THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 257, No. 21, Issue of November 10, pp. 13028-13033, 1982 Printed in U. S. A.

Import of Proteins into Mitochondria CYTOCHROME bz AND CYTOCHROME c PEROXIDASE ARE LOCATED IN THE INTERMEMBRANE SPACE OF YEAST MITOCHONDRIA*

(Received for publication, December 9, 1981)

Gunther Daum8, Peter C. Bohni, and Gottfried Schatzg From the Department of Biochemistry, Biocenter, Uniuersity of Basel, CH-4056 Basel, Switzerland

Yeast mitochondria were fractionated into inner membrane, outer membrane, matrix, and intermem- brane space. Identity and purity of each fraction were monitored by enzyme assays, dodecyl sulfate-poly- acrylamide gel electrophoresis, and immunological de- tection of characteristic mitochondrial polypeptides. Cytochrome bz and cytochrome c peroxidase were found to be components of the intermembrane space. The most reliable marker of the outer membrane was a major 29,000-dalton polypeptide component. The availability of submitochondrial fractions provides a basis for studying import of precursor polypeptides into isolated yeast mitochondria.

Isolated yeast mitochondria can take up in vitro synthesized mitochondrial precursor polypeptides by a process that is independent of protein synthesis, dependent on energy, and usually accompanied by the proteolytic “maturation” of the precursors (1-3). Up to now, however, uptake of precursors into mitochondria has only been inferred from the facts that the radiolabeled polypeptides became associated with mito- chondria and were rendered inaccessible to externally added proteases (I, 2). If this in vitro system indeed reflects mito- chondrial protein import as it occurs in living cells, different mitochondrial precursor polypeptides should be transported to their correct intramitochondrial location. This could not be verified previously because a suitable procedure for subfrac- tionating yeast mitochondria into distinct compartments was not available.

This paper describes a procedure for subfractionating yeast mitochondria into their four major compartments: matrix, inner membrane, intermembrane space, and outer membrane. While the yeast mitochondrial outer membrane (4, 5) and yeast mitoplasts (6) have been isolated before, comprehensive subfractionation of yeast mitochondria has not been reported. In particular, little information exists on the components of the yeast mitochondrial intermembrane space. With mam- malian mitochondria, the components of this space can be selectively released by digitonin ( 7 , 8); with yeast mitochon- dria, however, this detergent causes an almost parallel release of enzymes from the matrix, as well as the intermembrane space at any digitonin concentration tested.’

* This study was supported by Grants 3.172.77 and 3.606.80 from the Swiss National Science Foundation. 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.

$ Recipient of a European Molecular Biology Organization Long- term Fellowship. Present address, Institute of Biochemistry, Techni- cal University, Schlogelgasse 9, A-8010 Graz, Austria.

5 To whom requests for reprints should be addressed. ’ G. Daum, unpublished results.

The procedure presented in this paper takes advantage of a controlled osmotic shock to selectively release intermem- brane space compartments. The ease and specificity of this key step suggest that it might also be applicable to mitochon- dria from other yeasts and fungi. The subsequent separation of inner and outer membranes is very similar to that developed previously for mitochondria from Neurospora crassa (9).

AS outlined in the accompanying paper (3), the procedure described here has allowed a more detailed study of protein import into mitochondria, particularly into the intermem- brane space.

MATERIALS AND METHODS

Yeast Strain and Culture Conditions-The wild type S. cereuisiae strain D 273-10B (a; ATCC 25657) was grown aerobically on a medium containing per liter: 3 g of yeast extract (Difco), 1 g of glucose, 1 g of KHzP04, 1 g of NHdC1, 0.5 g of CaCL.2Ha0, 0.5 g of NaC1, 0.6 g of MgS04.H20, 0.3 ml of 1% FeCL, and 22 ml of 90% lactic acid. The final pH was adjusted to 5.5 with KOH. Cells were grown to the early logarithmic phase (120-130 Klett units). The yield was approximately 3 g (wet weight)/liter.

Preparation of Yeast Mitochondria-Cells were harvested by centrifugation (5 min at 3,000 X g), washed once with distilled water, suspended to 0.5 g, wet weight/ml in 0.1 M Tris.SO,, pH 9.4, 10 mM dithiothreitol, and incubated for 10 min a t 30 “C. They were then washed once with 1.2 M sorbitol and suspended in 1.2 M sorbitol, 20 mM KP,, pH 7.4, to give 0.15 g of cell, wet weight/ml. Zymolyase 5000 (5 mg/g of cell, wet weight) was added and the suspension was incubated a t 30 “C with gentle shaking. Conversion to spheroplasts was checked as described (10). After 50-60 min, all the cells had usually been converted to spheroplasts. Spheroplasts were harvested by centrifugation for 5 min a t 3000 rpm in a Sorvall GS-3 rotor at room temperature and washed twice with 1.2 M sorbitol. For homog- enization, spheroplasts were suspended in 0.6 M mannitol, 10 mM Tris- C1, pH 7.4, 0.1% bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride to a concentration of 0.15 g of spheroplasts (wet weight)/ml. After chilling on ice, spheroplasts were homogenized by 10-15 strokes in a tight-fitting Dounce homogenizer. From this point on, all opera- tions were carried out at 0-4 “C. All subsequent centrifugations were carried out in a Sorvall SS-34 rotor a t 2 “C.

The homogenate was diluted with 1 volume of the homogenization buffer and centrifuged for 5 min a t 3500 rpm. The supernatant was saved, and the pellet was rehomogenized as before and recentrifuged a t 3500 rpm. The supernatants were combined and crude mitochon- dria were sedimented at 9000 rpm for 10 min. The pellet was carefully resuspended in the homogenization buffer and the suspension was centrifuged for 5 min at 3500 rpm to remove residual cell debris. The supernatant was saved and centrifuged a t 9OOO rpm for 10 min. The mitochondrial pellet was washed twice by resuspension and recer,trif- ugation at 9OOO rpm for 10 min; for the last wash, bovine serum albumin and phenylmethylsulfonyl fluoride were omitted from the washing buffer. Mitochondria were resuspended in 0.6 M mannitol, 10 mM Tris-C1, pH 7.4, to give an approximate final concentration of 10 mg of protein/ml.

Isolation of the Mitochondrial Intermembrane Space-A suspen- sion of mitochondria (10-20 mg of protein/ml in 0.6 M mannitol, 10 mM Tris-C1, pH 7.4) was diluted with 5 volumes of 10 mM Tris-C1, pH 7.4, to a final mannitol concentration of 0.1 M. The suspension was

13028

Subfractionation of Yeast Mitochondria 13029

stirred very gently on a magnetic stirrer a t 0 "C for 20 min. The "shocked" mitochondria were sedimented a t 20,000 rpm in a Sorvall SS-34 rotor for 20 min. The supernatant contains the contents of the intermembrane space.

Isolation of Matrix and the Inner and Outer Mitochondrial Membrane-Shocked mitochondria (cF above) were resuspended in 10 mM Tris-C1, pH 7.4, to a protein concentration of about 2 mg/ml with 5 strokes in a loose-fitting Dounce homogenizer and left on ice to allow further swelling of the mitochondrial matrix space (11). After 5 min, "shrinking buffer" (one-third of the suspension volume), con- taining 1.8 M sucrose, 8 mM ATP, 8 mM MgC12, adjusted to pH 7.4 with KOH, was added. The suspension was mixed carefully by 3 strokes in the loose-fitting Dounce homogenizer and left on ice. After 5 min, 20-ml aliquots were exposed to ultrasonic irradiation for 3 X 5 s at 0 OC (Brownsonic, maximal output).

Total mitochondrial membranes were sedimented for 60 min at 35,000 rpm in a Beckman Type 40 rotor a t +2 " C . The supernatant represents the matrix fraction.

The mitochondrial membranes were vigorously resuspended in a minimal volume of 10 mM Tris-C1, pH 7.4, and layered on a 34-ml linear 30-5056 sucrose gradient buffered with 10 mM Tris-C1, pH 7.4. Gradient centrifugation was carried out in an SW-27 rotor at 20,000 rpm for 15 h. The small band of outer membrane on top of the gradient and the broad band of the inner membrane near the bottom were collected, diluted 3-fold with 10 mM Tris-C1, pH 7.4, and sedi- mented for 60 min at 35,000 rpm in a Beckman Type 40 rotor. Outer and inner mitochondrial membranes were resuspended in a minimal volume of 10 mM Tris-C1, pH 7.4.

Assays of Enzymes and Protein-Published procedures were used to assay fumarase (12), aconitase (12), cytochrome bl(13), cytochrome c peroxidase (14), myokinase (ll), cytochrome c oxidase (15), and kynurenine hydroxylase (4). Protein concentrations were assayed according to Lowry et al. (16) with crystalline bovine serum albumin as the standard.

Immunoreplica Technique-Proteins were transferred from SDSY- gels to Millipore nitrocellulose sheets by the method of Towbin et al. (17). After transfer, the Millipore sheets (about 10 X 15 cm) were incubated for 2 h a t 23 "C with serum buffer and then a t 4 "C overnight with 10-30 p1 of antiserum dissolved in 25 ml of serum buffer. After washing out excess antibody with serum buffer (3 X 25 ml; 30 rnin each a t 23 "C), immune complexes were decorated with "'I-protein A (2 X 10' cpm in 25 m1 of serum buffer; 2.2 X IO"' cpm/ mg of protein A) for 3 h a t 23 "C. After 3 further washes with serum buffer for 30 min each a t 23 "C, the Millipore sheets were dried and radioautographed.

Preparation of Antisera Against Cytochrome ba and the 29,000- dalton Protein of the Outer Membrane-A 29,000-dalton protein is the main protein of the mitochondrial outer membrane (18). This membrane was isolated by the procedure described above and elec- trophoresed on a semipreparative 12.5% SDS-gel slab (1-2 mg of protein/gel). Proteins were stained with Coomassie brilliant blue and the well resolved main protein band of apparent molecular weight 29,000 was cut out.

The protein was reisolated from the gel slices by electroelution. A 5% stacking gel was prepared at the cathode end of an electrophoresis tube and the cathode end was then closed with dialysis tubing. The gel slice with the 29,000-dalton protein to be isolated was layered on top of the stacking gel, the tube was fdled with electrophoresis buffer (19), and the anode end was closed with glass wool. The tube with the attached dialysis tubing was placed on an electrophoresis apparatus and subjected to electrophoresis a t 4 "C for 48 h a t 30 mA. Under these conditions, the protein migrated into the dialysis tubing. The dialysis tubing was then removed and the sample was dialyzed against double-distilled water for 20 h and against water/ethanol (70:30, v/v) for another 24 h. It was then lyophilized, dissolved in a small volume of phosphate-buffered saline, and stored in aliquots a t -20 "C. The protein isolated by this procedure migrated as a single band on a 12.5% SDS-gel. Samples of cytochrome 62 (kind gifts of Drs. F. Lederer and B. Matthews, CNRS, Gif-sur-Yvette, France and Wash- ington University, St. Louis, respectively) were subjected to SDS- polyacrylamide gel electrophoresis and reisolated as described for the 29,000-dalton outer membrane protein.

Antibodies against cytochrome bs and the 29,000-dalton protein were raised in pathogen-free male Chinchilla rabbits (2.5 kg in weight)

.' The abbreviations used are: SDS, sodium dodecyl sulfate; phos- phate-buffered saline, 0.14 M NaCI, 10 mM Nap,, pH 7.4; serum buffer, 5% newborn calf serum in phosphate-buffered saline.

which had been purchased from Hoechst Co., GFR. For the first injection, 250 pg of the protein antigen was dissolved in 300 pl of phosphate-buffered saline and mixed with 300 pl of Frend's complete adjuvant (Difco). The liquids were mixed with the aid of two con- nected syringes and injected subcutaneously in 5-6 portions. After 5 weeks, the first bleeding was taken from the ear vein and the animal was boosted intravenously with I00 pg of protein antigen dissolved in 300 pl of Freund's incomplete adjuvant. Three more boosts (100 pg of protein in 300 pl of phosphate-buffered saline, no Freund's adjuvant) were given a t intervals of 10 days and each time approximately 30 ml of blood was collected from the ear vein. Four more boosts (100 pg of protein each in 300 p1 of phosphate-buffered saline) were given in intervals of 20 days.

For the preparation of serum, blood was allowed to clot for 3 h a t room temperature. The clotted blood was triturated with a glass rod and the clot was removed by centrifugation on a Sorvall SS-34 rotor at 10,000 rpm for 10 min at 4 "C. The supernatant was collected and recentrifuged. The serum was stored in 3-ml aliquots at -20 "C.

For terminal bleeding, the rabbits were anesthetized intravenously with Numal, Roche (about 1 &kg), and then given 5 0 0 4 of Liquem- ine, Roche (5000 units of heparin/ml), intravenously to prevent blood clotting during the bleeding. Blood was taken b.v cutting the carotids and collected in a vessel which contained 500 pI of Liquemine. The blood was centrifuged for 15 min at 10,000 rpm in a Sorvall SS-34 rotor at 4 "C. The supernatant was saved and protamine sulfate (60 mg/100 ml of serum) was added to remove fibrinogen. The sample was mixed carefully and left overnight a t 4 "C. The precipitate was removed by centrifugation in a Sorvall SS-34 rotor a t 12,000 rpm for 10 min at 4 "C. Occasionally, the protamine-treated solution was left once again overnight at 4 "C and recentrifuged as described above. Samples of fibrinogen-free antisera were stored at -20 "C in 3-ml aliquots.

Characterization of Antisera-Antisera were tested by the im- munoreplica technique (17). Total mitochondrial proteins (1 mg) were electrophoresed on a SDS-12.5% polyacrylamide gel slab and electro- phoretically transferred to a nitrocellulose filter, and excess protein binding sites on the filter were blocked with serum buffer (cf. above). The filter was cut into strips which were then used for the immuno- logical test. Filter strips were incubated with antisera (0.05-10 p1) in 3 ml of serum buffer overnight, decorated with radioiodinated protein A ( IO6 cpm), and autoradiographed. This technique provides a simple and very sensitive method for checking the purity and the titer of antisera.

Miscellaneous-Protein A was obtained from Pharmacia Co. and iodinated by the chloramine-T procedure (20). I t was stored a t -30 "C in the presence of 30 mg of bovine serum albumin/mL Zymolyase was from Kirin Brewing Co., Tokyo. All other chemicals were of the highest purity available commercially. SDS-polyacrylamide gel elec- trophoresis in slab gels was performed according to Ref. 14. The preparation of an antibody against citrate synthase from yeast will be described elsewhere.'"

RESULTS

Selective Release of the Mitochondrial Intermembrane Space-When carefully isolated yeast mitochondria are ex- posed to media of decreasing tonicity, adenylate kinase (my- okinase) is progressively released from the organelles, whereas fumarase and aconitase are not released (Fig. 1). For example, exposure to 0.1 M mannitol for 20 min at 0 "C released between 90 and 98% of the myokinase, but less than 5% of the fumarase or aconitase. In a variety of mammals, adenylate kinase is a well established marker of the mitochondrial intermembrane space, whereas fumarase and aconitase are markers of the matrix space (11); exposure of yeast mitochondria to 0.1 M mannitol thus selectively solubilizes the intermembrane space contents. The particulate residue probably represents "mitoplasts" with an intact inner membrane since the full activities of aconitase and fumarase can only be measured after disruption of the inner membrane by detergents (not shown).

Cytochrome b2 (measured as L-lactate-ferricyanide reduc- tase) and cytochrome c peroxidase were released in parallel

H. Riezman, manuscript in preparation.

13030 Subfractionation of Yeast Mitochondria

with adenylate kinase; this suggests that they, too, aro com- ponents of the mitochondrial intermembrane space (Fig. 1). However, the experiment shown in Fig. 1 still leaves open the possibility that the easily released enzymes are merely loosely bound to the outer surface of the outer membrane. This was excluded by the demonstration that the activities of cyto- chrome b2 (measured with added cytochrome c as electron acceptor) and of cytochrome c peroxidase in intact mitochon- dria were very low if care was taken to preserve the integrity

100 I

0.6 0.5 O,‘+ 0.3 0.2 0.1

M MANNITOL

FIG. 1. Exposure of yeast mitochondria to 0.1 M mannitol releases the contents of the intermembrane space but not that of the matrix. Yeast mitochondria were incubated at 2.0 mg of protein/ml for 20 min a t 0 “C in the presence of the indicated final concentrations of mannitol and then reisolated by centrifugation (48,000 x g for 20 min). The activities of the enzymes listed in the figure were measured in the supernatants and with aliquots of the intact mitochondria (after suitable unmasking; see “Materials and Methods”). Cytochrome br (cyt b,) was measured as L-lactate-ferri- cyanide reductase (Ref. 13). CCPO, cytochrome c peroxidase; LDH, lactate dehydrogenase.

TABLE I Cytochrome b2 and cytochrome c peroxidase in intact yeast

mitochondria are inaccessible to externally added cytochrome c Enzymes were assayed in the presence of 0.6 M mannitol to prevent

rupture of the mitochondrial outer membrane during the assay. About 10 p g of mitochondrial protein were used per assay.

Activitv” with Activitv“ with

-fold -fold Cytochrome 62 0.047 0.595 12.7 6.29 6.43 1.02 Cytochrome c 0.021 0.298 14.2

oeroxidase

I‘ Micromoles of cytochrome c or ferricyanide reduced or oxidized per min per mg of mitochondrial protein.

TABLE I1 Cytochrome ba is inaccessible to antibody in intact yeast

mitochondria The experiment was identical with that outlined in Table I with

the following modifications: (a) only L-lactate-ferricyanide reductase was measured (b) the mitochondria (200 vg of protein) were prein- cubated in a final volume of 0.2 ml in 0.6 M mannitol. Where indicated, 50 p1 of cytochrome ba antiserum or 0.1% Triton X-100 was present. One-twentieth of the mixture was assayed.

Snecific activitv” Assay conditions Ta inhibition

Control +;::- -Triton X-100 6.29 6.56 None (4% stimulation) +Triton X-100 6.43 2.80 57

Micromoles of ferricyanide reduced/min X mg of protein.

I , , , , , I I ( ( I ,

1 2 3 L S 6 7 8 9 1011 1213

SPECIFIC A C T I V I T Y

BOTTOM FRACTION TOP

0 2

01

FIG. 2. Purification of the yeast mitochondrial outer mem- brane by sucrose gradient centrifugation. The total membrane fraction (about 100 mg of protein in 2 ml of 10 mM Tris-CI, pH 7.4) obtained after sonication of yeast mitochondria (see “Materials and Methods”) was layered onto a 34-ml linear 30-508 (w/v) sucrose gradient that was buffered with 10 mM Tris-CI, pH 7.4, and centrifuged for 15 h at 20,000 rpm in a Spinco SW 27 rotor a t +2 “C. Samples of 3 ml each were collected from the bottom and assayed for protein concentration and enzyme activity. CUT-OX, cytochrome oxidase; KYN-OH, kynurenine-hydroxylase.

6 K Y N U R E N I N E - H Y O R O X Y L A S E

4 3

20:-pJ 0

0 20 Lo 60 BO l o o

% O F PROTEIN

FIG. 3. Distribution of marker enzymes in subfractions of yeast mitochondria. Yeast mitochondria were subfractionated as outlined under “Materials and Methods” and each subfraction was assayed for the indicated enzymes. The ordinate denotes the relative specific enzyme activity, the specific activity in intact mitochondria being taken as 1.0. The abscissa denotes the percentage of mitochon- drial protein recovered in each subfraction. Actual recovery ranged from 85 to 103%; for each marker, the actual recovery value was taken as 100% for computing the values listed in the figure. A, matrix; B, inner membrane; C, intermembrane space; D, outer membrane.

of the outer membrane. In contrast, full activity of cytochrome b:! was observed even in intact mitochondria if added ferricy- anide was used as electron acceptor (Table I). Since ferricya- nide can readily penetrate across the outer, but not the inner,

Subfractionation of Yeast Mitochondria 13031

membrane whereas cytochrome c cannot penetrate across either of the two membranes ( l l ) , cytochrome bz and cyto- chrome c peroxidase must be located in the mitochondrial intermembrane space. A location of cytochrome bz on the outer face of the outer membrane is also excluded by the observation that an antibody cannot reach the cytochrome bz of intact mitochondria (Table 11).

Isolation of Matrix, Inner Membrane, and Outer Mem- brane-Severe hypotonic shock of the “mitoplasts” followed by ATP-induced shrinking (11) and subsequent sonication solubilized the contents of the matrix space. Inner and outer membrane could then be separated from each other by sucrose density gradient centrifugation (Fig. 2). To monitor separa- tion, cytochrome c oxidase (1 1) and kynurenine hydroxylase (4) were used as markers for inner and outer membrane, respectively. The results of the fractionation procedure are summarized in the diagrams of Fig. 3.

Polypeptide Composition and Purity of the Mitochondrial Subfractions-Fig. 4A shows the polypeptide pattern of an aliquot of each subfraction which had been derived from 100 pg of mitochondria. This presentation reveals the absolute distribution, rather than the enrichment, of a given band among the four subfractions. In contrast, Fig. 4B shows the

polypeptide pattern of equal amounts (protein basis) of each of the too membranes. The intermembrane space features a prominent band at 57 kilodaltons which was shown to be the apoprotein of cytochrome bz (see also below). In the lactate- grown cells used here, cytochrome bz is thus by far the most abundant polypeptide of the intermembrane space and can serve as a useful reference polypeptide for that space. The most prominent component of the outer membrane is a 29- kilodalton polypeptide which has been noted earlier in many different types of mitochondria (18, 21, 22). It is almost cer- tainly the protein forming the voltage-controlled pores (exclu- sion limit 5000-6000 daltons) across the mitochondrial outer membrane (23). Since these pores are probably the most characteristic feature of the mitochondrial outer membrane, a monospecific antibody against this polypeptide (see “Materials and Methods“) is a powerful tool for identifying outer membranes. The most useful polypeptide marker of the inner membrane is a prominent band of apparent molecular weight 34,000. We have identified it as the adenine nucleotide translocase by immune replication with a specific antiserum (kindly donated by Dr. P. Vignais, Grenoble, France).

Comparison of the gel patterns shown in Fig. 4 reveals that there is very little cross-contamination between intermem-

A

MIT IMSMAT OM IM - ”

I

-61

-4:

B I I

I

22-

FIG. 4. Polypeptide composition of submitochondrial fractions. A (left): an aliquot of each subfraction (de- rived from lOOpg of intact mitochondria) was electrophoresed on a 12.5% SDS- polyacrylamide gel slab and the sepa- rated polypeptide bands were stained with Coomassie brilliant blue. Arrows indicate the positions of the standard proteins bovine serum albumin (68 kilo- daltons), ovalbumin (45 kilodaltons), horse heart cytochrome c dimer (25 kilodaltons), and monomer (12.5 kilodal- tons). B (right): equal amounts (50 pg of protein) of inner and outer membrane were electrophoresed on an exponential 10-158 polyacrylamide gradient gel and stained as above. These fractions were derived in a separate experiment by H. Riezman”; only the peak fractions of outer membrane were collected after su- crose gradient centrifugation.

13032 Subfractionation of Yeast Mitochondria

TABLE 111 Summary of subfractionation of yeast mitochondria

Maximal Contamination" by

Fraction Actual Outer Inter- yield" mem- mem- mem- Matrix

Inner

brane brane

R 7 0 i7C B Outer membrane 40 25.1 18.4 0.1 Intermembrane space 254' 0.1 0.1 12.0 Inner membrane 412 10.6 1.5 16.7 Matrix 289 2.5 2.6 <O.l

Micrograms of protein obtained from 1 mg of mitochondrial protein.

Calculated from enzyme measurements. The specific activity of each marker enzyme in its appropriate fraction is taken as 100%. For example, contamination of intermembrane space by matrix was cal- culated as follows:

Specific activity of fumarase in intermembrane space Specific activity of fumarase in matrix x 100%

' For unexplained reasons, the protein assay of Lowry et al. seems to overestimate the protein content of the intermembrane space as compared to Coomassie blue staining after gel electrophoresis. For this reason, the contamination of outer membrane by intermembrane space is probably overestimated.

brane space and matrix. This is also borne out by Table I11 which gives the specific activity of typical marker enzymes for each mitochondrial subfraction (cL Fig. 3). On the other hand, measurements of marker enzymes (Table 111) reveal that the membrane fractions are significantly contaminated by other subfractions. Table I11 also lists the yields of each subfraction for a typical experiment.

Cross-contamination of the submitochondrial fractions was also checked by electrophoresing each fraction in an SDS- polyacrylamide gel slab and testing for the presence of typical markers by immune replication. The polypeptides tested for were citrate synthase (matrix), cytochrome c oxidase subunit V (inner membrane), cytochrome b2 (intermembrane space), and the major 29,000-dalton protein of the outer membrane. Although the immune replicas were not rigorously quantified, they essentially confirmed the results obtained by assaying marker enzymes: there was noticeable cross-contamination of the membrane fractions, but only little cross-contamination between matrix and intermembrane space (not shown).

DISCUSSION

This paper describes the isolation and characterization of the two membrane-bound soluble compartments of yeast mi- tochondria, as well as the two membranes themselves. We have been able to prepare matrix and intermembrane space fractions which are only slightly cross-contaminated by each other. This subfractionation procedure therefore has enabled us to show that cytochrome b? and cytochrome c peroxidase are located in the intermembrane space.

Our present data confirm, and provide a logical explanation for, some early observations by Somlo (25) who had found that mitochondria-bound L-lactate dehydrogenase was less inhibited by its antibody than the soluble enzyme. S o d o also had noted that the mitochondria-bound enzyme could not reduce externally added cytochrome c, whereas it readily reacted with ferricyanide. Since the different permeability properties of the two mitochondrial membranes were then not yet known, Somlo suggested alternate explanations for these phenomena.

Based on the results from either enzyme assays or immu- nological detection, we observed significant cross-contamina- tion of the two membrane fractions by each other and by the

soluble spaces. The outer membrane is approximately 20% contaminated with inner membrane. This contamination can be lowered by a careful fractionation of the linear sucrose gradient, but only at the expense of a lower yield of outer membrane. The inner membrane fraction contains significant amounts of matrix and outer membrane. This problem has also been encountered in previous efforts to subfractionate mammalian mitochondria (11). Reasons for this contamina- tion are unknown: matrix enzymes may become irreversibly trapped within inner membrane vesicle whereas outer mem- brane may remain attached to the inner membrane via "membrane junctions" which have been found in mitochon- dria from different sources (26).

To what extent are the mitochondria used to prepare these fractions contaminated by nonmitochondrial components? Immunoreplication tests with antiserum against the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase indicates that components of the soluble cytosol account for t 5 % of the mitochondrial protein. Contamination by nonmitochondrial membranes is likely, but difficult to estimate accurately be- cause a reliable marker of nonmitochondrial membranes is not yet available for yeast.

A comparison of the four mitochondrial subfractions (Fig. 4) has allowed us to identify three mitochondrial proteins (70, 45, and 14 kilodaltons) which co-fractionate with the 29-kilo- dalton marker polypeptide and are, thus, apparently compo- nents of the outer membrane.4 The mitochondrial intermem- brane space fraction consists mainly of the marker enzyme cytochrome b2 (58 kilodaltons) and two protein bands corre- sponding roughly to 31 kilodaltons, with 4 or 5 additional minor bands not visible in Fig. 4.4. The three protein bands corresponding to between 68 and 90 kilodaltons appear to be contaminants from the matrix fraction.5

While the isolation of subfractions from yeast mitochondria and the identification of protein components of these subfrac- tions is important in itself, the long term objective of this work was to learn more about how cytoplasmically made polypeptides are transported into mitochondria. As described in the accompanying papers, the results reported here have led to several new findings. One of these findings was that isolated mitochondria can transport in uztro-made precursor polypeptides to their correct intramitochondrial location (3, 27). Another finding was that a specific protease processing imported precursors is located in the matrix (28, see also Footnote 6). A particularly intriguing finding was that precur- sors of the intermembrane space enzymes cytochrome bz and cytochrome c peroxidase are processed to their mature size in two steps, one of them occurring in the matrix (27). This is one of the reasons why we attempted to establish the inter- membrane location of the mature forms of these two cyto- chromes as rigorously as possible.

Yeast is currently one of the most useful organisms for studying the biogenesis of mitochondria. The availability of defined mitochondrial subfractions from this organism should help to answer some unsolved questions on how mitochondria are made.

Acknowledgments-We wish to thank Greti Schneider, Simone Straumann, Wolfgang Oppliger, and Kitaru Suda for excellent tech- nical assistance. The antisera against the adenine nucleotide trans- locator and citrate synthase from yeast were kind gifts of Dr. Pierre Vignais (Grenoble) and Dr. Howard Riezman (Basel), respectively. Drs. Frangoise Lederer and Brian Matthews gave us valuable samples of cytochrome b2 which were used for preparing the cytochrome br

~. .

H. Riezman and R. Hay, unpublished results. "

'I R. Hay, unpublished results. " P. C. Bohni, manuscript in preparation.

Subfractionation of Yeast Mitochondria 13033

antisera. One of us (G. S.) also wants to express his gratitude to Lars Ernster for many enjoyable and instructive discussions on the mys- teries of the mitochondrial outer membrane.

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