THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 19. Issue of October 10, pp. 11760-11766, 1982 Prrnled in U.S.A.

Labeling of Succinate-Cytochrome c Reductase with lZ5I ACCESSIBILITY OF THE PEPTIDES TO THE AQUEOUS PHASES ON THE CYTOSOLIC AND MATRIX SIDES OF THE MITOCHONDRIAL MEMBRANE*

(Received for publication, September 2, 1981)

M. Patricia D’Souza and David F. Wilson From the Department of Biochemistry and Biophysics, University of Pennsyluania, Philadelphia, Pennsylvania 19104

Lactoperoxidase-catalyzed radioiodination was used to study the arrangement of the component peptides of succinate-cytochrome c reductase with respect to the aqueous phases on each side of the mitochondrial inner membrane. Mitochondria depleted of their outer mem- brane and inside-out vesicles purified from submito- chondrial particles by the lectin-affinity procedure (D’Souza, M. P., and Lindsay, J. G. (1981) Biochim Biophys. Acta 640, 463-472) were iodinated using im- mobilized preparations of lactoperoxidase. The labeled membranes were solubilized in detergent and the suc- cinate-cytochrome c reductase was purified by immu- noprecipitation with specific IgG. Analysis of the radi- oiodine distribution after sodium dodecyl sulfate-poly- acrylamide gel electrophoresis and comparison with peptide stain patterns show that bands 2 (64 kilodal- tons), 6 (30 kilodaltons), 9 (15 kilodaltons), and 11 (<lo kilodaltons) are labeled from the cytoplasmic surface of the membrane. Bands 1 (72 kilodaltons), 4 (48 kilo- daltons), and 8 (20 kilodaltons) appear to be labeled on the matrix side of the membrane, while bands 3 (52 kilodaltons), 5 (35 kilodaltons), 7 (25 kilodaltons), and 10 (11 kilodaltons) are labeled from both sides of the membrane. Tentative identification of the labeled bands suggests that band 1 is the large subunit of succinate dehydrogenase. Bands 3 and 4 represent pro- teins which have been referred to as core proteins I and II. Bands 5 and 6 are the proteins associated with cytochromes b and cl, respectively; band 7 is the Rieske iron-sulfur protein.

Isolated mitochondrial succinate-cytochrome c reductase is composed of succinate dehydrogenase and the cytochrome bc, complex. The cytochrome bcl complex is responsible for trans- ferring reducing equivalents from the ubiquinone redox level of the respiratory chain to cytochrome c, that is, energy- coupling site 11. It is an integral part of the inner membrane and contains four redox components: two b cytochromes ( b b ~ ~ and b565), cytochrome c, , and the Rieske iron-sulfur protein (1-4). The overall complex, succinate-cytochrome c reductase, is reported to contain 11 peptides (5). Other studies done on the isolated cytochrome bel complex have reported a total of nine (6), eight (7, 8), or seven peptides (9, 10).

The general approach to studying the orientation of pro- teins within a membrane is to have access to vesicles of defined sidedness. Polypeptides that are exposed on the ex-

* This work was supported by Grant GM 12202 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ternal surface of the vesicles can then be radiochemically modified with reagents that do not penetrate the membrane, for example, lactoperoxidase-catalyzed incorporation of ‘”1. Mitochondria depleted of their outer membranes provide a convenient source for labeling the cytoplasmic side of the membrane. On the other hand, obtaining vesicles in which the external surface is the matrix side of the membrane is difficult (11). Although some workers have used submitochondrial particles prepared by sonication procedures as populations of “inside-out” vesicles (see, for example, Refs. 6 and 9), other workers have suggested that such preparations are mixtures of inside-out and right-side-out vesicles (12, 13). Two studies have been reported (6, 9) of labeling of the peptides of the cytochrome bcl complex. Both studies utilized the chemical probe p-diazonium benzene sulfonate to label exposed sides of the membrane and assumed that submitochondrial particles are homogeneous preparations of inside-out vesicles. The labeling patterns reported differ in important points, both with respect to each other and to the results in this commu- nication (see “Discussion”).

In the present work, we have employed a membrane label (lZ5I) which has a different chemical specificity and also uti- lized inside-out vesicles purified by a lectin affinity procedure (13). The purified vesicles as assayed by enzymic methods are more than 90% inside-out, that is, more than 90% of the vesicles have the matrix side of the membrane as their external surface. The external surfaces of inside-out vesicles, right- side-out vesicles (mitoplasts), and a mixed population of ves- icles (submitochondrial particles) were labeled by the lacto- peroxidase-catalyzed iodination technique. The labeled mem- branes were solubilized with detergents and succinate-cyto- chrome c reductase was separated by specific immunoprecip- itation with IgG raised against the purified complex. The distribution of radioactivity between the constituent polypep- tides of the complex were then analyzed either by fluorogra- phy or liquid scintillation counting of SDSI-polyacrylamide gels. The data presented give information on the orientation of the peptides of succinate-cytochrome c reductase, together with tentative identification of some of the constituent pep- tides. An abstract of the work has been published (14).

MATERIALS AND METHODS

Biological Preparations-Mitochondria from pigeon breast mus- cle were prepared as described in (15).

Mitoplasts were prepared by swelling mitochondria in 10 mM Tris- phosphate, pH 7.4, for 5 min at 4 “C, followed by sedimentation at 1,500 x g. The protein was finally resuspended at 20 mg/ml into 0.13 M KCI, 20 mM MOPS, pH 7.2. Submitochondrial particles were prepared by sonicating mitoplasts that were suspended in 10 mM Tris- phosphate, pH 7.4, at 20 mg/ml of protein for three 30-s bursts at a

’ The abbreviations used are: SDS, sodium dodecyl sulfate; MOPS, 4-morpholinepropanesulfonic acid.

11760

Topography of Succinate-Cytochrome c Reductase 11761

power setting of 5 (Sonifier Cell Disrupter, Model W185). Vesicles were sedimented by centrifugation at 100,OOO X g for 40 min, and washed once before resuspending in 0.13 M KCI, 20 mM MOPS, pH 7.2, at 20 m g / d of protein. Inside-out vesicles were purified from mitoplasts by lectin affinity as described in Ref. 13. Calculations of the percentage of inside-out vesicles were based on the assumption that homogeneous inside-out preparations had only 5% of the cyto- chrome c oxidase activity of right-side-out vesicles or vesicles in the presence of detergent (13).

Succinate-cytochrome c reductase isolated and purified by Dr. Maria Erecinska (16) was used for production of antisera.

Preparation of Antisera-Rabbits were immunized with purified succinate-cytochrome c reductase by subcutaneous multiple site in- jections of 1 mg of protein in Freund's complete adjuvant. This treatment was repeated 4-6 weeks later with incomplete adjuvant and again 10-12 days later. Antiserum was collected after 2 weeks by bleeding from an ear vein. Purification of the IgG fraction was achieved by elution from DEAE-Affi-Gel Blue (Bio-Rad Laborato- ries) using the procedure recommended by the company and used in all the described experiments.

Labeling with '2sZ-The procedure was based on that described by David and Reisfeld (17). Carrier-free Na 'T was purchased from the Radiochemical Centre, Amersham, and Enzymo-Beads were obtained from Bio-Rad Laboratories. Mitoplasts, submitochondrial particles, or inside-out vesicles (each 0.5 mg of protein) in 0.13 M KC1, 20 mM MOPS medium, pH 7.2, were each incubated with Enzymo-Beads (immobilized preparations of lactoperoxidase and glucose oxidase (50 PI)), and 200 yCi of carrier-free Nalz5I (100 mCi/ml). The reaction was initiated by addition of 1% P-o-glucose (50 yl). After 30 min at 4 "C, the iodinated samples were washed 1x3) by suspension in 0.13 M KCl, 20 mM MOPS, pH 7.2, containing 1 mM KI and centrifugation at 100,OOO X g.

Preparation of Zmmunopreczpitates-Mitoplasts, submitochon- drial particles, or inside-out vesicles were resuspended in 0.1 M Na phosphate buffer, pH 7.2, to 1 mg of protein/ml containing 0.5% (w/ v) Na deoxycholate and 0.5% (v/v) Triton X-100. After incubation for 40 min at 4 "C, the samples were centrifuged in a Beckman Model B Microfuge for 2 min to pellet unsolubilized membrane and Enzymo- Beads. The supernatant fractions were made 0.75% with respect, to each detergent. Phenylmethylsulfonyl fluoride (1 mM) was added followed by anti-cytochrome c reductase IgG (0.4 mg/mg of mito- chondrial protein). The samples were incubated for 20 min at 25 "C and then the temperature was lowered to 0-4 "C. After 4 h, 0.2 ml of IO% (v/v) protein A fixed to Staphylococc~~~ cells (purchased from The Enzyme Center, Inc., Boston, MA) was added (18) and the samples were left overnight at 4 "C. The immunoprecipitates were collected by centrifugation for 2 min in a Beckman Microfuge. The pellets were washed three times in 0.1 M Na phosphate buffer, pH 7.2, containing 1% (w/v) Na deoxycholate, 1% (w/v) Triton X-100 and finally washed in 0.1 M Na phosphate buffer, pH 7.2.

SDS-Polyacrylamide Gel Electrophoresis-Protein samples were prepared for electrophoresis by suspending them in 10 mM Na phos- phate buffer, pH 7.4, containing 2% SDS, 2% /3-mercaptoethanol, 5% glycerol, and then denatured by placing them in a boiling water bath for 3 min. SDS-polyacrylamide gel electrophoresis was carried out by the method of Weber and Osborn (19) using 10% acrylamide gels with an acrylamide/bisacrylamide ratio of 37:l. After electrophoresis, pro- tein bands were visualized with Coomassie blue gel stain. Molecular weights were determined by co-electrophoresing six protein standards (M, = 14,200, 18,400, 24,000, 34,700, 45,000, and 68,000).

Determination of Radioactivity-Proteins labeled by 12'1 were electrophoresed on slab gels and identified by processing the gels for fluorography using the procedure of Bonner and Laskey (20). The dried gels were exposed to Kodak X-Omat R film for appropriate lengths of time at -70 "C.

Radioactivity was also determined by slicing cylindrical gels elec- trophoresed by the same procedure described above into 1-mm thick slices. The gel slices were digested for 16 h at 50 "C with 0.1 ml of 30% hydrogen peroxide. The radioactivity was measured by suspending these samples in Searle aqueous counting scintillant (Amersham) and counting in a Searle Delta 300 liquid scintillation counter.

Spectral Studies-Absorption spectra were recorded using a John- son Foundation dual wavelength scanning spectrophotometer pro- vided with a digital wavelength drive on one monochromator, the other being set at the reference wavelength. The operation of this instrument is described in Ref. 15.

in (13). Assays-Cytochrome c oxidase activity was assayed as described

Protein concentration was determined by the biuret reaction using bovine serum albumin (Fraction V from Sigma) as a standard.

RESULTS

Demonstration of Selective Immunoprecipitation of the Succinate-Cytochrome c Reductase by IgG against Succi- nate-Cytochrome c Reductase-The IgG fraction purified from rabbit serum previously injected with purified succinate- cytochrome c reductase was added to detergent-solubilized submitochondrial particles. The resulting immunoprecipitate was then resuspended in buffer and the difference in absor- bance between the dithionite-reduced sample and the un- treated sample was measured. In Fig. 1, this absorption spec- trum (spectrum B) is compared with that for the original suspension of mitochondrial particles solubilized in detergent (spectrum A ) . The immunoprecipitated material has an ab- sorption spectrum characteristic of the cytochrome be, com- plex, with a broad a maximum near 563 nm and a Soret maximum a t 430 nm. There is no evidence for cytochrome oxidase (a maximum at 605 and Soret maximum at 445 nm) in the immunoprecipitated material. The absorption differ- ence spectra of the supernatant fraction after immunoprecip- itation showed that over 95% of the cytochrome oxidase was present in this fraction while the cytochrome b content de- creased by at least 75%. The presence of hemoglobin in the IgG and the low protein concentrations used for immunopre- cipitation do not allow more precise measurement of the b cytochromes in the supernatant fraction. Control experiments were run to exclude possible artifacts. An oxidant, ferricya- nide, was added to the immunoprecipitate in case cytochrome oxidase was present, but this did not reveal the presence of any cytochrome oxidase. The latter would be spectrally un- detectable if it were in the reduced form prior to dithionite reduction. The slight trough observed at 580 nm in spectrum B is owing to hemoglobin absorption, a contaminant in the IgG fraction.

Composition of Succinate-Cytochrome c Reductase-Suc- cinate cytochrome c reductase was immunoprecipitated from submitochondrial particles with specific IgG and electropho- resed on SDS-polyacrylamide gels. The peptide bands were visualized by staining in Coomassie blue R and a photograph of one such gel is shown in Fig. 2A. For comparison, submi-

400 4 50 500 550 600 650 Wavelength (nml

FIG. 1. Absorption difference spectra of submitochondria1 particles and of succinate cytochrome c reductase purified by specific IgG. Submitochondrial particles prepared from pigeon breast muscle mitochondria by sonication (see "Materials and Meth- ods") were suspended in 50 mM phosphate buffer, pH 7.2, at 1.0 mg of protein/ml. The top spectrum (A) gives the difference in absor- bance between dithionite-reduced and oxidized suspensions of sub- mitochondrial particles. Spectrum B is the difference in absorbance between dithionite-treated and untreated suspensions of the fraction immunoprecipitated with anti-succinate-cytochrome c reductase IgG in the presence of 1% (w/v) Triton X-100, 1% (w/v) deoxycholate.

11762 Topography of Succinate-Cytochrome c Reductase

72,000

64,000 52,000 48,000

35,000 30,000 25,000

20,000

15,000 11,000

C l O ,000

C B A FIG. 2. Polypeptide composition of succinate-cytochrome c

reductase immunoprecipitated from membranes. Polyacryl- amide gel electrophoresis was carried out by the method of Weber and Osborn (19) on 10% (w/v) gels. Samples contained 100 pg of protein immunoprecipitated from detergent-solubilized submitochon- drial particles by IgC against succinate-cytochrome c reductase (A) and 100 pg ( B ) of protein from untreated submitochondrial particles. The purified complex is shown in C.

tochondrial particles (Fig. 2B) were co-electrophoresed on the same gel with the immunoprecipitated succinate-cytochrome c reductase. The apparent molecular weights of the 11 peptide components of the complex were calculated by comparison of their migrations with those of purified protein standards. The values obtained from eight different experiments that were averaged and numbered in order of decreasing molecular weight are: 1, 72,000, 2, 64,000; 3,52,000; 4,48,000; 5, 35,000; 6, 30,000, 7, 25,000; 8, 20,000; 9, 15,000, 10, 11,000; 11, <10,000. The intense Coomassie blue stain observed in the band 3 region can be attributed to the heavy chain of IgG (M, = 55,000). The polypeptide composition of a purified preparation of succinate-cytochrome c reductase (Fig. 2C) is also shown.

Inhibition of Respiratory Activity by Anti-succinate-Cyto- chrome c Reductase Antibody-Succinate-cytochrome c re- ductase antibody prepared against the complex inhibited both NADH and succinate oxidation in submitochondrial particles after a prior incubation of 20 min. The maximum inhibitions in respiratory rate that was observed with NADH and succi- nate as substrates were 43 and 50%, respectively (Table I). Maximal inhibition occurred with 260 pg of anti-succinate- cytochrome c reductase IgG/mg of submitochondrial parti- cles. These data indicate that, although the antibody binds to the succinate-cytochrome c reductase complex, it does not cause total inhibition of electron transfer. The fact that the antibody inhibits the respiratory rate when succinate is used as substrate rules out the possibility of nonspecific inactivation of NADH dehydrogenase by succinate-cytochrome c reduc- tase IgG. In control experiments, IgG purified from normal rabbit serum showed no inhibition of respiration. No inhibi-

tion of respiration was observed in mitochondria that were depleted of their outer membrane, indicating that inhibition occurs when IgG binds to that portion of the complex not accessible from the cytoplasmic side of the membrane.

In order to localize the site at which the IgG against succinate-cytochrome c reductase inhibited electron transfer, the “crossover” phenomenon was employed. The site of action of the antibody can be established by spectrophotometric measurements of the oxidation-reduction states of the electron carriers in the presence and absence of antibody. Carriers on the reducing substrate side of the inhibitory site become more reduced, and those on the oxygen side become more oxidized. Because the experiments must be carried out in very dilute suspension, submitochondrial particles were prepared in the presence of excess cytochrome c (20 PM). The spectra in Fig. 3 show absorption maximum a t 550 nm, characteristic of reduced cytochrome c. Absorption difference spectra of the aerobic steady state of NADH oxidation by control submito- chondrial particles (top spectrum) and those appropriately

TABLE I The effect of succinate-cytochrome c reductase IgG on the

respiration of submitochondrial particles Submitochondrial particles were prepared by sonication of mito-

plash (see “Materials and Methods”) in the presence of 10 PM cytochrome c and finally suspended in 0.25 M sucrose, 5 mM MOPS, 0.2 mM EDTA, pH 7.2, at 15 mg/ml. Samples (1 mg of protein in 1.5 ml) were incubated with 0.2 mg of anti-succinate-cytochrome c re- ductase (bel) IgG for 20 min at 25 “C, prior to measurement of the rate of 0 2 consumption in the presence of either 4 mM NADH or 10 mM succinate. The respiratory rates were measured at 25 “C.

Sample Inhihition of o: “IJLake 01’ uptake

nmoI/min/ mg protein

1 mg submitochondrial particles; 0.2 46.1

1 mg submitochondrial particles; 0.2 25.9 43.8

1 mg submitochondrial particles; 0.2 47.1

1 mg submitochondrial particles; 0.2 26.1 44.6

mg normal IgG; 4 mM NADH

mg bcl IgG; 4 mM NADH

mg normal IgG; 10 mM succinate

mg bc, IgC; 10 mM succinate

- .- -7”- -~ - 500 550 600

”

650

Wavelength (nm) FIG. 3. Absorption difference spectra of cytochrome c re-

duction in submitochondrial particles in the aerobic steady state. Submitochondrial particles were prepared by sonication of mitoplasts (see “Materials and Methods”) in the presence of 10 p~ cytochrome c and finally suspended in 50 RIM phosphate buffer, pH 7.2, to 1.0 mg of protein/ml. The upper spectrum gives the difference in absorbance between particles in the aerobic steady state when oxidizing NADH and particles in the absence of added NADH. The lower trace is the difference spectrum obtained for a similar experi- ment in which the particles were treated with anti-succinate-cyto- chrome c reductase IgC.

Topography of Succinate- Cytochrome c Reductase 11763

treated with IgG (bottom spectrum) were recorded. In the sample incubated with IgG against the succinate-cytochrome c reductase, there was a sizable decrease in absorption at 550 nm. The percentage decrease in reduction of cytochrome c was calculated from the relative absorbances and found to be approximately 48%. This is remarkably similar to the observed inhibition of respiration and it can be concluded that the antibody binds to that portion of the respiratory chain re- sponsible for transferring reducing equivalents to cytochrome c (cytochrome bcl complex) and not to cytochrome c oxidase. In keeping with this conclusion, the rate of oxidation of reduced cytochrome c was not affected by incubation with IqG.

The Sidedness of Submitochondrial Particles-It is com- monly assumed (21) that in submitochondrial particles the membranes are largely inverted with respect to those of intact mitochondria. In general, however, sonication produces a mix- ture consisting of right-side-out and inside-out vesicles, with the possibility of nonsealed membrane fragments. In order to estimate the homogeneity of populations of submitochondrial particles, we have assayed for the presence of a marker enzyme known to be located on a specific side of the membrane permeability barrier. Cytochrome c oxidase satisfies this cri- terion. Cytochrome c, the substate for this enzyme, has its binding site on the cytoplasmic side of the membrane and does not readily penetrate the lipid bilayer. Hence, assay of enzymic activity with externally added cytochrome c in the presence or absence of detergents (22) can provide a clear indication of the orientation of submitochondrial particles in any given preparation.

An experiment in which the rates of cytochrome c oxidation were measured in both the total population of submitochon- drial particles and purified inside-out particles as described in Ref. 13 was done in the presence and absence of dodecyl /?- maltoside. It can be seen (Fig. 4A) that, for the total popula- tion of particles, the rate of cytochrome c oxidation was stimulated from 83.5 to 135 nmol of 02/min/mg of protein on addition of detergent. The observed stimulation on addition of detergent is indicative of the population being 40-50% inverted. For purified inside-out vesicles (Fig. 4B), the rate of oxygen consumption was 25.9 nmol of Oa/min/mg of protein and was stimulated 5.3-fold (to 137 nmol of 02/min/mg of protein) on addition of 0.3% dodecyl /?-maltoside. The degree of stimulation of cytochrome c oxidase activity routinely

A B

1354\\ \ ‘,

136 8: \

\ \ \ FIG. 4. Preparation of a “purified” population of inside-out

vesicles. Submitochondrial particles were prepared by sonication of mitoplasts (see “Materials and Methods”). Particles (5 mg of protein in 2 ml) were treated with wheat germ agglutinin (2 mg) for 20 min a t 25 “C. They were then incubated with 0.4 ml of anti-wheat germ agglutinin IgG for 20 min at 25 “C and overnight a t 4 “C. Agglutinated vesicles were removed by centrifugation and discarded. The cyto- chrome c (cyt c) oxidase activity in both the untreated submitochon- drial particles ( A ) and the unagglutinated particles ( B ) was measured in the presence (dashed line) and absence (solid line) of detergent.

Y normal I q G - anti-WGA-IqG

0 O Y I

h

0 100 200 300 400

anti-WGA-IgG ( pl )

FIG. 5. Analysis of selective removal of right-side-out vesi- cles by enzymatic criteria. Standard preparations of mitoplasts were treated with wheat germ agglutinin (WGA) as described in Fig. 4. Aliquots containing 0.25 mg of protein were then treated with increasing quantities of anti-wheat germ agglutinin IgG for 20 min at 25 “C and then overnight at 4 “C. After centrifugation, the superna- tant and pellet fractions were monitored for the disappearance or appearance, respectively, of cytochrome c oxidase activity. Control samples were treated identically except that normal rabbit IgG was used instead of anti-lectin IgG.

ranged from 5-8-fold for the inside-out vesicles, indicating an 80-90% homogeneous population.

In an analogous experiment, the cytochrome c oxidase activity expressed in mitoplasts remained unchanged in the presence of dodecyl /?-maltoside (data not shown). The latter detergent was also reported to have no effect on cytochrome c oxidase activity in Ref. 23. Other data using 0.3% deoxycho- late have been reported by Wojtczak et al. (24) to break the membrane structure without activating or inhibiting the oxi- dase activity.

These conclusions require that the right-side-out vesicles be nearly quantitatively removed by lectin. Mitoplasts pre- pared from pigeon breast muscle mitochondria as described under “Materials and Methods” were therefore tested for their capacity to be quantitatively precipitated by wheat germ agglutinin and its respective antibody. The data are shown in Fig. 5 in which treatment with wheat germ agglutinin followed by immunoprecipitation with the rabbit anti-wheat germ ag- glutinin IgG elicits approximately 90% precipitation of the mitoplasts as assessed by the disappearance of cytochrome c oxidase activity from the supernatant fraction. In the absence of specific anti-lectin IgG, no precipitation was observed. The pellets were also assessed for residual cytochrome c oxidase activity after resuspension of the protein and these measure- ments confirm that neither the lectin nor the antiserum caused nonspecific inhibition of the enzymic activity.

Labeling and Immunoprecipitation of Succinate-Cyto- chrome c Reductase-The topography of the subunits of succinate-cytochrome c reductase to the cytoplasmic and ma- trix surface of the inner mitochondrial membrane was inves- tigated by chemically modifying the exposed peptide residues in mitoplasts and inside-out vesicles. This was followed by specific immunoprecipitation of the detergent-treated mem- branes with anti-succinate-cytochrome c reductase IgG. The resulting immunoprecipitates were analyzed by SDS-poly- acrylamide gel electrophoresis either by slicing cylindrical gels and preparing samples for liquid scintillation counting (Fig. 6) or by fluorography of dried slab gels (Fig. 7). Although the former method provides good resolution, inaccuracies can arise when slicing the gel into 1-mm slices, particularly if the gel is distorted by the pressure applied during cutting. The technique of slab gel electrophoresis followed by fluorography of the dried gel to reveal iodinated bands is somewhat superior to the slicing and counting technique in that direct compari- sons can be made visually of all samples co-electrophoresed

1 1764 Topography of Succinate-Cytochrome c Reductase

on a single gel. Both techniques ‘were used in the present study.

The subunits heavily labeled in mitoplasts (Fig. 6A) have M, = 64,000, 52,000, 35,000, 30,000, and 25,000. Polypeptides labeled to a lesser extent include those of M, = 20,000, 15,000, 11,000, and ~10,000. The subunits heavily labeled in mito- plasts as visualized by the densitometric scans (Fig. 7A) have M, = 52,000,35,000,30,000, 15,000, and 11,000. Similarly, those peptides labeled to a lesser extent have M, = 64,000, 25,000, 20,000, and 40,000.

t “ I

- 0 ’ ‘ -

35 I

0 ’ I >

I O 20 30 40 50 60 70

Migration distance from origin (mm)

FIG. 6. Labeling of succinate cytochrome c reductase iso- lated from mitochondria, submitochondrial particles, and in- side-out vesicles labeled with Iz5I. Labeling and immunoprecipi- tation of the complex were carried out as described under “Ma- terials and Methods.” Immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis on column gels. These were then sliced and digested and the radioactivity was measured. Samples were: A, succinate-cytochrome c reductase immunoprecipitated from mitoplasts (0.5 mg of protein); B, succinate-cytochrome c reductase

lectin affinity procedure (0.5 mg of protein); C, as in B but using immunoprecipitated from purified inside-out vesicles prepared by

submitochondrial particles (0.5 mg of protein) prepared by sonication of mitoplasts. Approximate molecular weights (in thousands) are indicated on C.

Orlgin [A]

A

h AA = 0.1

L

FIG. 7 . Densitometric scans of ‘2s1-labeled succinate cyto- chrome c reductase isolated from mitochondria, submitochon- drial particles, and inside-out vesicles. Labeling and immunopre- cipitation of the complex were performed as described in Fig. 5. Immunoprecipitates were co-electrophoresed on slab gels and proc- essed for fluorography. The dried gels were exposed to x-ray film for the appropriate length of time and densitometric scans of the devel- oped fiim are presented. The patterns are for the radiolabeled pep- tides of samples from (A) mitoplasts, (B) inside-out vesicles, (C) submitochondrial particles. Molecular weights (in thousands) are indicated.

The labeling pattern of succinate-cytochrome c reductase immunoprecipitated from inside-out vesicles (Fig. 6B) is clearly different and showed all peptides (M, = 72,000, 52,000, 35,000, 25,000, 20,000, and 11,000) to be significantly labeled with the possible exception of peptide M , = 48,000. The peptide labeling pattern as visualized by the densitometric scans (Fig. 78) showed bands of M, = 35,000, 20,000, and 11,000 as heavily labeled. Polypeptides of M , = 72,000, 52,000, 48,000, and 25,000 were also labeled, but to a lesser extent.

Immunoprecipitation of the cytochrome c reductase com- plex from submitochondrial particles which present an almost equal mixture of cytoplasmic and matrix surfaces of the mem- brane provides a good control. In these experiments, radioac- tivity was found to be associated with all 11 peptides. The labeling was unambiguous except in the case of peptide 11. Peptide 11 (Mr < 10,000) was observed to be labeled by the gel slicing method (Fig. 6C); however, in the set of densitom- eter scans shown (Fig. 7C), there is no clear maximum in this region. In other experiments (five out of eight), labeling was observed.

Deciding which polypeptides are “significantly” labeled in mitochondria or inside-out vesicles is made more difficult by the fact that the relative amount of radioactivity incorporated into each peptide varied in different experiments. The final assignments of the peptide labeling are based on a total of eight experiments, each employing both techniques.

We have assigned each of the labeled peptides to one of three categories: accessible to labeling from the cytoplasmic

Topography of Succinate

TABLE 11 Assignment of’ labeled peptides by category of location in

membrane

Band Location in mem- Proposed identification brane mass

1

2 3 4 5 6 7 8 9

10 11

kilodaltnns 72

64 52 48 35 30 25 20 15 11

4 0

Matrix

Cytoplasmic Transmembrane Matrix Transmembrane Cytoplasmic Transmembrane Matrix Cytoplasmic Transmembrane Cytoutasmic

Succinate dehydrogen- ase (large subunit)

? Core protein I Core protein I1 Cytochrome b Cytochrome CI

Fe-S ? ? ? ?

side of the membrane, accessible to labeling from the matrix side, and accessible to labeling from both sides. The data are summarized in Table 11. Bands 1 and 8 (AIr = 72,000 and 20,000) are assigned to the matrix side of the membrane. Band 1 represents the larger subunit of succinate dehydrogenase. The identity of this band was confirmed by co-electrophores- ing purified succinate dehydrogenase (data not shown). The lower molecular weight subunit of succinate dehydrogenase appears to migrate between bands 6 and 7 when electropho- resed with the bel complex. The molecular weight has been estimated a t 28 kilodaltons, which agrees with other reports (25 , 26). However, it does not appear to be labeled with lactoperoxidase under our conditions. Band 4 (M, = 48,000) appears to be labeled solely from the matrix side; it identifies closely with so-called core protein I1 (27). Bands 2, 6, 9, and 11 ( M , = 64,000, 30,000, 15,000, and 40,000) appear to be labeled only from the cytoplasmic side of the membrane. Band 6 closely corresponds in molecular weight to the peptide associated with cytochrome c1 ( 5 , 2 8 ) . Band 2 could be part of the “core protein” fraction reported in Ref. 27. The identities of bands 8 ,9 , and 11 are unknown. Bands 3, 5, 7, and 10 (M, = 52,000,35,000,25,000, and 11,000) appear to be labeled from both sides of the membrane. The molecular weight of band 3 suggests it is similar to so-called core protein I (27). Band 5 resembles in molecular weight the peptide associated with the two b cytochromes (29,30), and band 7 is probably the peptide of the Rieske iron-sulfur protein (4). The identity of band 10 is yet unknown.

DISCUSSION

The approach adopted for studying the orientation of suc- cinate-cytochrome c reductase relied on the impermeability of the membranes to lactoperoxidase. It was also necessary to ensure “intact” vesicle preparations that were not leaky to the chemical probe, thus permitting vectorial labeling. Lactoper- oxidase-catalyzed lZ5I iodination was selected in preference to other techniques because the conditions favoring vectorial labeling have been clearly defined (31). However, the proce- dure suffers from a fairly high degree of selectivity, in that only proteins with exposed tyrosine and possibly histidine residues are labeled. As a result, lack of labeling of a particular peptide may reflect absence of a tyrosine residue in the exposed region of the peptide rather than inaccessibility of the polypeptide per se.

Two important criteria for evaluation of the data must be considered. First, it is necessary to show that the antibody is specific for succinate-cytochrome c reductase, as failure to do so would result in precipitation of other labeled mitochondrial

-Cytochrome c Reductase 11765

proteins. This we have done by spectral analysis of the im- munoprecipitate which showed the presence of cytochrome bcl complex and absence of cytochrome c oxidase. In addition, SDS-polyacrylamide gel electrophoresis of the immunoprecip- itated material shows that it has a peptide content similar to that of the purified reductase. Data from both these experi- ments give support to the conclusion that the antibody is appropriately specific for the reductase. We have tried to eliminate as far as possible the problem of contaminating proteases by always working with the purified immunoglob- ulin G fraction and including the protease inhibitor phenyl- methylsulfonyl fluoride during all incubations.

The other important criterion concerns orientation of the cytoplasmic and matrix sides of the membrane to the labeling probe. Mitochondria depleted of their outer membranes (mi- toplasts) were used as a source for labeling the cytoplasmic surface. Two experimental checks done indicate that the mitoplasts had an essentially intact right-side-out membrane. First, they showed respiratory control (ratio of 1.8) with succinate/glutamate as substrate. Second, addition of deter- gent gave negligible (<lo%) stimulation of the rate of oxida- tion of added cytochrome c. Inside-out vesicles which were purified from submitochondrial particle preparations (13) were used as a source of membranes with the matrix side exposed. Our criteria for demonstrating the homogeneity of inside-out vesicles were based on activity of a marker enzyme: cytochrome c oxidase. The rate of oxidation of cytochrome c in inside-out vesicles was routinely stimulated 5-8 fold, indi- cating that the population was 80-95% inverted. Good evi- dence that the exposed (external) membrane surface of these particles is the matrix side in intact mitochondria is the clear difference in the labeling patterns observed between the two vesicle populations.

In this paper, the organization of the constituent peptides of succinate-cytochrome c reductase was studied by specifi- cally immunoprecipitating the complex from vesicles labeled from the cytoplasmic and matrix sides of the membrane. The data were analyzed by two techniques and the proposed location of the individual subunits was decided from a series of eight independent experiments, The results presented here demonstrate the transmembrane arrangement of succinate- cytochrome c reductase in the inner mitochondrial membrane.

The peptides with M, = 72,000 and 20,000 were both labeled from the matrix side of the membrane. The larger peptide most probably corresponds to the flavin-containing subunit of succinate dehydrogenase. Evidence that succinate dehydro- genase is located on the matrix side of the mitochondrial inner membrane was first provided by Klingenberg (32), who showed that submitochondrial particles but not intact mito- chondria exhibited antimycin-insensitive succinate-ferricya- nide reductase activity. Other data by Merli et al. (25) and Kenney et al. (26) have conclusively demonstrated that the larger flavin-containing subunit is located on the matrix side of the membrane. The peptide called band 3 (Mr = 52,000) appears to span the membrane by virtue of the fact that it is labeled from both the matrix and cytoplasmic sides of the membrane. This peptide corresponds to core protein I (27) and has been reported to be labeled from either the matrix side (9) or from both the matrix and the cytoplasmic sides ( 6 ) , using the chemical probe p-diazonium benzene sulfonate. In our studies, band 4 (M, 48,000), which corresponds to core protein I1 (27), is labeled only from the matrix side. This is consistent with data from Mendel-Hartvig and Nelson (9) and is in contrast to the report by Bell et al. (6) that core protein I1 is labeled from both the matrix and cytoplasmic sides.

The apoprotein(s) of the b cytochromes have been reported to have M,. - 35,000 (29,30). This corresponds in our study to

11 766 Topography of Succinate-Cytochrome c Reductase

band 5 which is labeled from both sides of the membrane. There are two b cytochromes (15, 33) and if their molecular weights are similar, this may mean either that each spans the membrane or that the two b cytochromes are each accessible on different sides of the bilayer. Electron paramagnetic reso- nance studies have indicated that the b cytochromes are closer to the cytoplasmic side than to the matrix side of the mem- brane (34, 35).

Cytochrome c1 (Mr = 30,000) is labeled from the cytoplasmic side of the membrane in our studies. This is in agreement with the reports of Bell et al. (6) and Mendel-Hartvig and Nelson (9). Erecinska et al. (5) have reported that, when photoaffin- ity-labeled cytochrome cis used to reactivate electron transfer in cytochrome c-deficient mitoplasts, photolysis results in its covalent binding to cytochrome cl. Since cytochrome c is impermeable to the membrane and in these experiments was added from the cytoplasmic side of the membrane, this is strong and independent support for the conclusion that cyto- chrome c1 is accessible to labeling from the cytoplasmic side of the membrane.

Band 7 (M, = 25,000) most probably represents the peptide of the Rieske iron-sulfur protein which is reported to have M, = 25,000 (4, 8). Labeling of the peptide of the Rieske iron- sulfur protein has been reported to occur from the cytoplasmic side of the membrane (6, 9). Bell et al. (6) examined the topography of this protein using [35S]diazobenzenesulfonate and observed stronger labeling from the cytoplasmic side than from the matrix side of the membrane. After comparing its labeling pattern with those peptides labeled from the matrix side, the authors concluded that the protein was accessible only to the cytoplasmic side. There are two possible reasons for our observation that band 7 is labeled from both sides of the membrane. 1) The protein is exposed to both surfaces, but at the matrix surface, band 7 is poorly reactive toward ["SI diazobenzenesulfonate, but reacts readily in the '"I-lactoper- oxidase systems used in the present study. 2 ) The smaller subunit of succinate dehydrogenase may have undergone par- tial proteolysis and co-migrated with band 7. The latter, although very difficult to completely exclude, seems unlikely in view of the precautions taken to purify the inside-out vesicles and of including protease inhibitors. We feel that the data indicate that band 7 is transmembranous. Little is known about the function or identity of the three lower molecular weight peptides, although various roles have been proposed, e.g. a peptide associated with cytochrome b (36) of M 14,500 and a peptide of M , = 13,000 associated with cytochrome c1 (36). The peptide of lowest molecular weight has been pro- posed to be the antimycin A-binding protein (36).

In summary, the data presented here show that succinate- cytochrome c reductase spans the nonaqueous core of the membrane and is asymmetrically oriented across the inner mitochondrial membrane. Tentative identification of certain bands has been attempted and it may be concluded that the peptide which corresponds to core protein I appears to be accessible from both sides of the membrane. The peptides which correspond to cytochrome b and the Rieske iron-sulfur protein are accessible to labeling from both the cytoplasmic and matrix sides of the membranes. Cytochrome c1 is labeled only from the cytoplasmic side of the membrane.

Acknowledgment-We are grateful to Dr. M. Erecinska for supply- ing purified preparations of succinate-cytochrome c reductase and for her many helpful suggestions during the course of this work.

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