Preparation and Some Properties of Yeast Mit,ochondria · Preparation and Some Properties of Yeast...

THE JOURNAL OF BIOLOGICAL CHE>I\~~STRY Vol. 241, No. 8, Issue of April 25, 196fi

Printed in U.S.A.

Preparation and Some Properties of Yeast Mit,ochondria

(Received for publication, July 2, 1965)

TO~MOKO OHNISHI,* KUMIKO KAWAGUCHI, AND BUNJI HAGIHARA

From the Department of Biochemistry, Medical School, Osaka University, Osaka, Japan

SUMMARY

Well preserved mitochondria were isolated from Saccharo- myces carlsbergensis by a procedure which involves digestion of the cell wall with snail gut juice. The yeast mitochondria thus prepared oxidized members of the tricarboxylic acid cycle, reduced nicotinamide adenine dinucleotide, D- and L-lactate, and tetrarnethyl-p-phenylenediamine reduced by ascorbate. Respiratory control in response to the addition of adenosiue diphosphate was observed with all of these substrates. The highest respiratory control ratio (5 to 6) was obtained with ar-ketoglutarate as substrate. The yeast mitochondria actively oxidized externally added NADH, and this oxidation likewise showed respiratory control. The ADP to oxygen or phosphorus to oxygen ratios observed in this system were about 0.9 with tetramethyl-p-phenylenediamine and lactate; 1.7 with succinate, NADH, citrate, and pyruvate plus catalytic amount of malate; and 2.5 with cu-ketoglutarate. These respirations were completely insensitive to Amytal and rotenone. It is suggested that phosphorylation Site I is absent from these yeast mitochondria. Effects of uncouplers and inhibitors of oxidative phosphorylation on yeast mitochondria were very similar to those observed with mammalian mitochondria. The P:O ratios in the respiration with NADH as a substrate showed constant values when measured between pH 5.4 and 7.5, and at a range of tonicity between 0.3 and 1.0 M sorbitol. The yeast mitochondria could be stored for a long period of time without appreciable loss of phosphorylation activity. The components of electron transport of isolated yeast mitochondria were studied by means of difference spectrophotometry. Electron micrographs of the isolated yeast mitochondria and mitochondria in sifu were also presented.

Yeast is one of the simplest organisms in which one can find mitochondria (l-6). This organism is suitable for the study of oxidative phosphorylation, since the physiological (7, 8) and genetic (9-12) state of the cell can be modified easily by chang- ing conditions of cultivation. However, studies of oxidative phosphorylation in cell-free yeast systems have lagged considerably behind those with the mammalian systems, owing mainly to

* Present address, Department of Physiological Chemistry, University of Stockholm, the Wenner-Gren Institute, Norrtulls- gatan 16, Stockholm S’a, Sweden.

the difficulty encountered in the preparation of yeast mitochondria, which are physiologically intact. The cell wall of yeast is quite resistant to mechanical breakage, and the preparations of mitochondria which have been isolated from this organism by the usual cell disruption procedures tend to be damaged. Thus, such mitochondria exhibit low efficiency of phosphorylation and poor control of respiration in response to added ADP. In 1959, Heyman-Blanchet, Zajdela, and Chaix (13, 14) reported that disruption of the cell wall by digestion with snail gut juice (15, 16) could be used successfully for the isolation of yeast mitochondria. Mitochondria thus isolated appeared fairly intact as judged by their spectral and morphological properties. How- ever, their oxidative and phosphcrylative activities have not yet been investigated. We have reported two procedures for the isolation of essentially intact yeast mitochondria in prelim- inary communications (17-19). The first method involves treatment with snail gut juice, and the other, a mechanical procedure, uses a specially designed mill-type glass homogenizer. The mitochondria prepared by either of these procedures possess high efficiency of phosphorylation and ratios of respiratory control. Duell, Inoue, and Utter (20) reported the isolation of yeast mitochondria from spheroplasts of Saccharomyces cerevisiue. They showed that mitochondria prepared by treatment with snail gut juice are more intact than mitochondria prepared by the Nossal shaker method, as judged by comparison of their respiratory control ratios and the distribution of protein and cytochrome oxidase activity in the cell-free homogenate prepared by the two procedures. In the present communication, we wish to report data relating to phosphorylating efficiencies of respiration with various substrates; the effect of inhibitors, pH, and tonicity on respiratory control and P:O ratios; and the stability of these activities during storage of mitochondria from Succhuro- myces carlsbergensis prepared by the snail gut juice procedure.

MATERIALS AND METHODS

Yeast Cells-S. carlsbwgensis was used in the present study, because its cell wall is highly sensitive to digestion by snail gut juice. The cells were first cultivated aerobically at 30” for 36 hours in a liquid medium containing potassium lactate (2.0%), yeast extract (0.5%), (NHI)zHPO~ (0.6%), and MgS04.7HzO (0.2%) at pH 4.4. Of this culture, 1 ml was inoculated into 1 liter of fresh medium of the same composition and incubated in a IO-liter bottle with vigorous shaking for 18 to 20 hours. When the cells were in the logarithmic growt,h phase, they were ml-

lected by centrifugation at 700 x g for 3 min and washed four times with distilled water.

Preparation of Protoplasts-The harvested cells were washed

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1798 Yeast Al’itochondria Vol. 241, Ko. 8

twice with a medium containing 1.3 .\I sorbitol, 0.1 m&r EDT.1,

and 10 mM Tris-maleate buffer (pH 5.7) (centrifugation at 5000 x g for 5 min) and suspended in the same medium at a concentration of about 0.5 g of wet cells per ml. Partially purified snail gut juice (18) was added to the suspension (1 to 1.5 mg of protein per ml of suspension), and the mixture was incubated at 30”. Within 1 to 1; hours of incubation, 80 to 90% of the yeast cells were degraded to protoplasts. The protoplasts could be distinguished easily from intact cells under a phase contrast microscope. The protoplasts were then sedimented by centrifugation at 4000 x g for 5 min and washed twice with the incubation medium containing no gut juice.

Preparation oj Mitochondria-The washed protoplasts were suspended (about 0.1 g per ml) in a medium (“mitochondrial preparation medium”) containing 0.65 M mannitol and 0.1 rn3f EDTA (adjusted to pH 6.5 by the addition of Tris base). This resulted in lysis of the protoplasts by osmotic shock. To ensure complete breakage, the suspension was dispersed with a Waring Hlendor for 30 set at medium speed. This suspension was centrifuged at 2100 x g for 5 min to remove cell debris, and the resultant supernatant fluid was then centrifuged at 8500 x g for 9 min. The sediment was washed two or three times with the mitochondrial preparation medium. The mitochondria had a reddish brown color. The procedure gave a yield of 5 to i mg of mitochondrial protein from 1 g of wet cells.

Assay oj Ozidative Phosphorylation-Oxygen consumption was measured according to Hagihara (21) in a closed cell equipped with a rotating platinum electrode. The reaction medium contained 0.5 to 0.65 M mannitol, 10 mM potassium phosphate buffer, 10 to 20 mM Tris-maleate buffer (pH 6.5), 0.1 mM EDTA, and 10 rnnl HCI. The reaction was carried out at 25”. P:O ratios were determined by two methods: (a) a graphic analysis of the polarographic tracings recorded in response to the addition of a definite amount of ADP as described by Chance and Williams (22), and (b) determination of incorporation of 32P into glucose 6-phosphate in the presence of hexokinase and glucose by the method of Hagihara and Lardy (23). Respiratory control ratios (RC ratios) were calculated from the polarographic tracings by the method of Chance and Williams (22) according to the equa- tion,

RC r atio _ State 3 respiration rate (in the presence of ADP)

State 4 respiratory rate (after exhaustion of ADP)

Diflerence Spectrophotometry-Difference spectra of respiratory carriers in yeast mitochondria were determined in a split beam spectrophotometeri (24, 25).

Determination oj Protein Content-Protein was determined by the biuret method (26) or by the method of Lowry et al. (27), with crystalline bovine serum albumin as standard.

Assay of A TPase nctivity-Yeast mitochondria were incubated at 25” in a reaction medium containing 0.65 M mannit.01, 0.2 mM EDTA, 20 mM Tris-maleate buffer (at the values of pH shown in the legends), and 1 mM BTP. The reaction was stopped by the addition of perchloric acid, and inorganic phosphate liberated was assayed by measuring the optical density at 310 rnp of the phosphomolybdic acid complex after extraction with butyl acetate (28). Liberation of inorganic phosphate was assayed at

1 This instrument was built at the Johnson Foundation. It was slightly modified by Hagihara in Osaka. Its construction will be described in more detail elsewhere.

I5 set, and 2, 4: and 7 min after the addition of mitochondria. The reaction rate during this time was essentially constant.

Reagents-Oligcmycin and antimycin ;1 were dissolved in absolute methanol. 2,4-IXnitrophenoI and pentachlorophenol were used in aqueous solutions at alkaline pH. Tri-n-butyl tin chloride was dissolved in absolute methanol and diluted with water.

RESULTS

Electron Jlicroscopy of Yeast Cell and Isolated Mitochondria- Fig. I shows an electron micrograph of a thin section of a yeast cell which had been fixed with 4% potassium permanganate; cells growing exponentially with lactate as the carbon source were used. As can be seen, such cells did show typical mitochondrial profiles. In Fig. 2 is shown an electron micrographs of a thin section of yeast mitochondria, isolated as described in this paper and fixed in buffered osmium tetroxide. Practically no contamination by other subcellular components was observable, and the internal structure of the mitochondria was fairly well preserved.*

Respiratory Control-As shown in Table I, these yeast mitochondria were able to oxidize various members of the tricarboxylic acid cycle, such as citrate, isocitrate, a-ketoglutarate, succinate, and pyruvate (plus a catalytic amount of malate). In addition, externally added NADH, I)- and L- lactate, ethanol, and tetramethyl-p-phenylenediamine reduced by ascorbate were also actively oxidized. With all of these substrates, the respiratory rate was dependent on the presence and absence of ADP. The respiratory patterns obtained with the polarographic method with cY-ketoglutarate and KADH as substrates are shown in Fig. 3. It is evident that the addition of ADP accelerated the respiratory rate with both substrates. After a while, the rate of respiration declined to the same level as before ADP addition and was accelerated again upon further addition of ADP, indicating that all of the ADP added initially had been phosphorylated to ATP. A respiratory control ratio of 5.0 to 6.0 was obtained with a-ketoglutarate as substrate (Fig. 3a) ; this value is as high as that usually obtained with carefully prepared mammalian mitochondria.

i\nother remarkable feature of the yeast system was that externally added NADH was oxidized with a rather high RC ratio of 3.0 to 3.6 (Fig. 3b). This has never been observed with mammalian mitochondria. As reported previously (18), respiratory control could also he demonstrated clearly with substrates other than a-ketoglutarate and NADH, although the observed RC ratios were lower than 2.0 (Table I).

Phosph,orylation EfFciency-In the experiments recorded in Table I, the efficiency of phosphorylation was calculated from the polarographic respiratory tracings according to Chance and Williams (22) and expressed in terms of BDP:O ratios. The efficiency could also be expressed as P:O ratios. Table II shows P:O ratios in the presence of an ATP-trapping system as determined by the method of Hagihara and Lardy (23). It can be seen that the P:O ratios were in good agreement with the cor-

2 This electron micrograph was kindly taken by Dr. T. Oda of Okayama University.

3 This electron micrograph was kindly taken by Dr. Y. Shina- gawa of Kyoto University, by use of the slightly modified procedure of Shinagawa and Uchida (29).

4 More detailed studies of the morphology of yeast mitochondria will be published elsewhere.


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Issue of April 25, 1966 T. Ohnishi, K. Kawaguchi, and B. Hagihara

FIG. 1. Electron micrograph of a section of a yeast cell. The organisms were cultured in a lactate medium and harvested in the log phase.

responding ADP:O ratios. It is clear from the results reported in Tables I and II that the ADP: 0 or P :0 ratio was about 1.8 and 1.0 with succinate and with tetramethyl-p-phenylenediamine plus ascorbate as substrates, respectively. These data indicate the presence of two phosphorylation sites in succinate oxidation and one site in tetramethyl-p-phenylenediamine oxidation, just as in the case with mammalian mitochondria. In contrast to mammalian mitochondria, however, the ADP:O or P:O ratio with yeast mitochondria never exceeds 2.0 with externally added NADH or with NAD-linked substrates such as citrate, pyruvate plus malate, etc. With D- and L-lactate, which are not oxidized by mammalian mitochondria, a P:O ratio of about 1.3 was obtained. However, this ratio was lowered to 1 .O or even less when antimycin A was added to the reaction mixture. It seems likely that only one phosphorylation site is involved in the oxidation of D- and L-lactate, and this site is the same as the one involved in tetramethyl-p-phenylenediamine oxidation. The P : 0 ratios ex- ceeding 1.0 with lactate as substrate in the absence of antimycin

A probably are caused by oxidation of pyruvate derived from la&ate. The P:O ratio obtained in the oxidation of a-ketoglutarate was definitely higher than that obtained with the other NAD-linked substrates. This high ratio (2.3 to 2.6) suggests the involvement of a substrate level phosphorylation site in ar-ketoglut.arate oAxidation.

Eflect of Inhibitors on the Electron Transfer Systems-KCN (2.1 x 10-Z M) inhibited the respiration almost completely with every substrate, just as in the case of mammalian mitochondria. Antimycin A (2.3 x 10Vg mole per mg of protein) (22, 30) blocked respiration with various substrates such as cr-ketoglutarate, NADH, and succinate, while it gave only partial inhibition of respiration with u- and L-lactate and had no effect on the tetramethyl-p-phenylenediamine-ascorbate system (cf. Table II).

Fig. 4 shows difference spectra of the mitochondrial suspension at various states of respiration with citrate as substrate. Partial reduction of cytochromes b and c + cl, and of flavoprotein, was observed in State 4 (Curve b). Pyridine nucleotide was reduced


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Yeast Mitochondria Vol. 241, No. 8

FIG. 2. Electron micrograph of isolated yeast mitochondria

considerably in this state, although this is not illustrated in the figure. When oxygen was exhausted (State 5), all the known respiratory carriers were extensively reduced (Curve c). The extent of the reduction was essentially the same as that obtained by the addition of dithionite, except in the case of the cytochrome b type and of flavoproteins. When antimycin A (2.0 x 10eg mole per mg of protein) was added to the sample in State 5, followed by aeration, practically all the cytochrome b (563, 532, 431 mp) and some flavoprotein (450 to 460 rnp) remained reduced (Curve d). These results show that the behavior of yeast mitochondria is very similar to that of mammalian mitochondria. As will be reported elsewhere, the content of respiratory carriers was quite variable depending on the strain of yeast, growth condi-

tions, and growth phase. Leakage of cytochrome c from mitochondria during preparation appeared to be negligible, because no cytochrome c was detected in the supernatant fluid remaining after the sedimentation of the mitochondria; addition of cytochrome c caused no acceleration of the respiration of isolated mitochondria.

Fig. 5A shows difference spectra of yeast mitochondria in n-lactate respiration, with antimycin A present both in the sample and in the reference cuvettes. On addition of n-lactate to the sample cuvette, while oxygen was present (about 1 min), very slight reduction of cytochrome a and partial reduction of cytochrome c + cl was observed (Curve 6). When oxygen was exhausted, extensive reduction of flavoprotein (460 mp), cyto-


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Issue of April 25, 1966 T. Ohnishi, K. Kawaguchi, and B. Hagihara 1801

chrome c + cl (550, 520, 421 mp), and cytochrome a (605, 444 rnp) occurred (Curve c). It was confirmed, by reduction with dithionite, that the reduction of cytochromes c + cl and a was complete at this stage.

The result of the same type of experiment with L-lactate is shown in Fig. 5B. On addition of L-lactate to the sample suspension under aerobic conditions, there occurred a partial reduction of cytochrome c + c1 and flavoprotein and a well marked reduction of a cytochrome of the cytochrome b type (presumably cytochrome bz, 557, 528, 424 mp) (31, 32). In State 5, flavoprotein (460 mp), cytochrome c + cl (550, 520, 421 mp), and cytochrome a (605, 444 rnp) were completely reduced (Curve c’). This is similar to the case with n-lactate. These results show

that the electron transfer pathways of D- and n-lactate respiration do not involve the antimycin A-sensitive site in yeast mito-

chondria.

TABLE I

RC ratio, ADP:O ratio, and Qo, in various substrate oxidations

The reaction medium contained 0.65 M mannit.01, 10 m&r potas-

sium phosphate buffer (pH 6.5), 10 mM Tris-maleate buffer (pH 6.5), 10 rnM KCI, and 0.1 mM EDTA. Substrates were added at a final concentration of 6 mM, except in the cases of NADH and tetramethyl-p-phenylenediamine when 560 and 100 uM were used, respectively.

Substrate Qo*= (State 3) RC ratio

a-Ketoglutarate. .......... Citrate. .................. Isocitrate ................. Pyruvate + malate. ...... Ethanol, .................. NADH. ...................

Succinate ................. n-Lactate ................. L-Lactate ................. Tetramethyl-p-phenylene-

diamine + ascorbate. ... cy-Glycerophosphate. ......

0.26-0.41 5.5-6.0 2.3-2.6 0.20.45 1.5-1.7 1.7-1.8 0.14-0.20 1.2-1.5 1.6-1.8 0.15-0.35 1.6-2.0 1.6-1.8

0.11-0.30 1.4-1.5 1.6-1.8 0.60-1.10 3.0-3.6 1.5-1.8 0.25-0.38 1.4-1.7 1.61.8

0.50-0.75 1.1-1.5 0.9-1.1 0.150.20 1.1-1.3 0.9-1.1

o.s(M.70 0.08-0.30

1.2-1.5 0.8-1.0

-

I _-

SDP:O ratio

0 Microatoms of oxygen uptake per min per mg of protein.

Time -)

FIG. 3. Tracings of respiratory patterns with a-ketoglutarate &KG) or NADH as substrate. The reaction medium contained 0.5 M mannitol, 10 rnM potassium phosphate buffer (pH 6.5), 20 mM Tris-maleate buffer (pH 6.5), 10 mrvr KCl, and 0.1 mrvr EDTA. a, final protein concentration, 0.28 mg per ml of reaction mixture; b, 0.13 mg per ml. Other additions were indicated by arrows in the figure. The reaction was carried out at 25”. Yeast Mt., yeast mitochondria.

TABLE II

O&dative and phosphorylative activities with various substrates as

assayed by isotopic procedure

The reaction medium contained 0.65 M mannitol, 10 mM potassium phosphateJ2P (1.08 X 106 cpm per ml of reaction medium), 10 rnM Tris-maleate (pH 6.5), 10 mM KCl, and 0.1 m EDTA. Hexokinase (300 units per ml), 17 mM glucose, 0.62 mM MgC12, and 827 p~ ADP were added.

The concentration of mitochondrial protein was 0.27 mg per ml in a-ketoglutarate and citrate, 0.19 mg per ml in NADH, succinate, and n-lactate, and 0.42 mg per ml in L-lactate oxidation. Other conditions were the same as those in Table I.

Substrate

a-Ketoglutarate..

Citrate. NADH. . . Succinate . D-Lactate................... n-Lactate + lO-+M antimycin

A. . . L-Lactate................... L-Lactate + lo-” M antimycin

A. Tetramethyl-p-phenylenedi-

amine + ascorbate.. Tetramethyl-p-phenylenedi-

amine + ascorbate + 10V~ antimycin A. .

- Oz uptake

?Wp&WlSfd

158.0

137.0 193.1 132.5 162.0

160.0

138.5

117.5

129.5

147.0

Pi Uptake

362.0 2.30 255.0 1.86 315.0 1.63 227.0 1.71 208.0 1.28

162.0 1.00 183.0 1.32

107.0 0.91

144.3 1.12

147.0 1.00

I

450 500 Wave S$h in mp

P:O ratio

.I 605

w>-

-I 600

FIG. 4. Difference spectra of yeast mitochondria at various states of respiration with citrate as a substrate. a, base-line. The sample and reference cuvettes contained yeast mitochondria which were suspended in the reaction medium containing 0.65 M mannitol, 10 mM phosphate, 10 mM Tris-maleate buffer (pH 6.6), 10 mM KCl, and 0.1 mM EDTA. The incubation was carried out under aerobic conditions. Final concentration of mitochondrial protein was 3.5 mg per ml of reaction mixture. b, 5 II~M citrate was added to the sample cuvette under aerobic conditions (State 4). c, after exhaustion of oxygen in the sample cuvette (State 5). d, antimycin A (2.0 X lo+ mole per mg of protein) was added to the sample cuvette, and the mixture was aerated with oxygen.

It was observed that Amytal (3.0 X 10” M) and rotenone (1.5 x 10-g mole per mg of protein) did not inhibit the respiration of externally added NADH or NAD-linked substrates, such as

ar-ketoglutara,te and citrate. This feature is in sharp con-

trast to mammalian mitochondrial and submitochondrial sys-


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1802 Yeast MitochondGa Vol. 241, Ko. 8

621

I& ao~=0.01 -7

LL&LL~--L~ 500 550

Wave Length in mp

FIG. 5. Difference spectra of yeast mitochondria in D- and L-

lactate respiration in the presence of antimycin A. A, Curve a, base-line. Both sample and reference cuvettes cont,ained 5 mar citrate and antimycin A (2.0 X 1OW’ mole per mg of protein) and were aerated. Other conditions are the same as those in Fig. 4. Curve b, n-lactate was added to the sample cuvette under aerobic conditions (State 4). Curve c, after depletion of oxygen in the sample cuvette by n-lactate oxidation (State 5). B, Curve a’, base-line, similar to Curve a in Fig. 5. Curve b’, n-lactate was added to the sample cuvette under aerobic conditions (State 4). Curve c’, when oxygen in the sample cuvette was exhausted by L-lactate oxidation (State 5).

TABLE III

Effect of I,J-dinitrophenol and Mg++ on mitochond,ial ATPase activity

Reaction medium contained 0.65 Mmannitol, 0.2 mM EDTA, 20 mM Tris-maleate buffer at pH indicat,ed, and 1 mrvr ATP. Final protein concentration of mitochondria was 1.92 mg per ml. The initial ATPase activity was assayed by the procedure described under “Materials and Methods.”

PH

5.7

7.2

-

Concentration of Mg++

M

0.0 0.0

1.0 X 10-z

1 .o x 10-z

0.0

0.0 1.0 x 10-z 1.0 x 10-a

Concentration of Z&dinitrophenol

M

0.0 1.0 x lo-”

0.0 1.0 x 10-S

0.0 8.6 1.0 x 10-s 8.2

0.0 57.0

1.0 x 10-5 67.5

ipecific activity

mpmoles Pi/ min/mg pro1ein

2.5 7.6

15.2

24.8

terns (33) and appears to be a characteristic property of yeast mitochondria.

E$ect of Uncou,plers and Iizhibitors of the Energy Transfer Sys- tem-When 10-J M 2,4-dinitrophenol was added, the respiration in the absence of iZDP (State 4) was accelerated to the level of the respiratory rate in the presence of ADP (State 3) (34, 35).

Acceleration was also observed in a reaction medium which con-

tained no inorganic phosphate. Similar uncoupling by 2,4-

dinitrophenol was observed with various substrates except a-ketoglutarate (cf. Fig. 9;.

In the absence of both 2,4-dinitrophenol and Mg*, yeast mitochondria exhibited only slight ATPase activity as shown in

Table III. At pH 5.7, 2,4-dinitrophenol (lo-” nf) accelerated ATPase activity approximately S-fold, while at pH 7.2, 2,4- dinitrophenol gave no acceleration. In the range between pH 5.7 and pH 7.2, acceleration of ATPase activity by 2,4-dinitrophenol diminished gradually with increasing pH. Increase of the 2, ii-dinitrophenol concentration to 10W4 M did not accelerate the ATPase activity further. Activation of ATPase activity by aIg++ was more pronounced than that by 2,4-dinitrophenol, in particular at the higher pH values. The Mg++-activated ATPase was accelerated further by the addition of 2,4-dinitrophenol.

Fig. 6 shows the effect of various concentrations of pentachlorophenol on the P:O ratio and respiratory rate in State 3 (Fig. 6a), as well on the respiratory rate in State 4 (Fig. 6b), and on the ATPase activity (Fig. 6~). At concentrations higher than 1OV M, a decrease of the P:O ratio was observed, and complete uncoupling of phosphorylation occurred at 10e4 M pentachlorophenol (Fig. 6~). Acceleration of State 4 respiration occurred paralleling the uncoupling of oxidative phosphorylation (Fig. 6, a and b). However, at 1OV M pentachlorophenol, the respiratory rate was less than optimal, an effect which is presumably

due to a secondary inhibitory effect of pentachlorophenol on the respiratory system. Pent.achlorophenol stimulated the ATPase activity in a manner similar to that caused by 2,4-dinitrophenol;

maximum acceleration of ATPase activity was observed at lo+

M pentachlorophenol, which caused neither inhibition of phosphorylation nor acceleration of respiration.

a.0 7.0

FIG. 6. The effect of various concentrations of pentachlorophenol (PCP) on oxidation, phosphorylation, and ATPase activity. a, Qo, (microatoms of oxygen uptake per min per mg of protein) and P: 0 ratio in State 3 respiration with NADH as substrate; b, Qo, in State 4 respiration; c, ATPase activity. a and b, the reaction medium contained 0.5 M mannitol, 10 m&r potassium phosphate, 10 mM Tris-maleate buffer (pH 6.3), 10 mM KCl, and 0.2 rnM EDTA. NADH, 520 PM, was added as substrate. Final protein concentration of mitochondria was 0.20 mg per ml of reaction mixture. c, the reaction medium contained 0.5 M mannitol, 0.2 mM EDTA, 20 mM Tris-maleate buffer (pH 6.3), and 1 mM ATP. Final protein concentration of mitochondria was 0.91 mg per ml of reaction mixture. The same preparation of mitochondria was used in the three experiments described under a, 6, and c.


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Fig. 7 illustrates the effect of oligomycin on the respiration of yeast mitochondria with NADH (Curve a) or citrate (Curve b) as substrate. Oligomycin depressed the State 3 respiratory rate to the State 4 level. This effect is similar to that observed with mammalian mitochondria, except that the amount of oligomycin required for maximal inhibition appears to be considerably higher in the case of yeast, approximately 20 pg per mg of mitochondrial protein. 2,4-Dinitrophenol or pentachlorophenol relieved the inhibition of respiration by oligomycin, just as in the case of mammalian mitochondria. Oligomycin exerted similar effects on the respiration with other substrates except a-ketoglutarate (cf. Fig. 9).

As shown in Fig. 8, tri-n-butyl tin chloride at a concentration of 2 X lo+ M (1.5 X 1O-8 mole per mg of protein) partially inhibited State 3 respiration and also partially abolished phosphorylation. The effect is like that of oligomycin. However, at concentrations higher than 1 X 1O-5 M (7.5 X 10M8 mole per mg of protein), tri-n-butyl tin chloride reacted as a true uncoupler (Fig. 8), and at even higher concentrations (1 X lop4 M) it also inhibited respiration. Thus, in yeast mitochondria, tri-n- butyl tin chloride acts as a true uncoupler in a wide range of concentration, while with mammalian mitcchondria an inhibition of energy transfer is the predominant effect of this compound (36, 37).

As shown in Fig. 9, addition of 1 x 1OV M 2,4-dinitrophenol stimulated only slightly the State 4 respiration with cr-ketoglutarate, and subsequent addition of ADP further accelerated the respiration. However, the P : 0 ratio, as calculated according to Chance and Williams (22), decreased in the presence of 2,4- dinitrophenol. When 6 mM malonate was added to inhibit the oxidation of succinate, the stimulation of State 4 respiration with cy-ketoglutarate by 2,4-dinitrophenol became almost negligible. Increasing concentrations of 2,4-dinitrophenol did cause a lower- ing of the P:O ratio, but, the regulation of respiratory rate by ADP was always retained. With cr-ketoglutarate as substrate, the P:O ratios obtained in the presence of various concentrations of 2,4-dinitrophenol were consistently higher by 1 unit than those obtained in the oxidation of NADH, citrate, or pyruvate plus malate. These findings indicate that the phosphorylation at substrate level is not uncoupled by 2,4-dinitrophenol, in contrast to the respiratory chain phosphorylation (38). The data also

Time -

FIG. 7. Effect of oligomycin on the oxidation of NADH and citrate. a, final protein concentration of mitochondria, 0.23 mg per ml of reaction mixture; b, 0.32 mg per ml. Additions are indicated by 0~~0’1~‘s in the figure. Other experimental conditions were the same as those in Fig. 3, except that 0.65 M mannitol was used in this experiment. PCP, pentachlorophenol; yeast Mt., yeast mitochondria.

tJ5minl Time F

FIG. 8. Effect of tri-n-butyl tin chloride (TBTC) on the oxidation of NADH. Final protein concentration of mitochondria in a and b was 0.13 mg per ml of reaction mixture. Abbreviations and other conditions were the same as those in Fig. 7.

Time -

FIG. 9. Effect of 2,4-dinitrophenol (DATP) on the oxidation of cu.ketoglutarate (a-KG). Final protein concentration of mitochondria was 0.37 mg per ml of reaction mixture. Other conditions were the same as those in Fig. 7. Yeast Mt, yeast mitochondria.

suggest that the phoaphorylation site associated with the substrate level phosphorylation is rate-limiting in the over-all rate of State 4 respiration with a-ketoglutarate in yeast mitcchondria. This conclusion is further supported by measurement with the split beam spectrophotometer, where no reduction of electron carriers, except pyridine nucleotide, was observed in State 4 respiration with cY-ketoglutarate. The effect of oligomycin was apparently similar to that of 2,4-dinitrophenol, except that the State 3 respiration was partially inhibited. Tri-n-butyl tin chloride exerted essentially the same effect on a-ketoglutarate respiration as 2,4-dinitrophenol or oligomycin, except that tri-n- butyl tinchloridedid not accelerate the State 4 respiration even in the absence of malonate.

Efect of pH and Tonicity on NADH Oxidation-Fig. 10 shows the effect of pH on the respiratory activity, respiratory control, and phosphorylation efficiency accompanying the oxidation of externally added NADH by yeast mitochondria. It can be seen that the highest RC ratio (3.5 to 3.7) was obtained at pH 6.3 to 6.9 in the presence of 0.75 M sorbitol. The respiratory rate, on the other hand, reached a maximal value at pH 6.6 and remained constant up to pH 7.5. In the pH range tested (pH 5.4 to 7.5), the P:O ratio was essentially unchanged. It seems from these data that the optimal pH for respiration of yeast mitochondria is a little lower than that of mammalian mitochondria. Further- more, it was noted that there is a relationship between the optimal pH of respiration and the tonicity of the reaction medium.


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1804 Yeast Mitochondria Vol. 241, No. 8

4.0 -

,3.0 - F 2

z 2.0 -

P/O

RC ratio

-. -lz

A’ -ll/=

,l344--D-

Qo2 in state III

P/

- 1.0

FIG. 10. Effect of pH on RC, P:O ratio, and Qo, on NADH oxidation at a fixed tonicity (0.75 M sorbitol). Final concentration of mitochondria was 0.19 mg per ml of reaction medium. Final concentrations of NADH and ADP were 520 and 189.5 pM, respectively.

1.0 0.3 05 0,6 0.7 Q8 1.0

'0

Cont. Sorbitol (M )

FIG. 11. Effect of tonicity on RC, P:O ratio, and Qo, accompanying NADH oxidation at pH 6.6. Experimental conditions were the same as those in Fig. 10.

FIG. 12. Effect of storage of yeast mitochondria on a-ketoglutarate (or-KG) oxidation. Final concentration of mitochondria was 0.33 mg of protein per ml of reaction medium. Other conditions were the same as those in Fig. 3a. Time of storage: fresh mitochondria (I), 2 days (2), 3 days (S), 5 days (4). Mt, yeast mitochondria.

In general, a decrease in tonicity caused a shift of optimal pH to higher values, and the reverse occurred when tonicity was in- creased. As shown in Fig. 11, tonicity had an effect on respiratory control in NADH oxidation. The highest RC ratio was obtained with 0.55 to 0.75 M sorbitol at pH 6.6. This concentration of sorbitol is much higher than that used with mammalian mitochondria (39). The P:O ratio and respiratory rate in

State 3 were almost constant within a wide range of tonicity, i.e. 0.3 to 1.0 M sorbitol.

Stability of Yeast Mitochondria-Yeast mitochondria when prepared in a sufficiently intact state appeared to be rather stable. Respiratory control of NADH oxidation was retained after storage of mitochondria in an ice bath for 3 days, even though the RC ratio declined during this period; however, only a slight decrease of the P:O ratio was observed during this period. After 5 days of storage, respiratory control was completely lost, but some phosphorylation activity was still detectable. Similar stability of respiratory control and phosphorylation efficiency was also observed in the oxidation of certain other substrates. However, respiratory control was more stable when a-ketoglutarate was the substrate (Fig. 12). Even after 5 days of storage, when respiratory control was no longer observable in NADH respiration, control of respiration by ADP was still clearly demonstrable with ar-ketoglutarate. But the P:O ratio declined to less than one-half of the original value. This suggests that the predominant step regulating the rate of cY-ketoglutarate respiration is different from that involved in the respiration with NADH and other substrates. There was a lag in the response of the respiratory activity of aged mitochondria upon addition of ADP. This occurred with all substrates tested, and the delay became more pronounced as the period of storage was prolonged (cf. References 40 to 42). The lag period could not be abolished by the addition of ATP to the mitochondria.

DISCUSSION

Snail gut juice containing various digestive enzymes was first used for the preparation of yeast mitochondria by Heyman- Blanchet et al. (13, 14). This method seemed to be much milder than a variety of mechanical procedures which had been used previously for the isolation of yeast mitochondria. The yeast mitochondria prepared by these authors were reported to be in a fairly intact state, because leakage of cytochrome c could not be detected during isolation (13) and partial preservation of the internal structure of the mitochondria was confirmed by electron microscopy (14). Heyman-Blanchet et al. examined the cytochrome content of their particles spectrophotometrically, but not the respiratory and phosphorylative properties of their preparations.

We have modified the original method of Heyman-Blanchet et al. and isolated what would seem to be fairly intact mitochondria from S. carlsbergensis, with only slight contamination by other subcellular fractions. The procedure described in this paper accomplishes a more complete degradation of yeast cells into protoplasts than the original method. The introduction of 1.3 M sorbitol into the suspending medium affords effective pro- tection of the protoplasts against osmotic lysis. Thorough wash- ing of the protoplasts was essential to obtain good results, because the gut juice even after partial purification was found to be toxic to mitochondria. When suspended in the “mitochondrial preparation medium” described here, the washed protoplasts were easily broken by osmotic shock, and only a brief period of blend- ing was necessary to accomplish complete breakage of protoplast membranes. Conditions of pH, tonicity, and composition of preparation media have to be carefully selected to obtain mitochondria in a relatively undamaged state.

Electron microscopy showed that our mitochondria were practically free from contamination by other subcellular components and still retained cristae-like internal structures, although these


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appeared to have been damaged considerably. It is not certain at present whether this damage occurred during the isolation or the fixation procedure. The physiological intactness of the preparation is indicated by the respiratory control in the presence and absence of ADP. The efficiency of phosphorylation (ADP:O or P:O ratio) of the yeast mitochondria was also almost as high as that of mammalian mitochondria when succinate was the substrate. The retention of the capacity for oxidation and phosphorylation of yeast mitochondria exposed to long periods of storage (Fig. 12) is comparable to that observed with certain preparations of mammalian mitochondria (39, 43).

It is generally accepted that externally added NADH is not readily oxidized by intact mammalian mitochondria owing to the impermeability of the mitochondrial membrane to NADH (44). In such mitochondria, NADH is oxidized at a high rate only when either the mitochondrial membrane is disintegrated (45, 46) or cytochrome c is added (47), but in both cases the phosphorylating efficiency is decreased. However, the present preparation of yeast mitochondria can oxidize added NADH at a rapid rate, and this oxidation appears to be tightly coupled to phosphorylation, as indicated by the following findings: (a) the oxidation reveals a high degree of respiratory control, as already noted by Duel1 et al. (20); (b) the efficiency of phosphorylation obtained with externally added NADH was as high as that observed with substrates oxidized by means of intramitochondrial NAD, such as citrate, ethanol, pyruvate plus malate, etc.; (c) the oxidation of NADH in the absence of ADP can be accelerated by uncoupling agents, e.g. 2,4-dinitrophenol or pentachlorophenol; and (d) the addition of cytochrome c scarcely enhances the rate of oxidation of NADH. Recent studies have revealed that oxidation of external NADH by mitochondria isolated from plant sources is also accompanied by respiratory control (48). It would appear that the inability of mitochondria to oxidize external NADH cannot be regarded as a general criterion of their intactness. This is in agreement with studies with liver mitochondria by Maley (49).

The data presented in this paper further indicate that the regulation of the respiratory rate involved in a-ketoglutarate oxidation by yeast mitochondria may differ from that operating in the oxidation of other substrates. Thus, the oxidation of a-ketoglutarate was accompanied by a considerably higher RC ratio than that observed with the other substrates (Table I), and the respiratory control was also more resistant to storage (Fig. 12). In addition, 2,4-dinitrophenol did not accelerate the rate of respiration in the absence of ADP, and, when ADP was present, the respiration was insensitive to oligomycin. It is therefore con- ceivable that a-ketoglutarate oxidation in yeast mitochondria is regulated principally by the substrate level phosphorylat.ion site, which is insensitive to both 2,4-dinitrophenol and oligomycin (38).

The ADP:O or P:O rat,ios obtained for yeast mitochondria were 0.7 to 1.0 with tetramethyl-p-phenylenediamine; 1.6 to 1.8 with succinate, NADH, and NAD-linked substrates including citrate and pyruvate plus malate; and 2.3 to 2.7 with a-ketoglutarate. Thus the values obtained with tetramethyl-p- phenylenediamine and succinate were identical with those reported for mammalian mitochondria. However, the values obtained with a-ketoglutarate and other NAD-linked substrates were about 1 unit lower than those obtained with mammalian mitochondria. Similar indications have been reported by Lynen and Konigsberger (50) and Vitols and Linnane (5). These

observations suggest that Site I phosphorylation (cf. Reference 51) is lacking in the present yeast mitochondria. In support of this possibility, it was found that the oxidation of NADH and NAD-linked substrates in yeast mitochondria was not inhibited by Amytal and rotenone. Since these reagents are well known inhibitors of electron transport in the phosphorylation Site I region of mammalian mitochondria, this finding strongly suggests that the NADH dehydrogenase in yeast mitochondria is different from that of mammalian mitochondria. By analysis of the oxidation-reduction states of electron carriers in starved bakers’ yeast cells, Chance (52) has suggested the existence of three phosphorylation sites in ethanol oxidation. Therefore, it may be premature at present to decide whether phosphorylation Site I is absent originally in yeast mitochondria or whether this site is so unstable that it has been lost during the preparation of the mitochondria.

The present yeast mitochondria oxidized both D- and n-lactate, but n-lactate was oxidized at a higher rate than n-lactate. These findings are in accordance with those reported by Vitols and Linnane (5), Roy (53), and Gregolin and Scalella (54) with yeast mitochondria prepared by mechanical procedures. The oxidation of both isomers does not seem to involve the antimycin A-sensitive site, because this inhibitor caused only a slight decrease in the oxidation rate and phosphorylation efficiency. The P:O ratio accompanying the oxidation of both D- and L-lactate was 0.8 to 1.0 in the presence of antimycin A. It seems likely that the phosphorylation site operating in lactate oxidation is Site III, which is also associated with tetramethyl-p-phenylenediamine oxidation.

Duel& Inoue, and Utter (20) reported that pretreatment of yeast cells with 2-mercaptoethylamine and EDTA greatly in- creased their sensitivity to digestion by snail gut juice. This made the enzymic procedure suitable for the preparation of mitochondria from cells in the stationary growth phase as well. In- tactness of yeast mitochondria isolated by their method was supported by density gradient centrifugation analysis of the cell lysate. The oxidation of various substrates by their preparation clearly showed acceleration in response to the addition of ADP, but efficiency of phosphorylation and deceleration of respiration on exhaustion of added ADP was not reported in their paper; hence, it is difficult to make a detailed comparison of the oxidative and phosphorylative characteristics of the two types of preparation.

Acknowledgments-We wish to express our thanks to Dr. T. Oda of Okayama University and Dr. Y. Shinagawa of Kyoto University for taking electron micrographs of yeast cells and mitochondria; to Dr. L. Ernster of Stockholm University and Dr. R. Sato of Osaka University for having read the manuscript and for their criticism and helpful discussions; and to Miss H. Komatsu and Mrs. M. Hamada (Yamanaka) for their excellent technical assistance. One of us (T. 0.) wishes to express her appreciation to Dr. T. Ohnishi of Waseda University for en- couragement and advice throughout this work. Thanks are also due to the Institute for Fermentation, Osaka, for supplying sev- eral strains of yeast.

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Tomoko Ohnishi, Kumiko Kawaguchi and Bunji HagiharaPreparation and Some Properties of Yeast Mitochondria

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