsson and Dallner (5) have concluded that 90% of the total phosphatidylethanolamine was localized on the external surface of the inner membrane of rat liver mitochondria, by using phospholipase Az treatments; but unfortunately, the first point in the kinetics of these authors was 10 min, and at this time, practically, the major part of phosphatidylethanolamine is already degraded. In a very recent paper (29) which appeared at the same time as our preliminary report (ll), the phospholipase Az from N. naja venom was also used to determine the asymmetric orientation of phospholipids in beef heart mitochondrial membrane. In this work, the disorganization of either the structure or the functions was not studied. Cardiolipin was hydrolyzed in mitoplasts while in our conditions, it is not affected, which permits its precise location and obviously proves that the membrane does not become permeable to the phospholipase. This is the most important prerequisite when using phospholipases in sidedness experiments. Other results of these authors differ from ours: they locate phosphatidyl-

GAUTHERON

choline rather on the outer half of inner membrane and phosphatidylethanolamine in a more internal position; but they also observed an attack of both these phospholipids by the phospholipase either from the outer or the inner face of the membrane. Several reasons can explain the differences between their results and ours: we work at 30°C instead of 37°C and in the absence of Ca2+ to avoid membrane disorganization, with lower concentration of phospholipase in the presence of twofold higher concentration in proteins. Besides, we work with pig heart vesicles instead of beef and we do not use the same type of preparation for mitoplasts: ours keep a good structural integrity as shown in freeze-fracturing. The decrease of fracture faces (convex or concave) after 15-min treatments of mitoplasts with phospholipase from N. naja venom and the increase of cross sections are in agreement with the hydrolytic profiles of external phosphatidylethanolamine (biphasic) and -choline. In contrast to Packer et al. (28) with beef heart, the spherical structure of mitoplasts is preserved in our preparations and no aggregated particles could be seen. The appearance of aggregates and clusters of particles in phospholipase-treated inverted vesicles is concomitant with the disappearance of cardiolipin. The role of this phospholipid thus appears very important for the hydrophobic environment and in the maintenance of the smooth internal particlefree areas in the fracture faces. It seems difficult to correlate the appearance of clusters and aggregates to the hydrolysis of phosphatidylethanolamine in view of the effects of phospholipase on the structure of mitoplasts and inverted vesicles. After a 15-min treatment, the same amount of phosphatidylethanolamine is hydrolyzed in mitoplasts or inverted vesicles, although the morphological disturbance is very different, and besides, at this time, if no cardiolipin at all was hydrolyzed in mitoplasts, already about 25% of cardiolipin was degraded in inverted vesicles. The activation of cytochrome c oxidase, in mitoplasts, after phospholipase treat-

LIPID-PROTEIN

INTERACTIONS

IN HEART

ment, suggests that the added reduced cytochrome c is more accessible to the enzyme after phospholipid hydrolysis. Moreover, the degradation of about 60% of phospholipids and particularly of a part of cardiolipin in inverted vesicles does not induce any loss of activity of cytochrome c oxidase. These results are in agreement with the transmembrane location of the enzyme and suggest that this enzyme is not affected by the general distribution of phospholipi’ds but only by a small specific fraction: the phosphatidylcholine and cardiolipin which are not hydrolyzed and thus must be protected, may be forming an annulus around the enzyme. The rotenone-sensitive NADH-cytochrome c reductase seems strongly dependent on cardiolipin: in mitoplasts, the phospholipase induces an activation of the enzyme probably due to a better accessibility of NADH, and of cytochrome c, as in the case ad cytochrome c oxidase but the degradation of cardiolipin in inverted vesicles is followed by the rapid loss of enzymatic activity (Fig. 7b). Moreover, the addition of pig heart mitochondrial total lipids or of cardiolipin liposomes restored the rotenone-sensitive NADH-cytochrome c reductase activity which was inactivated by lipid depletion. This is well in agreement with the presence of a phospholipid annulus around the enzyme (32) but with an exclusive cardiolipin requirement indicating a major orientation in the inner half of inner membrane. In any case, the observed effects cannot be due to proteolytic enzymes contaminating the phospholipase preparations since their absence has been controlled. In contrast with us, Machinist and Singer (33) reported that the sensitivity of the NADH-cytochrome c reductase to rotenone was unaffected by phospholipa.se treatment. In agreement with our results, Awasthi (34) related a partial release of NADH-dehydrogenase from beef heart electron particles, concomitant to cardiolipin hydrolysis. The release of malate dehydrogenase, a reputed soluble matricial enzyme from intact mitoplasts, might be related to protein movements similar to those described in rat liver mitochondria (35,36); however

MITOCHONDRIAL

INNER

MEMBRANE

317

20% of the activity is always detected in the membrane; this proves as we reported before (8) that the particulate form of malate dehydrogenase is strongly associated with the inner membrane. However this particulate form is sensitive to phospholipase in inverted vesicles, since only 3% of the total activity remained after phospholipase attack, and was not completely recovered in supernatant fluids, indicating a denaturation of the enzyme. All these results show that the particulate malate dehydrogenase is strongly associated to the mitochondrial inner membrane and tightly dependent on phospholipids, especially cardiolipin. ACKNOWLEDGMENTS We thank E. Frey for the photographs and drawings and B. Duclot for producing a good manuscript. Financial support was obtained from the Centre National de la Recherche Scientifique and from the DBlkgation G&&ale % la Recherche Scientifique (contract Membranes Biologiques 77-7-02’77). REFERENCES 1. ZWAAL, R. F. A., ROELOFSEN, B., AND COLLEY, C. M. (1973) Biochim. Biophys. Acta 300,159182. 2. HARMON, H. .I., HALL, J. D., AND CRANE F. L. (1974) Biochim. Biophys. Acta 344, 119-155. 3. COLEMAN, R., AND BRAMLEY, T. A. (1975) Biochim. Biophys. Acta 382, 565-575. 4. NILSSON, 0. S., AND DALLNER, G. (1975) FEBS Lett. 58, 190-193. 5. NILSSON, 0. S., AND DALLNER, G. (1977) Biochim. Biophys. Acta 464,453-458. 6. FEO, F., CANUTO, R. A., GARCEA, R., AND BROSSA, 0. (1978) Biochim. Biophys. Acta 504, l-14. 7. MCINTYRE, J. O., BOCK, 0. H. G., AND FLEISCHER, S. (1978) Biochim. Biophya Acta 513,225-267. 8. COMTE, J., AND GAUTHERON, D. C. (1978) Biochimie 60, 1299-1305. 9. COMTE, J., MA~TERRENA, B., AND GAUTHERON, D. C. (1976) Biochim. Biophys. Acta 419, 271284. 10. MA’I’ROUCH, H., AND GODINOT, C. (1977) Proc. Nat. Acad. Sci. USA 74, 4185-4189. 11. COMTE, J., HARB, J. S., AND GAUTHERON, D. C. (1979) XIth International Congress of Biochemistry, Toronto, p. 340. 12. CRANE, F. L., GLENN, J., AND GREEN, D. E. (1956) Biochim. Biophys. Acta 135, 599-613. 13. GODINOT, C., VIAL, C., FONT, B., AND GAUTHERON, D. C. (1969) Eur. J. Biochem. 8, 385-394.

318

HARB,

COMTE,

14. MA’I’STERRENA, B., COMTE, J., AND GAUTHERON, D. C. (1974) Biochim. Biophys. Acta 367, 115126. 15. GODINOT, C., AND GAUTHERON, D. C. (1979) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 55F, pp. 112-114, Academic Press, New York. 16. PHILLIPS, A. H., AND LANGDON, R. G. (1962) J. Biol. Chem. 237.2652-2660. 17. OCHOA, S. (1955) in Methods in Enzymology (Kaplan, N. O., and Colowick, S. P., eds.), Vol. 5, pp. 807-809, Academic Press, New York. 18. Dr PIETRO, A., GODINOT, C., BOUILLANT, M.L., AND GAUTHERON, D. C. (1975) Biochimie 57, 959967. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 20. BACHMANN, L., AND SCHMI?T-FUMIAN, W. W. (1973) in Freeze-Etching Technique and Application (Benedetti, E. L., and Favard, P., eds.), pp. 63-79, Societe Francaise de Microscopie Electronique, Paris. 21. BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOOR, H., M~HLETHALER, K., NORTHCOTE, D. H., PACKER, L.,SATIR, B., SATIR, P., SPETH, V., STAEHLIN, E. A., STEERE, R. L., AND WEINSTEIN, R. S. (1975) Science 190, 54-56. 22. VALE, M. G. P. (1977) Biochim. Biophys. Acta 471, 39-48. 23. SANDRA, A., AND PAGANO, R. E. (1978) Biochemistry 17, 332-338. 24. SUNDLER, R., ALBERT, S. A., AND VAGELOS, P. R. (1978) J. Biol. Chem. 253, 5299-5304.

AND

GAUTHERON 25. MARINETTI, G. V., SENIOR, A. E., LOVE, R., AND BROADHURST, C. P. (1976) Chem. Phys. Lipids 17,353-362. 26. CRAIN, R. C., MARINETTI, G. V., AND O’BRIEN, D. F. (1978) Biochemistry 17,4186-4192. 27. CRAIN, R. C., AND MARINETTI, G. V. (1979) Biochemistry 1, 2407-2414. 28. PACKER, L., MEHARD, C. W., MEISSNER, G., ZAHLER, W. L., AND FLEISCHER, S. (1974) Biochim. Biophys. Acta 363, 159-181. 29. KREBS, J. J. R., HAUSER, H., AND CARAFOLI, E. (1979) J. Biol. Chem. 254, 5308-5316. 30. BISHOP, D. G., OP DEN KAMP, J. A. F., AND VAN DEENEN, L. L. M. (1977) Eur. J. Biochem. 80, 381-391. 31. BISHOP, D. G., BEVERS, E. M., VAN MEER, G., OP DEN KAMP, J. A. F., AND VAN DEENEN, L. L. M. (1979) Biochim. Biophys. Acta 551, 122-128. 32. HERON, C., CORINA, D., AND RAGAN, I. C. (1977) FEBS Lett. 79.399-403. 33. MACHINIST, J. M., AND SINGER, T. P. (1965) J. Biol. Chem. 240, 3182-3190. 34. AWASTHI, Y. C., RUZICKA, F. J., AND CRANE, F. L. (1970) Biochim. Biophys. Acta 203, 233248. 35. WAKSMAN, A., RENDON, A., CREMEL, G., PELLICONE, C., AND GOUBAULT DE BRUGIERE, J. F. (1977) Biochemistry 16.4703-4707. 36. RENDON, A., AND PACKER, L. (1976) in Mitochondria, Biogenesis and Membrane structure (Packer, L., and Gomez-Puyon, A., eds.), pp. 151-154, Academic Press, New York.