- Email: [email protected]
O and respiratory control ratios
[19]
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149
[19] P r e p a r a t i o n o f Y e a s t M i t o c h o n d r i a ( S a c c h a r o m y c e s cerevisiae) with Good P/O and Respiratory Control Ratios B y BERNARD GUI~RIN, PIERRE LABBE a n d MARIE SOMLO
As described in a previous volume of Methods in Enzymology, 1"the foremost experimental obstacle in the preparation of intact yeast mitochondria is the need for efficiently breaking the refractory cell wall without extensively damaging the liberated mitochondria." Two methods have been used and described in the literature: (1) mechanical cell rupture 1-5 and (2) cell wall digestion by snail gut juice. 6-1° This article gives an account of new modifications of the two methods, the advantages over the previously described versions being an increased yield, greater simplicity, and shorter manipulation time. Futher advantages are (1) use of the mechanical methods for studying proton translocation and oligomycin-sensitivity of oxidative phosphorylation in S. cerevisiae mutants (p. 153), as well as application toCandida lipolytica (p. 152), and (2) use of the enzyme method for the study of mitochondrial membrane permeability and localization of loosely bound enzymes (p. 157). Since we have not experimented with other enzymatic methods not using the snail enzyme, these are not discussed in this article. As emphasized by Mattoon and Balcavage, 1 both the mechanical and the enzymatic methods have specific advantages and drawbacks. The main advantage of the mechanical method is speed; this is of critical importance in certain cases, e.g., when a long physiological study of freshly prepared mitochondria is planned. The preparation of mitochondria by the mechanical methods described in this article takes only 2 hr, as compared to about 6 hr for methods involving enzymatic cell wall digestion. The main advantage of the latter over the mechanical method is that mitochondria of higher integrity are obtained. In fact, as is shown by the systematic comparison below, Qo2 values and respiratory control 1 j. R. Mattoon and W. X. Balcavage, this series, Vol. 10 [26]. 2 M. Guarnieri, J. R. Mattoon, W. X. Balcavage, and C. Payne, Anal. Biochem. 34, 39 (1970). 3 G. Schatz and E. Racker, Biochem. Biophys. Res. Commun. 22, 579 (1966). 4 C. Spencer, S. A. Symons, and R. V. Brunt, Arch. Mikrobiol. 75, 246 (1971). 5 B. Mackler and B. Haynes, Biochim. Biophys. Acta 292, 88 (1973). 6 E. A. Duell, S. I n o u r , and M. F. Utter, J. Bacteriol. 88, 1762 (1964). 7 T. Ohnishi, K. Kawaguchi, and B. Hagihara, J. Biol. Chem. 241, 1797 (1966). 8 L. Kovac, G. S. P. Groot, and E. Racker, Biochim. Biophys. Acta 256, 55 (1972). 9G. Schatz and L. Kovac, this series, Vol. 31 [65]. 10 M. Briquet, N. Sabadie-Pialoux, and A. Goffeau, Arch. Biochem. Biophys. 174, 684 (1976). METHODS IN ENZYMOLOGY,VOL. LV
Copyright O 1979by Academic Press. Inc. All fights of reproduction in any formreserved. ISBN 0-12-181955-8
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PREPARATION OF CONTROLLED YEAST MITOCHONDRIA
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ratios are higher for mitochondria obtained by the enzymatic method, although the mechanical method yields similar P/O ratios and properly oriented mitochondrial membranes (as shown by respiratory proton translocation studies H) (see Table I). With regard to the purity and morphology of the mitochondria obtained, representative electron micrographs have been published for the enzymatic methods t7,12,13) and for the mechanical methods, apart from those shown here. 11,14 Methods of Preparation Mechanical Method The mechanical methods quoted above 1-3 produced relatively low yields of mitochondrial protein. The methods described in this article produce considerably higher yields, as will be shown. Furthermore, the Braun Shaker, which is simpler than a colloid mill, is a widely used homogenizer for relatively small-scale preparations (from 1 to 20 g cell protein, i.e., from 10 to 200 g wet weight). For larger-scale preparations (from 50 to 300 g cell protein) the use of the Dyno Mill homogenizer is recommended. Two different small-scale methods and one large-scale method are described. Yeast Culture Conditions. The general effects of carbon source and aeration, as well as the effects of physiological state, are well described elsewhere. 1 The wild-type strain PS 194 or Yeast Foam is grown aerobically in a glucose-containing preculture (yeast extract Difco 1%, Difco Bactopeptone I%, glucose 2%) until the stationary phase. Erlenmeyer flasks (6liter) containing 800 ml of culture medium (1% Difco yeast extract, 0.12% ammonium sulfate, and carbon source--either 1% ethanol, or 0.5% glucose, or 0.1% glucose plus 0.4% galactose) are inoculated with 4 ml of preculture. The cultures are vigorously shaken at 28 ° (70 oscillations per minute) and harvested about 24 hr later: for the ethanol medium at 1.61.8 g cell protein per liter; for the glucose or galactose medium at 1.8-1.9 g cell protein per liter. Final growth is 3 g cell protein per liter in the ethanol medium and 2 g cell protein per liter in the glucose or galactose medium. Absorbancy in a Klett nephelometer was standardized against grams cell protein per liter. Cell protein was determined by the biuret method modified by Strickland. 15 11 M. Somlo, R. A. Reid, and M. Krupa, Biochem. J. 162, 51-59.(1977). 12 p. S. Perlman and H. R. Mahler, Arch. Biochem. Biophys. 136, 245 (t970). 13 M. Petzuch, C.R. Hebd. Seances Acad. Sci. 273, 105 (1971). 14 W. X. Balcavage and J. R. Mattoon, Biochim. Biophys. Acta 153, 521 (1968). 15 L. H. Stickland, J. Gen. Microbiol. 5, 698 (1951).
152
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The main difference between the two small-scale methods involves the breaking conditions. In method 1, the firmly sedimented predried pellet is broken directly with a relatively large amount of glass beads, at high homogenizing speed, without addition of an osmotic stabilizer, taking advantage of the high internal cellular osmotic pressure. In method 2, a yeast suspension in osmotic stabilizer (sorbitol) is broken with a small amount of glass beads, at low homogenizing speed. The mitochondrial yields are 30 and 20 mg/g cell protein, respectively, for our smallscale methods 1 and 2 and 100 mg/g cell protein for the large-scale method, as compared to the yields of previously published mechanical methods: 0.25 mg/g cell protein, 1 4 mg/g, 2 and 10 mg/g. a
Small-Scale Method 1 Besides strains of S. cerevisiae, this type of preparation was successfully applied to a strain of Candida lipolytica (Saccharomycopsis lipolytica). TM The method was systematically compared to the enzymatic method 16 (see Table I). Immediately after harvesting, the cells are washed once with cold distilled water and kept ice-cold, not longer than 1 week.
Preparation of Mitochondria Reagents. Suspending buffer (employed after breakage), final concentrations: 0.65 M sorbitol, 10 mM Tris-HC1, 0.1 mM EDTA, 0.1% crystalline bovine serum albumin, pH 6.5 (KOH). Higher concentrations of serum albumin and EDTA do not improve the quality of the mitochondria.
Glass Beads. Size 0.45-0.50 mm. B. Braun, Melsungen, West Germany, catalog No. 54170 (2886). The beads are used as delivered without further purification. Braun Homogenizer MSK. Glass flasks of 75-ml capacity. Homogenizer cooled with liquid CO2. One gram cell protein per 30 g glass beads. One-half to two g cell protein per flask can be employed. Centrifuge. Sorvall model RC-2B, rotor SS 34, polycarbonate or polypropylene tubes. Glass Homogenizer. Potter-Elvehjem, Teflon pestle, 5-ml capacity. Procedure (0°). The cell pellet (2 g protein) is dried on filter paper sheets and introduced with a spatula into the Braun flask (containing the appropriate amount of glass beads). Breaking for 20 sec is carried out at high speed (4000 oscillations per minute). The resulting highly viscous slurry is quickly transferred to a beaker kept in crushed ice containing 80 ml of 'hsuspending medium." After rapid mixing, the pH is immediately ~6 H. Chambon and P. Labbe, Biochimie. 58, 837 (1976).
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adjusted to 6.5 (dilute KOH). The beads are decanted, and the supernatant is centrifuged for 10 min at 1500 g. The supernatant is carefully decanted, and the pH is checked again and adjusted to 6.5 if necessary. The supernatant is centrifuged for 10 min at 9500 g, and the fluffy layer, if present, is discarded. The mitochondrial pellet is suspended in 20 ml of "suspending buffer" and centrifuged again for 10 min at 9500 g. The washed pellet is resuspended in a minimal volume of suspending buffer (30-50 mg protein per milliliter). The total yield is 60 mg/2 g cell protein.
Assay for Oxidative Phosphorylation. A Clark-type oxygen electrode (oxygen sensor, Beckman Instruments) was used with a 5-ml reaction chamber (30°). 16 The reaction medium (final concentrations) was as follows: 0.65 M sorbitol, 0.36 mM EDTA, 10 mM KHePO4, 10 mM TrisHCI, 10 mM KC1, 0.3% bovine serum albumin, substrate I0 mM. The pH of the medium was adjusted to 6.5 (KOH). Oxygen uptake rates were determined after successive addition of mitochondria (1-3 mg protein according to substrate), substrate, and known small amounts of 0.02 M ADP. Respiratory control and ADP/O ratios were calculated as by Chance and Williams.17a Small-Scale Method 2 The mitochondria resulting from this type of preparation were successfully used for respiratory-driven proton translocation,ll and Fig. 1 is the corresponding electron micrograph. The same type of preparation was also used for determination of oligomycin-sensitivity of oxidative phosphorylation in mutants.17b Immediately after harvesting, cells are washed in the following medium: 0.65 M sorbitol, 0.1 mM Na-EDTA, pH 6.5. The yeast was kept ice-cold in pellets containing 1.3 g or a multiple of 1.3 g cell protein (determined by absorbancy of the suspension), not longer than ! week. The use of 1.3-g aliquots (_+ 10%) with constant amounts of glass beads and a constant breaking volume secures reproducible yields and mitochondria of good quality.
Preparation of Mitochondria Reagents. Breaking medium (final concentrations): 0.65 M sorbitol (Roquette Fr6res, Lille, France), 0.1 mM Na-EDTA, 0.1% bovine serum albumin, crystallized and lyophylized (Sigma Chem. Co., St. Louis, Missouri), pH 6.5. 17a B. Ch ance and G. R. Williams, Adv. Enzymol. 17, 65 (1956). 17b M. Somlo, Arch. Biochem. Biophys. 182, 518 (1977).
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FIG. 1. Isolated mitochondria from S. cerevisiae strain PS 194. (A)× 15,000, (B) ×45,000. Pellets were fixed in 1% osmium tetroxide in phosphate buffer (pH 7.2), dehydrated, and embedded in Epon. Ultrathin sections were stained by uranyl acetate and lead citrate.
Glass Beads. See method 1. Braun Homogenizer MSK. Each flask (75-ml capacity) was used with 1.3 g total cell protein, equivalent to 800 ml culture, and 20 g glass beads. Centrifuge. See method 1. Glass Homogenizer. See method 1. Procedure (0°). Yeast cells are suspended in aliquots of 25 ml "breaking m e d i u m " per 1.3 g cell protein with 20 g glass beads and broken for 2 min at low speed (2000 oscillations per minute). The homogenate is decanted and the beads rinsed with 5 ml breaking medium. The pH after breakage is 6.35 and left as such. Combined washings are
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PREPARATION OF CONTROLLED YEAST MITOCHONDRIA
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centrifuged for 10 min at 1500 g. The supernatant is carefully decanted (it contains about 40% of the total cell protein). After twofold dilution with breaking medium to facilitate further fractionation, the supernatant is centrifuged again for 10 min at 1500 g. The carefully decanted supernatant is then centrifuged for 10 min at 9500 g, and the abundant fluffy layer is discarded completely by simply inverting the centrifuge tube immediately after the centrifugation. The pellet is gently suspended in a Potter-Elvejhem homogenizer in breaking medium (about 20 ml/aliquot) and centrifuged again for 10 min at 9500 g. The fluffy layer is again completely discarded and the mitochondria suspended in a Potter-E1vejhem homogenizer at 10-15 mg/ml. The total yield is 25 mg/1.3 g cell protein.
Assay for Oxidative Phosphorylation (30°). A Clark electrode (Gilson oxygraph) with 1.7-ml reaction chamber was used. Reaction medium was 0.65 M sorbitol, 2 mM Na-EDTA, 10 lnM KHzPO4, 3 mM substrate (pH adjusted to 6.3 with NaOH). Other details as in method 1. Large-Scale Method The use of the Dyno Mill model KDL for the large-scale preparation of yeast submitochondrial particles was recently described.'8 We have adapted '° the utilization of this continuous grinder to the preparation of acceptor-controlled mitochondria from kilogram (wet weight) quantities of S. cerevisiae. The flexibility of this equipment is very satisfactory; flow rate, cellular concentration, breaking buffer composition, size of glass beads, stirring rate, and temperature are readily controlled.
Yeast Culture Conditions. Strain PS 194 is grown aerobically (30 °) in a fermenter in 50 liters of either of the following three media: (1) synthetic "H3 ''z° slightly enriched by adding ZnSo4, 7H20 plus CuSo4, 5HzO (0.7 mg/liter each), and nicotinamide (2 mg/liter); (2) Difco yeast nitrogen base (synthetic medium), 0.7%; or (3) the natural medium described above for the small-scale methods. The carbon source was 2% ethanol; the air flow rate was 50 liters/min. The medium was inoculated with 1 or 2 liters of a stationary-phase preculture grown in the same medium as the large culture (15 mg cell protein per liter). Foaming is prevented by polypropylene glycol Pz000 (0.1 ml/liter of medium) added before sterilizing. The cells are harvested at 2 g cell protein per liter (maximal growth in this ,8 D. Deters, U. Muller, and H. Homberger, Anal. Biochem. 70, 263 (1976). ,9 p. L a b b e and H. C h a m b o n , Anal. Biochem. 8 1 , 4 1 6 (1977). 2o B. Ephrussi, P. P. Slonimski, Y. Yotsuyanagi, and J. Tavlitzki, C.R. Tray. Lab. Carlsberg, Ser. Physiol. 26, 87 (1956).
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medium is 3 g/liter) with a Sharpies continuous ultracentrifuge. The resuiting yeast cake is washed once in cold distilled water. The yeast pellets were kept ice-cold not longer than 1 week.
Preparation of Mitochondria Reagents. The breaking buffer is the same as the suspending buffer for small-scale method 1. Glass Beads. See small-scale method 1. Dyno Mill Model KDL. Willy A. Bachofen, 4005 Basel, Utengasse 15, Switzerland. Equipped w i t h a 600-ml continuous flow breakage glass chamber, four 6.4-cm polyurethane rotating agitator disks, and a 0.05mm dynamic slit separator. The chamber is filled with 520 ml of glass beads, and 10 min before breakage the mill is cooled to - 5 ° by circulation of a cold mixture of ethylene glycol/water (50:50,v/v) from an 80-litercapacity cold bath (flow rate 300 liters/hr). Cooling is continued throughout the breakage. A peristaltic pump drives the yeast through the mill chamber. Any commercial variable pump allowing a flow rate from 50 to 300 ml/min and equipped with silicone tubing of 5 mm internal diameter is convenient. Centrifuge. Sorvall model RC5, rotor GS 3 with 0.5-liter polycarbonate bottles and 00430 caps, as well as rotor SS 34. Procedure (0°). Before breakage the cells are washed twice with breaking buffer and resuspended in breaking buffer to yield a 60% suspension (wet weight). Careful adjustment of the suspension to pH 6.5 is necessary (KOH) because of strong endogenous acidification by the yeast cells. Breakage must be carried out immediately afterward. All subsequent operations are given for 1 liter of yeast suspension in breaking buffer (600 g wet weight represents 60 g cell protein). The yeast suspension, kept ice-cold with constant magnetic stirring, is driven through the precooled mill by the peristaltic pump (60-80 ml/min flow rate). When approximately 200 ml of the yeast suspension have been introduced into the breakage chamber, the driving motor is switched on (stirring shaft speed 3000 rpm), while pumping continues at the same flow rate. Broken cells exit through a tubing to a 3-liter glass beaker kept in crushed ice. Estimation of cell breakage (usually 90%) is made by phase microscopy. The breakage of 1 liter of suspension takes 10-15 min. The temperature of the broken cells gradually rises from 10° to 27 °. Immediately after the yeast suspension has passed through the mill, the glass beads are washed
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with 0.5 liter cold breaking buffer at a flow rate of 300 ml/min, while the mill is still stirring. When no more liquid is collected at the chamber exit, the driving motor is switched off. To the 1.5 liter of the broken cells collected into the 3-liter beaker (1 liter of 60% suspension plus 0.5 liter wash) 1.5 liter of cold breaking buffer is immediately added and the pH is adjusted rapidly to 6.5 (KOH), since the pH has fallen to about 6.0. The homogenate is centrifuged at 1500 g for 15 min (GS 3 rotor) and the supernatant carefully decanted; the pellet is resuspended with 0.5 liter of breaking buffer and centrifuged again. The combined supernatants (if necessary adjusted to pH 6.5) are centrifuged at 9000 rpm for 30 min (GS 3 rotor) to collect the mitochondria. The supernatant (and, if desired, the fluffy layer) is discarded. Fat deposits are wiped off. Mitochondrial pellets are resuspended in 2 liters of breaking buffer and centrifuged again as above. The yield of (unpurified) mitochondrial protein is 7-8 g protein/600 g (wet weight) cells, or about 100 mg protein/l g cell protein.
Assay for Oxidative Phosphorylation. The assay is the same as for method 1, except that the EDTA concentration is increased to 2 mM (see General Remarks).
Enzymatic Method The method described here is adapted from previously published procedures. 6-8 In particular we stress some critical points that we found important for obtaining mitochondria with good R C. The main interest of this relatively laborious procedure is that high yields (130 mg mitochondrial protein per gram cell protein) of purified mitochondria with good R C are obtained that can be employed for further research in which integrity and purity are essential, such as studies of membrane permeability 21 and localization of loosely bound enzymes.
Yeast Culture Conditions. Saccharomyces cerevisiae strain Yeast Foam is grown aerobically (27 °) in the following medium: 1% Difco yeast extract, 0.1% KH2PO4, 0.12% (NH4)2SO4; carbon source, 2% lactate or ethanol. The medium is inoculated from exponential-phase cells pregrown in the same medium (0.75 mg protein per liter). Final growth in this medium is reached at 3 g cell protein per liter. Cells are harvested at 2 g cell protein per liter and washed twice with cold distilled water. 2~G. Arselin-Dechateaubodeau,M. Gu6rin, and B. Gu6rin,Biochimie 58, 601 (1976).
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Preparation of Mitochondria Reagents Buffers (final concentrations) 1. 0.1 M Tris-HCl, 0.5 M 2-mercaptoethanol, pH 9.3 2. 0.01 M Tris-HC1, 0.5 M KC1, pH 7.0 3. 0.01 M citrate-phosphate, 1.35 M sorbitol, 1 mM EGTA, pH 5.8 4. 0.01 M Tris-maleate, 0.75 M sorbitol, 0.4 M mannitol, 2 mM EGTA, 0.1% bovine serum albumin, pH 6.8 5. 0.01 M Tris-maleate, 0.6 M mannitol, 2 mM EGTA, 0.2% bovine serum albumin, pH 6.8 Snail gut juice from Helix pomatia (Industrie Biologique Fran~aise, Gennevilliers, France) Homogenizer. Overhead Waring Blendor, commercial model 5011, container capacity 100 ml Centrifuge. Sorvall model RC-2B, rotor SS 34, and continuous flow system (Sorvall KSB) adapted on rotor SS 34. Procedure (Described for 2 g Cell Protein = 20 g Wet Weight). The washed cells are suspended in 80 ml of buffer 1 and incubated for 10 min at 32 °. The cells are collected by centrifugation for 5 min at 1500 g, suspended in 3 liters of buffer 2, and centrifuged with the continuous flow system at 8000 rpm (flow rate 400 ml/min). This highly efficient washing step is essential for complete elimination of mercaptoethanol. Cells are suspended in 40 ml of buffer 3, then 5 ml of snail enzyme are added, and the mixture is shaken for 60-90 min at 32 ° under N2 atmosphere. Spheroplast formation, controlled by intermittent visual observation, 9 was found to be more reproducible under anaerobic conditions. The spheroplast suspension is centrifuged for 10 min at 1200 g and the supernatant discarded. All the following manipulations are carried out at 0 °. The spheroplast pellet is washed twice with 40 ml of buffer 4 by identical centrifugation. The final pellet is then suspended in 80 ml of buffer 5 and homogenized in the Waring blendor three times for 5 sec each at low speed. It is important to use the 100-ml container, closed with Parafilm foil below the container cap, in order to prevent foaming and drastic aeration. The homogenized suspension is distributed among six polypropylene tubes (50-ml, rotor SS 34). The volume of each tube is adjusted to 40 ml with fresh buffer 5 and the suspension centrifuged for 10 min at 800 g. Supernatants are carefully decanted. The pellets are resuspended with a
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Teflon pestle, collected into two centrifuge tubes in buffer 5, and centrifuged again for l0 min at 800 g. The combined supernatants are centrifuged for 10 min at 12,000 g. The supernatants are removed; there is no fluffy layer over the mitochondrial pellets. The centrifuge tubes are wiped to eliminate adhering lipid. Mitochondrial pellets are homogenized gently in a Potter-Elvejhem homogenizer with a Teflon pestle with a small volume of buffer 5 containing no serum albumin. The suspension is distributed among six centrifuge tubes; the volume of each tube is adjusted to 40 ml with the same buffer and centrifuged for l0 min at 800 g. The supernatant is centrifuged for l0 min at 12,000 g. The pellets are resuspended in a small volume of buffer 5 without serum albumin in two tubes and centrifuged for l0 min at 800 g. The supernatant is centrifuged for 10 min at 12,000 g, and the resulting purified mitochondrial pellet is used for the experiments.
Assay for Oxidative Phosphorylation. The assay is the same as for small-scale mechanical method 1. General Remarks Because of the varying genetic background of yeast laboratory strains, the methods described in this article might have to be slightly adjusted to suit each strain or culture condition. For instance, susceptibility to the snail enzyme is known to be somewhat variable, and a preliminary study should be carried out 9 to establish optimal enzyme concentration and incubation time. For the mechanical methods, the extraction yield of mitochondria also depends on the genetic background of the strains. When the yield is lowered, mitochondria might be of apparently poorer quality. In such cases we found it useful to increase the concentration of EDTA and/or serum albumin in the respiration medium. When endogenous respiration is relatively high, respiratory control is often poor. In this case two to three additional washings at 9500 g for 10 min will often produce considerable improvement. In the case of strains growing sluggishly on ethanol, the extraction yield of mitochondria will be lowered and mitochondrial respiration poorly controlled. In this case it is advisable to adopt the 0.5% glucose or the 0.1% glucose plus 0.4% galactose medium.
PREPARATION OF CONTROLLED YEAST MITOCHONDRIA
149
[19] P r e p a r a t i o n o f Y e a s t M i t o c h o n d r i a ( S a c c h a r o m y c e s cerevisiae) with Good P/O and Respiratory Control Ratios B y BERNARD GUI~RIN, PIERRE LABBE a n d MARIE SOMLO
As described in a previous volume of Methods in Enzymology, 1"the foremost experimental obstacle in the preparation of intact yeast mitochondria is the need for efficiently breaking the refractory cell wall without extensively damaging the liberated mitochondria." Two methods have been used and described in the literature: (1) mechanical cell rupture 1-5 and (2) cell wall digestion by snail gut juice. 6-1° This article gives an account of new modifications of the two methods, the advantages over the previously described versions being an increased yield, greater simplicity, and shorter manipulation time. Futher advantages are (1) use of the mechanical methods for studying proton translocation and oligomycin-sensitivity of oxidative phosphorylation in S. cerevisiae mutants (p. 153), as well as application toCandida lipolytica (p. 152), and (2) use of the enzyme method for the study of mitochondrial membrane permeability and localization of loosely bound enzymes (p. 157). Since we have not experimented with other enzymatic methods not using the snail enzyme, these are not discussed in this article. As emphasized by Mattoon and Balcavage, 1 both the mechanical and the enzymatic methods have specific advantages and drawbacks. The main advantage of the mechanical method is speed; this is of critical importance in certain cases, e.g., when a long physiological study of freshly prepared mitochondria is planned. The preparation of mitochondria by the mechanical methods described in this article takes only 2 hr, as compared to about 6 hr for methods involving enzymatic cell wall digestion. The main advantage of the latter over the mechanical method is that mitochondria of higher integrity are obtained. In fact, as is shown by the systematic comparison below, Qo2 values and respiratory control 1 j. R. Mattoon and W. X. Balcavage, this series, Vol. 10 [26]. 2 M. Guarnieri, J. R. Mattoon, W. X. Balcavage, and C. Payne, Anal. Biochem. 34, 39 (1970). 3 G. Schatz and E. Racker, Biochem. Biophys. Res. Commun. 22, 579 (1966). 4 C. Spencer, S. A. Symons, and R. V. Brunt, Arch. Mikrobiol. 75, 246 (1971). 5 B. Mackler and B. Haynes, Biochim. Biophys. Acta 292, 88 (1973). 6 E. A. Duell, S. I n o u r , and M. F. Utter, J. Bacteriol. 88, 1762 (1964). 7 T. Ohnishi, K. Kawaguchi, and B. Hagihara, J. Biol. Chem. 241, 1797 (1966). 8 L. Kovac, G. S. P. Groot, and E. Racker, Biochim. Biophys. Acta 256, 55 (1972). 9G. Schatz and L. Kovac, this series, Vol. 31 [65]. 10 M. Briquet, N. Sabadie-Pialoux, and A. Goffeau, Arch. Biochem. Biophys. 174, 684 (1976). METHODS IN ENZYMOLOGY,VOL. LV
Copyright O 1979by Academic Press. Inc. All fights of reproduction in any formreserved. ISBN 0-12-181955-8
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ratios are higher for mitochondria obtained by the enzymatic method, although the mechanical method yields similar P/O ratios and properly oriented mitochondrial membranes (as shown by respiratory proton translocation studies H) (see Table I). With regard to the purity and morphology of the mitochondria obtained, representative electron micrographs have been published for the enzymatic methods t7,12,13) and for the mechanical methods, apart from those shown here. 11,14 Methods of Preparation Mechanical Method The mechanical methods quoted above 1-3 produced relatively low yields of mitochondrial protein. The methods described in this article produce considerably higher yields, as will be shown. Furthermore, the Braun Shaker, which is simpler than a colloid mill, is a widely used homogenizer for relatively small-scale preparations (from 1 to 20 g cell protein, i.e., from 10 to 200 g wet weight). For larger-scale preparations (from 50 to 300 g cell protein) the use of the Dyno Mill homogenizer is recommended. Two different small-scale methods and one large-scale method are described. Yeast Culture Conditions. The general effects of carbon source and aeration, as well as the effects of physiological state, are well described elsewhere. 1 The wild-type strain PS 194 or Yeast Foam is grown aerobically in a glucose-containing preculture (yeast extract Difco 1%, Difco Bactopeptone I%, glucose 2%) until the stationary phase. Erlenmeyer flasks (6liter) containing 800 ml of culture medium (1% Difco yeast extract, 0.12% ammonium sulfate, and carbon source--either 1% ethanol, or 0.5% glucose, or 0.1% glucose plus 0.4% galactose) are inoculated with 4 ml of preculture. The cultures are vigorously shaken at 28 ° (70 oscillations per minute) and harvested about 24 hr later: for the ethanol medium at 1.61.8 g cell protein per liter; for the glucose or galactose medium at 1.8-1.9 g cell protein per liter. Final growth is 3 g cell protein per liter in the ethanol medium and 2 g cell protein per liter in the glucose or galactose medium. Absorbancy in a Klett nephelometer was standardized against grams cell protein per liter. Cell protein was determined by the biuret method modified by Strickland. 15 11 M. Somlo, R. A. Reid, and M. Krupa, Biochem. J. 162, 51-59.(1977). 12 p. S. Perlman and H. R. Mahler, Arch. Biochem. Biophys. 136, 245 (t970). 13 M. Petzuch, C.R. Hebd. Seances Acad. Sci. 273, 105 (1971). 14 W. X. Balcavage and J. R. Mattoon, Biochim. Biophys. Acta 153, 521 (1968). 15 L. H. Stickland, J. Gen. Microbiol. 5, 698 (1951).
152
ORGANELLES AND MEMBRANES
[19]
The main difference between the two small-scale methods involves the breaking conditions. In method 1, the firmly sedimented predried pellet is broken directly with a relatively large amount of glass beads, at high homogenizing speed, without addition of an osmotic stabilizer, taking advantage of the high internal cellular osmotic pressure. In method 2, a yeast suspension in osmotic stabilizer (sorbitol) is broken with a small amount of glass beads, at low homogenizing speed. The mitochondrial yields are 30 and 20 mg/g cell protein, respectively, for our smallscale methods 1 and 2 and 100 mg/g cell protein for the large-scale method, as compared to the yields of previously published mechanical methods: 0.25 mg/g cell protein, 1 4 mg/g, 2 and 10 mg/g. a
Small-Scale Method 1 Besides strains of S. cerevisiae, this type of preparation was successfully applied to a strain of Candida lipolytica (Saccharomycopsis lipolytica). TM The method was systematically compared to the enzymatic method 16 (see Table I). Immediately after harvesting, the cells are washed once with cold distilled water and kept ice-cold, not longer than 1 week.
Preparation of Mitochondria Reagents. Suspending buffer (employed after breakage), final concentrations: 0.65 M sorbitol, 10 mM Tris-HC1, 0.1 mM EDTA, 0.1% crystalline bovine serum albumin, pH 6.5 (KOH). Higher concentrations of serum albumin and EDTA do not improve the quality of the mitochondria.
Glass Beads. Size 0.45-0.50 mm. B. Braun, Melsungen, West Germany, catalog No. 54170 (2886). The beads are used as delivered without further purification. Braun Homogenizer MSK. Glass flasks of 75-ml capacity. Homogenizer cooled with liquid CO2. One gram cell protein per 30 g glass beads. One-half to two g cell protein per flask can be employed. Centrifuge. Sorvall model RC-2B, rotor SS 34, polycarbonate or polypropylene tubes. Glass Homogenizer. Potter-Elvehjem, Teflon pestle, 5-ml capacity. Procedure (0°). The cell pellet (2 g protein) is dried on filter paper sheets and introduced with a spatula into the Braun flask (containing the appropriate amount of glass beads). Breaking for 20 sec is carried out at high speed (4000 oscillations per minute). The resulting highly viscous slurry is quickly transferred to a beaker kept in crushed ice containing 80 ml of 'hsuspending medium." After rapid mixing, the pH is immediately ~6 H. Chambon and P. Labbe, Biochimie. 58, 837 (1976).
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adjusted to 6.5 (dilute KOH). The beads are decanted, and the supernatant is centrifuged for 10 min at 1500 g. The supernatant is carefully decanted, and the pH is checked again and adjusted to 6.5 if necessary. The supernatant is centrifuged for 10 min at 9500 g, and the fluffy layer, if present, is discarded. The mitochondrial pellet is suspended in 20 ml of "suspending buffer" and centrifuged again for 10 min at 9500 g. The washed pellet is resuspended in a minimal volume of suspending buffer (30-50 mg protein per milliliter). The total yield is 60 mg/2 g cell protein.
Assay for Oxidative Phosphorylation. A Clark-type oxygen electrode (oxygen sensor, Beckman Instruments) was used with a 5-ml reaction chamber (30°). 16 The reaction medium (final concentrations) was as follows: 0.65 M sorbitol, 0.36 mM EDTA, 10 mM KHePO4, 10 mM TrisHCI, 10 mM KC1, 0.3% bovine serum albumin, substrate I0 mM. The pH of the medium was adjusted to 6.5 (KOH). Oxygen uptake rates were determined after successive addition of mitochondria (1-3 mg protein according to substrate), substrate, and known small amounts of 0.02 M ADP. Respiratory control and ADP/O ratios were calculated as by Chance and Williams.17a Small-Scale Method 2 The mitochondria resulting from this type of preparation were successfully used for respiratory-driven proton translocation,ll and Fig. 1 is the corresponding electron micrograph. The same type of preparation was also used for determination of oligomycin-sensitivity of oxidative phosphorylation in mutants.17b Immediately after harvesting, cells are washed in the following medium: 0.65 M sorbitol, 0.1 mM Na-EDTA, pH 6.5. The yeast was kept ice-cold in pellets containing 1.3 g or a multiple of 1.3 g cell protein (determined by absorbancy of the suspension), not longer than ! week. The use of 1.3-g aliquots (_+ 10%) with constant amounts of glass beads and a constant breaking volume secures reproducible yields and mitochondria of good quality.
Preparation of Mitochondria Reagents. Breaking medium (final concentrations): 0.65 M sorbitol (Roquette Fr6res, Lille, France), 0.1 mM Na-EDTA, 0.1% bovine serum albumin, crystallized and lyophylized (Sigma Chem. Co., St. Louis, Missouri), pH 6.5. 17a B. Ch ance and G. R. Williams, Adv. Enzymol. 17, 65 (1956). 17b M. Somlo, Arch. Biochem. Biophys. 182, 518 (1977).
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FIG. 1. Isolated mitochondria from S. cerevisiae strain PS 194. (A)× 15,000, (B) ×45,000. Pellets were fixed in 1% osmium tetroxide in phosphate buffer (pH 7.2), dehydrated, and embedded in Epon. Ultrathin sections were stained by uranyl acetate and lead citrate.
Glass Beads. See method 1. Braun Homogenizer MSK. Each flask (75-ml capacity) was used with 1.3 g total cell protein, equivalent to 800 ml culture, and 20 g glass beads. Centrifuge. See method 1. Glass Homogenizer. See method 1. Procedure (0°). Yeast cells are suspended in aliquots of 25 ml "breaking m e d i u m " per 1.3 g cell protein with 20 g glass beads and broken for 2 min at low speed (2000 oscillations per minute). The homogenate is decanted and the beads rinsed with 5 ml breaking medium. The pH after breakage is 6.35 and left as such. Combined washings are
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centrifuged for 10 min at 1500 g. The supernatant is carefully decanted (it contains about 40% of the total cell protein). After twofold dilution with breaking medium to facilitate further fractionation, the supernatant is centrifuged again for 10 min at 1500 g. The carefully decanted supernatant is then centrifuged for 10 min at 9500 g, and the abundant fluffy layer is discarded completely by simply inverting the centrifuge tube immediately after the centrifugation. The pellet is gently suspended in a Potter-Elvejhem homogenizer in breaking medium (about 20 ml/aliquot) and centrifuged again for 10 min at 9500 g. The fluffy layer is again completely discarded and the mitochondria suspended in a Potter-E1vejhem homogenizer at 10-15 mg/ml. The total yield is 25 mg/1.3 g cell protein.
Assay for Oxidative Phosphorylation (30°). A Clark electrode (Gilson oxygraph) with 1.7-ml reaction chamber was used. Reaction medium was 0.65 M sorbitol, 2 mM Na-EDTA, 10 lnM KHzPO4, 3 mM substrate (pH adjusted to 6.3 with NaOH). Other details as in method 1. Large-Scale Method The use of the Dyno Mill model KDL for the large-scale preparation of yeast submitochondrial particles was recently described.'8 We have adapted '° the utilization of this continuous grinder to the preparation of acceptor-controlled mitochondria from kilogram (wet weight) quantities of S. cerevisiae. The flexibility of this equipment is very satisfactory; flow rate, cellular concentration, breaking buffer composition, size of glass beads, stirring rate, and temperature are readily controlled.
Yeast Culture Conditions. Strain PS 194 is grown aerobically (30 °) in a fermenter in 50 liters of either of the following three media: (1) synthetic "H3 ''z° slightly enriched by adding ZnSo4, 7H20 plus CuSo4, 5HzO (0.7 mg/liter each), and nicotinamide (2 mg/liter); (2) Difco yeast nitrogen base (synthetic medium), 0.7%; or (3) the natural medium described above for the small-scale methods. The carbon source was 2% ethanol; the air flow rate was 50 liters/min. The medium was inoculated with 1 or 2 liters of a stationary-phase preculture grown in the same medium as the large culture (15 mg cell protein per liter). Foaming is prevented by polypropylene glycol Pz000 (0.1 ml/liter of medium) added before sterilizing. The cells are harvested at 2 g cell protein per liter (maximal growth in this ,8 D. Deters, U. Muller, and H. Homberger, Anal. Biochem. 70, 263 (1976). ,9 p. L a b b e and H. C h a m b o n , Anal. Biochem. 8 1 , 4 1 6 (1977). 2o B. Ephrussi, P. P. Slonimski, Y. Yotsuyanagi, and J. Tavlitzki, C.R. Tray. Lab. Carlsberg, Ser. Physiol. 26, 87 (1956).
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medium is 3 g/liter) with a Sharpies continuous ultracentrifuge. The resuiting yeast cake is washed once in cold distilled water. The yeast pellets were kept ice-cold not longer than 1 week.
Preparation of Mitochondria Reagents. The breaking buffer is the same as the suspending buffer for small-scale method 1. Glass Beads. See small-scale method 1. Dyno Mill Model KDL. Willy A. Bachofen, 4005 Basel, Utengasse 15, Switzerland. Equipped w i t h a 600-ml continuous flow breakage glass chamber, four 6.4-cm polyurethane rotating agitator disks, and a 0.05mm dynamic slit separator. The chamber is filled with 520 ml of glass beads, and 10 min before breakage the mill is cooled to - 5 ° by circulation of a cold mixture of ethylene glycol/water (50:50,v/v) from an 80-litercapacity cold bath (flow rate 300 liters/hr). Cooling is continued throughout the breakage. A peristaltic pump drives the yeast through the mill chamber. Any commercial variable pump allowing a flow rate from 50 to 300 ml/min and equipped with silicone tubing of 5 mm internal diameter is convenient. Centrifuge. Sorvall model RC5, rotor GS 3 with 0.5-liter polycarbonate bottles and 00430 caps, as well as rotor SS 34. Procedure (0°). Before breakage the cells are washed twice with breaking buffer and resuspended in breaking buffer to yield a 60% suspension (wet weight). Careful adjustment of the suspension to pH 6.5 is necessary (KOH) because of strong endogenous acidification by the yeast cells. Breakage must be carried out immediately afterward. All subsequent operations are given for 1 liter of yeast suspension in breaking buffer (600 g wet weight represents 60 g cell protein). The yeast suspension, kept ice-cold with constant magnetic stirring, is driven through the precooled mill by the peristaltic pump (60-80 ml/min flow rate). When approximately 200 ml of the yeast suspension have been introduced into the breakage chamber, the driving motor is switched on (stirring shaft speed 3000 rpm), while pumping continues at the same flow rate. Broken cells exit through a tubing to a 3-liter glass beaker kept in crushed ice. Estimation of cell breakage (usually 90%) is made by phase microscopy. The breakage of 1 liter of suspension takes 10-15 min. The temperature of the broken cells gradually rises from 10° to 27 °. Immediately after the yeast suspension has passed through the mill, the glass beads are washed
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with 0.5 liter cold breaking buffer at a flow rate of 300 ml/min, while the mill is still stirring. When no more liquid is collected at the chamber exit, the driving motor is switched off. To the 1.5 liter of the broken cells collected into the 3-liter beaker (1 liter of 60% suspension plus 0.5 liter wash) 1.5 liter of cold breaking buffer is immediately added and the pH is adjusted rapidly to 6.5 (KOH), since the pH has fallen to about 6.0. The homogenate is centrifuged at 1500 g for 15 min (GS 3 rotor) and the supernatant carefully decanted; the pellet is resuspended with 0.5 liter of breaking buffer and centrifuged again. The combined supernatants (if necessary adjusted to pH 6.5) are centrifuged at 9000 rpm for 30 min (GS 3 rotor) to collect the mitochondria. The supernatant (and, if desired, the fluffy layer) is discarded. Fat deposits are wiped off. Mitochondrial pellets are resuspended in 2 liters of breaking buffer and centrifuged again as above. The yield of (unpurified) mitochondrial protein is 7-8 g protein/600 g (wet weight) cells, or about 100 mg protein/l g cell protein.
Assay for Oxidative Phosphorylation. The assay is the same as for method 1, except that the EDTA concentration is increased to 2 mM (see General Remarks).
Enzymatic Method The method described here is adapted from previously published procedures. 6-8 In particular we stress some critical points that we found important for obtaining mitochondria with good R C. The main interest of this relatively laborious procedure is that high yields (130 mg mitochondrial protein per gram cell protein) of purified mitochondria with good R C are obtained that can be employed for further research in which integrity and purity are essential, such as studies of membrane permeability 21 and localization of loosely bound enzymes.
Yeast Culture Conditions. Saccharomyces cerevisiae strain Yeast Foam is grown aerobically (27 °) in the following medium: 1% Difco yeast extract, 0.1% KH2PO4, 0.12% (NH4)2SO4; carbon source, 2% lactate or ethanol. The medium is inoculated from exponential-phase cells pregrown in the same medium (0.75 mg protein per liter). Final growth in this medium is reached at 3 g cell protein per liter. Cells are harvested at 2 g cell protein per liter and washed twice with cold distilled water. 2~G. Arselin-Dechateaubodeau,M. Gu6rin, and B. Gu6rin,Biochimie 58, 601 (1976).
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Preparation of Mitochondria Reagents Buffers (final concentrations) 1. 0.1 M Tris-HCl, 0.5 M 2-mercaptoethanol, pH 9.3 2. 0.01 M Tris-HC1, 0.5 M KC1, pH 7.0 3. 0.01 M citrate-phosphate, 1.35 M sorbitol, 1 mM EGTA, pH 5.8 4. 0.01 M Tris-maleate, 0.75 M sorbitol, 0.4 M mannitol, 2 mM EGTA, 0.1% bovine serum albumin, pH 6.8 5. 0.01 M Tris-maleate, 0.6 M mannitol, 2 mM EGTA, 0.2% bovine serum albumin, pH 6.8 Snail gut juice from Helix pomatia (Industrie Biologique Fran~aise, Gennevilliers, France) Homogenizer. Overhead Waring Blendor, commercial model 5011, container capacity 100 ml Centrifuge. Sorvall model RC-2B, rotor SS 34, and continuous flow system (Sorvall KSB) adapted on rotor SS 34. Procedure (Described for 2 g Cell Protein = 20 g Wet Weight). The washed cells are suspended in 80 ml of buffer 1 and incubated for 10 min at 32 °. The cells are collected by centrifugation for 5 min at 1500 g, suspended in 3 liters of buffer 2, and centrifuged with the continuous flow system at 8000 rpm (flow rate 400 ml/min). This highly efficient washing step is essential for complete elimination of mercaptoethanol. Cells are suspended in 40 ml of buffer 3, then 5 ml of snail enzyme are added, and the mixture is shaken for 60-90 min at 32 ° under N2 atmosphere. Spheroplast formation, controlled by intermittent visual observation, 9 was found to be more reproducible under anaerobic conditions. The spheroplast suspension is centrifuged for 10 min at 1200 g and the supernatant discarded. All the following manipulations are carried out at 0 °. The spheroplast pellet is washed twice with 40 ml of buffer 4 by identical centrifugation. The final pellet is then suspended in 80 ml of buffer 5 and homogenized in the Waring blendor three times for 5 sec each at low speed. It is important to use the 100-ml container, closed with Parafilm foil below the container cap, in order to prevent foaming and drastic aeration. The homogenized suspension is distributed among six polypropylene tubes (50-ml, rotor SS 34). The volume of each tube is adjusted to 40 ml with fresh buffer 5 and the suspension centrifuged for 10 min at 800 g. Supernatants are carefully decanted. The pellets are resuspended with a
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Teflon pestle, collected into two centrifuge tubes in buffer 5, and centrifuged again for l0 min at 800 g. The combined supernatants are centrifuged for 10 min at 12,000 g. The supernatants are removed; there is no fluffy layer over the mitochondrial pellets. The centrifuge tubes are wiped to eliminate adhering lipid. Mitochondrial pellets are homogenized gently in a Potter-Elvejhem homogenizer with a Teflon pestle with a small volume of buffer 5 containing no serum albumin. The suspension is distributed among six centrifuge tubes; the volume of each tube is adjusted to 40 ml with the same buffer and centrifuged for l0 min at 800 g. The supernatant is centrifuged for l0 min at 12,000 g. The pellets are resuspended in a small volume of buffer 5 without serum albumin in two tubes and centrifuged for l0 min at 800 g. The supernatant is centrifuged for 10 min at 12,000 g, and the resulting purified mitochondrial pellet is used for the experiments.
Assay for Oxidative Phosphorylation. The assay is the same as for small-scale mechanical method 1. General Remarks Because of the varying genetic background of yeast laboratory strains, the methods described in this article might have to be slightly adjusted to suit each strain or culture condition. For instance, susceptibility to the snail enzyme is known to be somewhat variable, and a preliminary study should be carried out 9 to establish optimal enzyme concentration and incubation time. For the mechanical methods, the extraction yield of mitochondria also depends on the genetic background of the strains. When the yield is lowered, mitochondria might be of apparently poorer quality. In such cases we found it useful to increase the concentration of EDTA and/or serum albumin in the respiration medium. When endogenous respiration is relatively high, respiratory control is often poor. In this case two to three additional washings at 9500 g for 10 min will often produce considerable improvement. In the case of strains growing sluggishly on ethanol, the extraction yield of mitochondria will be lowered and mitochondrial respiration poorly controlled. In this case it is advisable to adopt the 0.5% glucose or the 0.1% glucose plus 0.4% galactose medium.