[8] Cytochrome-c oxidase from Saccharomyces cerevisiae

June 3, 2017 | Autor: Kevin Sevarino | Categoria: Saccharomyces cerevisiae, Cytochrome c oxidase
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[8]

CYTOCHROME-C OXIDASE FROM S. c e r e v i s i a e

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[8] C y t o c h r o m e - c O x i d a s e f r o m

Saccharomyces cerevisiae By

ROBERT 0.

POYTON, BRADLEY GOEHRING, MARTIN DROSTE,

KEVIN A . SEVAR1NO, L A R R Y A . A L L E N , a n d X I A O - J I A N Z H A O

Introduction Cytochrome-c oxidase, the terminal member of the respiratory chain, catalyzes the concerted transfer of four electrons from ferrocytochrome c to molecular oxygen, with the simultaneous pumping of protons across the mitochondrial inner membrane from the matrix to the cytoplasmic side. ~ Four redox-active metal centers (heme a, heine a3, CUA, and CUB), embedded in a multisubunit complex, constitute the catalytically active holoenzyme. Heme a3 and Cu~ are bridged in the resting fully oxidized form and constitute the binuclear reaction center, l The protein matrix surrounding the metal centers consists of several polypeptide subunits. In both prokaryotic and eukaryotic cytochrome-c oxidases, subunit I binds the heine a, heine a3, and Cu~ redox centers. 2 Subunit II binds CUA and also participates in cytochrome-c binding, presumably at a site close to CUA.X Subunit III may modulate the proton pumping activity of subunits I and II? In prokaryotes, these three subunits constitute the complete polypeptide contribution to the holoenzyme. In eukaryotes, the number of subunits comprising an active cytochrome-c oxidase molecule is significantly greater than three. The three largest subunits (I-III) in eukaryotic cytochrome-c oxidases are encoded by the mitochondrial genome. They possess primary sequence homology to the three subunits of some prokaryotic cytochrome-c oxidases. 4 All additional subunits are nuclear genome products. As mentioned later, these are currently thought to function in the regulation of catalysis or in the assembly of the holoenzyme,s In recent years, cytochrome-c oxidase from Saccharornyces cerevisiae has emerged as a useful model for studying both the crosstalk between nuclear and mitochondrial genomes ~ and subunit structure-function relaG. T. Babcock and M. Wikstrom, Nature 356, 301 (1992). 2 R. A. Capaldi. Ann. Rev. Biochem. 59, 569 (1990). 3 M. Brunori, G. Antonini, F. Malatesta, P. Sarti, and M. Wilson, Fur. J. Biochem. 169, 1 (1987). 4 M. Saraste, Q. Rev. Biophys. 23, 331 (1990). R. O. Poyton, C. E. Trueblood, R. M. Wright, and L. E. Farrell, Ann. N e w York Acad. Sci. 550, 289 (1988). ~' R. O. Poyton and J. E. McEwen, Ann. Rev. B i o d w m . 65, in press (1996).

MEIItODS IN ENZYMOLOGY.VOL. 26(1

Copyright !c 1995by AcademicPress. Inc. All rightsof reproduction in an} foun reserved.

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ANALYSTS OF OXIDATIVE PHOSPHORYLATION COMPLEXES SUBUNIT

GENOME

GENE

M

COX1

II

M

COX2

III

M

COX3

IV

N

COX4

V

N

COX5a, COX5b

i

N

COX6

~ ~,~,,

N N

Vl

Vll,Vlla V lll

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COX7, COX9 COX8

FIG. 1. G e n e subunit relationships for cytochrome-c oxidase from S. cerevisiae. LeJhPurified cytochrome-c oxidase analyzed by S D S - P A G E . Subunit polypeptides are designated. Middle: The genome that encodes each subunit is designated (M for mitochondrial: N for nuclear). Right: The n a m e of the structural genes for each subunit. Both the COX5a and C O X 5 b genes, which encode the Va and Vb isoforms of subunit V, are listed next to subunit V. Subunits VII and VIIa cannot be resolved on this gel system so the n a m e s of their genes, C O X 7 and COX9, appear next to one another.

tionships in eukaryotic cytochrome-c oxidases, v Active preparations of yeast cytochrome-c oxidase contain a total of nine subunits: three subunits (I, II, and III) encoded by mitochondrial genes (COXI, COX2, and COX3, respectively), and six subunits (IV, Va or Vb, VI, VII, VIIa, and VIII) encoded by nuclear genes (COX4, COX5a or COX5b, COX6, COX7, COX9, and COX& respectively)5 (Fig. 1). COX5a and COX5b encode interchangeable isoforms, Va and Vb, of subunit V. 5 The other subunits are specified by unique genes present in a single copy on their respective genomes. All three mitochondrially coded subunits and all six nuclear coded subunits have primary sequence homology to subunits in mammalian cytochrome-c oxidases. 8 Genetic analysis of gene disruption strains have shown that subunits IV, VI, VII, and VIIa are essential; strains that lack them have no cytochromes aa3 or cytochrome-c oxidase activity¢ In con7 R. A. Waterland, A. B. Basu, B. Chance, and R. O. Poyton, J. Biol. Chem. 266, 4180 (1991). T. E. Patterson, C. E. Trueblood, R. M. Wright, and R. O. Poyton, in " C y t o c h r o m e Systems: Molecular Biology and Bioenergetics" (S. Papa, B. Chance, and L. Ernster, eds.), p. 253, Plenum Publishing, New York, 1987.

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CYTOCHROME-C OXIDASK FROM S. cerevisiae

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trast, a strain that lacks subunit VIII has normal levels of cytochromes aa~ but reduced activity (80% of wild type). 5 Studies with genetically altered yeast strains have also shown that an isoform of subunit V is essential and that isozymes with Va and Vb possess different turnover numbers, different TN ...... values with cytochrome c, and different numbers of CO vibrators that can be visualized by infrared spectroscopy] '9 Together, these genetic studies have established that each of the six nuclear-coded subunits is an essential component of the holoenzyme. 5 It appears that subunits V and VIII function to regulate catalysis and that subunits 1V, VI, VII, and VIIa may function in assembly. It has been suggested that three additional polypeptides are present in yeast cytochrome-c oxidase preparations.~° At least two of these have partial sequence homology to polypeptides found in mammalian cytochrome-c oxidases. A strain that carries a disruption in the gene for one of these polypeptides has diminished cytochrome-c oxidase activity but retains optically detectable cytochrome-c oxidase] ~Insofar as the nine-subunit enzyme is fully active it is not yet clear if these polypeptides are bona fide subunits of the holoenzyme, polypeptides required for holoenzyme assembly, or adventitious contaminating polypeptides that copurify with it. This chapter concentrates on methods for the isolation and characterization of the nine-subunit yeast holocytochrome-c oxidase and its subunits as well as procedures for producing and analyzing mutant cytochrome-c oxidases.

General P r o c e d u r e s Protein Determination

Routine protein assays during the purification of cytochrome-c oxidase or its subunits are carried out by the Lowry assay ~2 in the presence of 0.4% sodium deoxycholate, the Bradford assay, ~3 or by an assay that uses bicinchoninic acid. ~4Bovine serum albumin (fraction V) is used as a calibration standard. ') L. A. Allen. X-J. Zhao. W. Caughey, and R. O. Poyton, d. Biol. Chem. 270, 110 (1994). ioj. M. T a a n m a n and R. A. Capaldi, J. Biol. Chem. 267, 22,481 (1992). t J A. E. P. LaMarche, M. 1. Abate, S. H. P. Chan. and B. L. Trumpower, J. Biol. Chem. 267, 22,473 (1992). i: O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall. J. Biol. Chem. 193, 265 (1951 ). ~ M. M. Bradford, Anal. Biochem. 72, 248 (I976). ~4 p. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia. F. H. Gartner. M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olsen, and D. C. Klenk. Anal. Biochem. 150, 76 (1985).

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ANALYSIS OF OXIDATIVE PHOSPHORYLATION COMPLEXES

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Heine A Determination

Heme A in purified cytochrome-c oxidase preparations is determined from the difference in absorbance between 605 and 630 nm after reduction with dithionite, using an extinction coefficient (A605 nm -- A630 . . . . reduced) of 21.5 mM t cm 1.~ For turbid suspensions, heine A content is determined from room-temperature difference spectra of reduced minus oxidized samples, using an Aminco DW-2000 double-beam dual-wavelength spectrophotometer and an extinction coefficient (A605 A630 nrn) of 16.5 mM 1cm 1.7 For submitochondrial particle preparations, difference spectra are recorded from a sample reduced with dithionite and another oxidized with ferricyanide. 9 For whole cells, difference spectra are recorded from cell suspensions (0.45 to 0.50 g wet weight/ml), which are oxidized with 0.1% (v/v) H202 or reduced with 5 mg/ml solid sodium dithionite.7 Heme A content can also be determined as its pyridine hemochrome, using an extinction coefficient at 587 nm of 26.0 mM i cm-l.16 n m

--

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) is performed as a modification17 of the method described by Merle and Kadenbach. 18 Gels are poured and run in Hoefer Mighty Small II electrophoresis units. The gels consist of a 5.5-cm-long separating gel [0.1% (w/v) SDS, 16% (w/v) 32:1 acrylarnide:bisacrylamide, 3.6 M urea, 10% (v/v) glycerol, 0.4 M Tris-HC1, pH 8.8] and a 0.5-cm-long stacking gel [0.1% (w/v) SDS, 3.5% (w/v) 32 : 1 acrylamide : bisacrylamide, 0.125 M TrisHC1, pH 6.8]. The running buffer consists of 0.025 M Tris base, 0.192 M glycine, 0.1% SDS, pH 8.4. Samples (for example, mitochondria, cytochrome-c oxidase, or subunits) are denatured, reduced, and solubilized in protein dissociation buffer (10 mM NaPO4, pH 6.8, 2% (w/v) SDS, 20 mM dithiothreitol, 4% (v/v) glycerol). The samples are heated at 37° for 30 min, boiled for 2 min, loaded onto the gel, and run at 110 V (constant voltage) for 3 hr. After electrophoresis, the gels are stained in 25% (v/v) 2-propanol, 10% (v/v) acetic acid, 0.05% (w/v) Coomassie blue R-250 for at least 2 hr, and destained in 10% (v/v) 2-propanol, 10% (v/v) acetic acid. ts T. E. Patterson, Ph.D. Dissertation, University of Colorado, Boulder (1990). ~6T. L. Mason, R. O. Poyton, D. C. Wharton, and G. Schatz, J. Biol. Chem. 248, 1346 (1973). t7 S. D. Power, M. A. Lochrie, K. A. Sevarino, T. E. Patterson, and R. O. Poyton, J. Biol. Chem. 259, 6564 (1984). is p. Merle and B. Kadenbach, Eur. J. Biochem. 105, 499 (1980).

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Assays Two types of assay can be used to measure the activity of yeast cytochrome-c oxidase. One, a polarographic assay, follows the rate of oxygen consumption by cytochrome-c oxidase in the presence of excess reducing equivalents provided by the redox dye N, N, N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD). The other is a spectrophotometric assay, which follows the rate of cytochrome-c oxidation. Both assays are applicable to crude cell fractions, mitochondria, mitochondrial subfractions, or cytochrome-c oxidase preparations. When p e r f o r m e d at ionic strengths below 100 raM, both assays are biphasic, with changing cytochrome-c concentrations. ~9 Because the spectrophotometric assay is more conducive to steadystate rate analysis 19 and more reliable in our hands, we r e c o m m e n d it for general use. For routine assays a saturating concentration of 32/xM is used.

Solutions Assay Buffer. 40 m M KPO4, p H 6.7, 0.5% Tween 80 Cytochrorne c. For routine studies horse heart cytochrome c (type III or VI from Sigma Chemical) is used. However, for more detailed studies of cytochrome-c oxidase mutants, we r e c o m m e n d the use of the physiological substrate; either iso-1 or iso-2 yeast cytochrome c. 9 Prior to use, cytochrome c is reduced as follows: A few grains of solid dithionite are added to a 10ml aliquot of a 32/~M cytochrome-c solution in assay buffer; the sample is then vortexed for 1 to 2 rain to remove excess dithionite and absorbance at 550 nm is determined ( m e a s u r e m e n t 1). To determine the extent of reduction a 1-ml aliquot of this solution is treated first with a few more grains of solid dithionite and absorbance at 550 nm is determined (measurement 2). This sample is then treated with excess potassium ferricyanide (added as a solid or a 1.2 M solution) and absorbance at 550 nm is determined ( m e a s u r e m e n t 3). The percent of reduction is determined from the difference in absorbance at 550 nm of m e a s u r e m e n t 1 minus m e a s u r e m e n t 3 divided by the difference in absorbance of m e a s u r e m e n t 2 minus measurement 3. The cytochrome-c solution should be at least 85% reduced for use. Procedure A 1-ml aliquot of reduced cytochrome-c solution is placed in a microcuvette and the initial absorbance at 550 nm is recorded. A small aliquot of enzyme (5 to 10 ~1) is added, the solution is mixed immediately, and the change in absorbance at 550 nm is followed. Sufficient enzyme should be t,*S. H. Speck, D. Dye, and E. Margoliash, Proc. Natl. Acad. Sci. U.S.A. 81, 347 (1984).

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ANALYSIS OF OXIDATIVE PHOSPHORYLAT1ON COMPLEXES

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added to complete the oxidation of cytochrome c within 3 min. At the end of the incubation period, 10 ttl of 1.2 M potassium ferricyanide is added to complete the oxidation of cytochrome c. Activity is expressed as the first-order rate constant, K (in sec ~). This is determined from a semilog plot of absorbance (at various times) minus absorbance of totally oxidized cytochrome c versus time. An appropriate control should be run to correct for any autoxidation of reduced cytochrome c.

Purification of Holoenzyme Two methods r'2° for the isolation of a nine-subunit yeast cytochrome-c oxidase are described. They are applicable to either laboratory strains of S. cerevisiae or to commercially grown baker's yeast (e.g., Fleischman's, Red Star, or Budweiser). These methods use submitochondrial particles (SMPs) as starting material. Both methods involve (1) detergent solubilization of SMPs; (2) ammonium sulfate fractionation of the detergent extracts: and (3) chromatography. Method 1 is generally used 2° when 10 to 50 mg of highly purified holoenzyme are required. Method 2 is used 17when larger amounts of enzyme (0.1 to 1 g) are required. A modification of method 2 has been devised for the micropurification of cytochrome-c oxidase. :l

Preparation of Subrnitochondrial Particles The following protocol > is appropriate for preparing mitochondria and submitochondrial particles from 0.5 to 50 pounds (wet weight) of yeast. It makes use of a Dyno-Mill cell disintegrator (W. A. Bachofen, Machinenfabrick, Basel, Switzerland, also available from Owen Mills, Maywood, N J) to break cells. For smaller mitochondrial and submitochondrial particle preparations, we use methods that remove the cell wall enzymatically? 2,> Pressed cakes of yeast are suspended at 1 lb/liter of 50 mM K2HPO4, 0.9% (w/v) KC1, I mM E D T A , pH 8.4 (KPE buffer). The suspension is pumped at 6 liters/hr through the 0.6-liter continuous flow cell of a model KD-L Dyno-Mill cell disintegrator filled with acidwashed glass beads (0.25 to 0.5 mm in diameter). The Dyno-Mill is operated at a speed of 3000 rpm with a jacket temperature of - 1 8 ° and a distance piece of 0.02 mm at the flow cell exit. The broken cell suspension is collected and immediately centrifuged for 20 rain at 12,000g ...... . The _~0C. George-Nascimento and R. O. Poyton. J. Biol. Chem. 256, 9363 (1981). ~/ M. G. Cumsky. C. Ko, C. E. Trueblood, and R. O. Poyton, Proc. Natl. Acad. Sci. U.S.A. 82, 2235 (1985). :2 G. Daum, P. C. Bohni. and G. Schatz, .I. Biol. Chem. 257, 13028 (1982). > E. E. McKee and R. O. Poyton..I. Biol. Chem. 259, 9320 (1984).

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supernatant from this step is pumped at a flow rate of 80 ml/min through a TZ-28 zonal reorienting continuous-flow rotor (Sorvall-Dupont, Newton, CT) run at 19,000 rpm. The pelleted mitochondria are scraped from the rotor wall with a rubber spatula, suspended in KPE buffer with a glassTeflon homogenizer, and centrifuged for 60 rain at 94.000g ..... . The pellet of mitochondria is resuspended to 20 mg protein/ml in KPE buffer and stored at - 7 0 ° until ready for use. Frozen SMPs can be stored for up to two years without any noticeable loss of cytochrome-c oxidase activity. When needed, the mitochondrial suspension is thawed slowly on ice and sonicated in 150-ml aliquots with a Branson Sonifier (model W 185) run at a power setting of 60 W. Each 150-ml aliquot is sonicated for 60 sec in a rosette recirculating cell (Branson Instruments, Danbury, CT). After sonication, the suspension is centrifuged for 180 min at 94,000g ..... . The supernatant is discarded and the pellet (submitochondrial particles) is suspended to 20 mg of protein/ml of KPE buffer by homogenization with a glass-Teflon homogenizer.

Isolation of" Cytochrome-c Oxidase: Method 1 This method 2° is convenient for preparing 10 to 50 mg of highly purified cytochrome-c oxidase. All steps are carried out at 4 to 6 °. Step 1: Solubilization of SMPs. Cytochrome-c oxidase is released from submitochondrial particles by cholate solubilization as follows. A 20% cholate solution (use recrystallized cholic acid adjusted to a pH of 7.8 with 1 N KOH) is added to a suspension of SMPs (20 mg protein/ml of KPE) at a final concentration of 3 mg cholate per milligram of SMP protein. Ammonium sulfate (176 g/liter of suspension) is added, the pH is adjusted to 7.4, and the suspension is stirred gently overnight (8 to 10 hr). Step 2: Ammonium Sulfate Fractionation. The submitochondrial particle cholate extract is centrifuged for 20 rain at 25,500g ...... . The supernatant is retained and has added to it 94 g of ammonium sulfate per liter. If necessary, the pH is adjusted to pH 7.4 with 1 M H3PO4. This suspension is incubated for 10 rain and then centrifuged at 48,000gm~,x for 15 rain. The supernatant is discarded and the green pellet is suspended, by homogenization with a glass-Teflon homogenizer, in 0.25 M sucrose, 10 mM Tris-C1, pH 7.4, 0.5% cholate (STC) (15 ml for each gram of submitochondrial particle protein processed in step 1). This solution is clarified by centrifugation at 94,000g ...... for 15 rain. The pellet is discarded and the supernatant is brought to 24% saturation by the addition of a saturated (at 4 °) and neutralized solution of ammonium sulfate. If necessary, the pH is adjusted to pH 7.4 with 1 M Tris base and the solution is incubated for 10 rain. The precipitate is removed by centrifugation at 48,500g ..... for 15 min and discarded. The supernatant is

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adjusted to 41% ammonium sulfate by the addition of a saturated solution of ammonium sulfate, incubated for 10 min, and centrifuged for 15 min at 48,500gma×. The pellet is dissolved in 0.2 M NaPO4, pH 7.0, containing 4% Triton X-100 (add 1 ml for each gram of submitochondrial particle protein processed in step 1). If the solution is turbid, it is clarified by centrifuging for 10 min at 110,560gmax. The supernatant is diluted with 9 volumes of STC. Saturated ammonium sulfate is added to give a solution with 32% saturation. The pH is adjusted to 7.4 with 1 M Tris base, if necessary, and the solution is centrifuged at 94,000gmax for 15 min. The supernatant is brought to 41% saturation by the addition of saturated ammonium sulfate, adjusted to a pH of 7.4, if necessary, and incubated for 10 min. It is then centrifuged at 48,250gmax for 15 min. This pellet (P6) is dissolved in 1% Triton X-100, 20 mM NaPO4, pH 7.0 (TP buffer), and centrifuged at 110,560gmaxfor 15 min to clarify. The pellet is discarded. For P6 fractions with heme A/protein ratios in excess of 6 nmol of heme A/mg protein, no further ammonium sulfate fractionations are required prior to the chromatography step. Those P6 fractions with heme A/protein ratios of less than 6 nmol of heme A/rag of protein are further purified by two additional ammonium sulfate fractionations. These P6 fractions (10 to 20 mg protein/ml) are diluted with 5 volumes of STC and brought to 31% ammonium sulfate by the addition of a saturated' and neutralized solution of ammonium sulfate. The suspension is adjusted to pH 7.4 with 2 M KOH, stirred for 10 min, and then centrifuged at 28,000 rpm in a Spinco 30 rotor (94,000gmax). The supernatant is collected, adjusted to 38% saturation with ammonium sulfate, and centrifuged as in the previous step. This pellet (P6A) is dissolved in TP buffer. Step 3: Detergent Exchange Chromatography. Resuspended P6 or P6A pellets (10 to 20 mg protein/ml) are desalted by passage through a column of Sephadex G-25 (coarse), equilibrated with TP buffer, or by dialysis against 1000 volumes of TP buffer. An aliquot of the desalted material, containing up to 30 mg protein is concentrated to 0.5 ml by dialysis against solid sucrose and then subjected to gel filtration on a Sephadex G-100 column (1.5 × 90 cm) equilibrated and run with 10 mM NaPO4, pH 6.8, 0.5% potassium cholate. The column is run at a hydrostatic pressure of 18 cm and monitored for absorbance at 422 and 280 nm. Those fractions that are included in the column and that contain absorbance at 422 nm contain cytochrome-c oxidase. When pooled they should account for 60 to 70% of the protein loaded and should have a heme A/protein ratio of 9 to 10 nmol/mg. This step may be substituted by ion-exchange chromatography on DEAE cellulose 16 or by hydroxylapatite chromatography. 24 24R. O. Poyton and G. Schatz,J. Biol. Chem. 250, 752 (1975).

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Isolation of Cytochrome-c Oxidase: Method 2 This method ~7is preferable when large amounts of enzyme (0.1 to 1 g) are required. It can also be scaled down for micropurification2~ (see later). All operations are carried out at 4 to 6° unless otherwise indicated. Step 1: Solubilization of SMPs. Submitochondrial particles, suspended at 20 mg protein/ml (designated as the V20 fraction) in KPE buffer, are extracted by the addition of 0.3 volumes of 20% (w/v) Fisher cholic acid (unrecrystallized, adjusted to pH 8.3 with 4 N KOH) and 184 g/liter of ammonium sulfate. The pH is adjusted to 7.2 with 4 N KOH and the suspension stirred for 12 to 16 hr. Step 2: Ammonium Sulfate Fractionation. The detergent solubilized SMP extract is centrifuged for 20 rain at 27,578gma~. Ammonium sulfate (84 g/ liter) is added to the supernatant and the solution centrifuged as was done earlier. The pellet is resuspended to a final volume of 0.3 x V20 in STC. Saturated ammonium sulfate (0.428 volumes), neutralized with NH4OH, is then added and the suspension is centrifuged at 17,000 rpm (34,858gma×) in a Sorvall SS-34 rotor. Crude cytochrome-c oxidase is precipitated with saturated ammonium sulfate (0.296 volumes) and centrifuged as in the previous step. The pellet is resuspended to a final volume of 0.1 x V20 in 50 mM potassium 3-(N-morpholino)propanesulfonic acid (MOPS), 2 mM EDTA, pH 7.5 (ME buffer), containing 2% potassium cholate (Sigma). This opalescent solution is centrifuged for 10 rain at 127,000g~,×, and the clear, green-brown, supernatant is retained for chromatography on octyl Sepharose. Step 3: Octyl-Sepharose Chromatography and Detergent Exchange. An octyl-Sepharose (Pharmacia) column (bed volume of 0.2 x V20) is formed in a 50-ml disposable syringe column equilibrated with ME containing 2% potassium cholate (ME-2% cholate). The green-brown enzyme solution obtained in step 2 is applied to the column under a hydrostatic pressure equal to 1 x bed height. The column is first washed with 5 column volumes of ME-2% cholate at a hydrostatic pressure of 5x bed height, and then washed successively with 5 bed volumes of ME-2% cholate containing 84 g/liter ammonium sulfate, 4 column volumes of ME-2% Tween 20 containing 34 g/liter ammonium sulfate, and 5 column volumes of ME-2 M urea. After these washes are completed cytochrome-c oxidase is eluted with ME-3% Triton X-100 at a hydrostatic pressure of 1 x the bed height. The Triton X-100 in the eluted cytochrome-c oxidase solution is replaced with sodium-cholate by "exchange centrifugation" as follows. Aliquots (25 ml) of the dark-green cytochrome-c oxidase fraction collected from the octyl-Sepharose column are centrifuged for 12 to 16 hr through a 3-ml pad of ME containing 1% cholate and 50% sucrose 194,000g..... The dark-green cytochrome-c oxidase band in the sucrose layer is removed by aspiration

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ANALYSIS OF OXIDATIVE PHOSPHORYLATION COMPLEXES

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and desalted by passing it through a column (bed volume of 7× sample volume) of Bio-Rad P-6 (coarse) gel, which has been equilibrated with ME-0.5% cholate. The desalted enzyme is concentrated by centrifugation at 194,000gm~,x for 14 to 16 hr. The pelleted enzyme is resuspended in a small volume of ME-0.5% cholate and incubated at 4 ° for 8 hr in order to allow it to dissolve completely. The clarified solution is then adjusted with ME-0.5% cholate to 200 nmol (heine A) cytochrome-c Oxidase/ml and frozen at - 7 0 ° for future use. Cytochrome-c oxidase produced by this method usually has a heine A/ protein ratio of greater than 9 nmol heme A/rag protein. However, the enzyme loses a substantial portion of its enzymatic activity during octylSepharose chromatography. This loss occurs during elution with the M E - 2 M urea buffer. Enzyme prepared in the absence of this step has a specific activity [K(min i m g i × 10 2)] of 10 to 12, a value that is comparable to enzyme prepared by method 1. Despite the loss of activity that results from urea treatment, this step is included in the large-scale purification procedure because it is very effective in removing residual Tween 20, which is bound to the resin and to the holoenzyme and because it facilitates the subsequent elution of the holoenzyme in a small volume of ME-3% Triton X-100 buffer. It is, therefore, very useful for enzyme preparations that are going to be processed further for the isolation of subunits, as discussed later. We emphasize, however, that this step can be omitted for other applications.

Microisolation of Cytochrome-c Oxidase: Method 2A This method is a modification of method 22~ and is applicable to much smaller amounts of submitochondrial particles than either of the two methods already described. Generally the starting material is 5 to 10 mg of submitochondrial particle protein isolated from enzymatically lysed yeast cells. Submitochondrial particles are extracted with 0.3 volume of 20% cholate, followed by overnight precipitation at 4 ° with 50 mg of ammonium sulfate per milliliter. After centrifugation at 27,600g ...... the supernatant is subjected to octyl-Sepharose chromatography (0.2 ml bed volume) as described earlier except that cytochrome-c oxidase is eluted in 1 ml of buffer containing 5% rather than 3% Triton X-100. The detergent exchange and desalting steps are accomplished by ultracentrifugation of the enzyme through the buffers described earlier in a microfuge tube, supported by an O ring on the rim of a centrifuge tube that had been filled with the same buffer, This procedure typically yields 10 to 20 ~g of purified enzyme.

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Purification of S u b u n i t s Subunit polypeptides are purified from holocytochrome-c oxidase preparations purified by method 2. The enzyme is first fractionated into subunit pools using mixtures of organic solvents, and then subunits are isolated either by gel permeation chromatography (subunits I, II, and III) or reversed-phase H P L C (subunits IV, V, VI, VII, VIIa, and VIII).

Pur(fication of Subunits 11, IV, V, WL Wla, VIH Step 1: Solvent Prefractionation of Holoenzyrne. Purified holoenzyme is adjusted to 200/~M (heine A) with extraction buffer I (50 mM MOPS, 2 mM EDTA, 0.5% sodium cholate, adjusted to pH 7.5 with 4 N sodium hydroxide) and sequentially extracted with mixed organic solvents as follows. A l-ml aliquot of holoenzyme is mixed with an equal volume of 80% acetonitrile and stirred for 1 hr at 4 ° in two 1.6-ml polypropylene centrifuge tubes. The suspension is centrifuged for 10 rain in an Eppendorf microfuge centrifuge and the supematant reserved. The pellet is reextracted with 1 ml of buffer I and 1 ml of 80% acetonitrile, sonicated briefly in a Megason bath sonicator (Ultrasonic Instruments Int., Farmingdale, NY) to disperse the protein, and stirred for 1 hr at 4 °. Following centrifugation, the supernarant is pooled with the first extract and the pellet reextracted. The final supernatant pool ($4,6) is lyophilized and processed for the isolation of subunits IV and VI (as described later); the pellet (PI) is suspended in 200 ~1 of water and lyophilized. The dry pellet is then suspended in 1 ml of buffer II [1.25% (v/v) triethylamine. 1.25% (v/v) trifluoroacetic acid] and extracted with 1 ml of mixed solvent [0.05% (v/v) triethlyamine, 0.05% (v/v) trifluoroacetic acid in 1 : 1 acetonitrile :n-propanol]. The suspension is stirred for 4 hr at 4 °, centrifuged, and the supernatant reserved as before. The pellet is reextracted twice more with the same solvents; the first time for 4 hr and the second, overnight. The pooled supernatants (Ss.7) are lyophilized directly and the pellet (P2) is suspended in 200/xl of water and then lyophilized. Step 2: Purification of Subunits IV, V, VI, VII, Vlla, and VIH Using Reversed-Phase HPLC. The general scheme for the HPLC purification of subunits involves dissociation of the enriched subunit pools ($4,(~ or $5.7) with guanidine hydrochloride: filtration through a Millipore HA 0.45-/xm filter: and reversed-phase chromatography on a Waters Associates/xBondapak C~s column. The solvents used for chromatography are A: 0.05% triethylamine, 0.05% trifluoroacetic acid. 5% acetonitrile; and B: 0.05% triethylamine, 0.05% trifluoroacetic acid in either acetonitrile, for $4.~, or a 1 : 1 mixture of acetonitrile and n-propanol, for Ss.7. To purify subunits IV and VI, a lyophilized $4.~,fraction from 100 nmol

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ANALYSIS OF OXIDATIVE PHOSPHORYLATION COMPLEXES

[8]

of cytochrome-c oxidase (200 nmol heme A) is dissociated by adding 1 volume (1 ml per 100 nmol heine A) of 6 M guanidine hydrochloride-10 mM dithiothreitol and incubated for 4 hr at room temperature. The dissociated sample is then acidified with 1/10 volume of 10% triftuoroacetic acid, filtered through a Millipore HA 0.45 /xm filter to remove any insoluble material and injected onto a/xBondapak C18 column (3.9 m m × 30 cm) equilibrated with solvent A. After 5 min of isocratic elution with solvent A at 0.5 ml/ rain, a linear gradient at 0.5 ml/min from 0 to 60% B in 60 rain (+1% B/ min) is initiated. Absorbance at 240 nm is monitored continuously and peak fractions are analyzed by SDS-PAGE. Subunit IV elutes at about 57 min and subunit VI elutes at about 65 rain (Fig. 2). Cholic acid and free heme elute after these two subunits (Fig. 2). Prior to reequilibration and the next injection and after the last protein peak is eluted, 1 ml of dimethyl sulfoxide is injected to remove any denatured, precipitated protein. To purify subunits V, VII, VIIa, and VIII a lyophilized $5,7 fraction is dissolved in 2 ml 0.05% trifluoroacetic acid, 8 M guanidine hydrochloride, and then treated with 20/xl of 1 M dithiothreitol (DTT). This extract is allowed to stand for 2 hr at room temperature and centrifuged as before to remove precipitated heine A, prior to chromatography. A 0.5-ml aliquot of the dissolved S~,7fraction is injected onto a/xBondapak Cls column (3.9 mm × 3.0 cm) equilibrated with solvent A. After chromatography for 5 rain at 0.5 ml A/rain, a linear gradient at 0.5 ml/min from 0 to 70% B in 115 rain is initiated. The order of elution is subunit VIII, residual subunit

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RETENTION

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FJ6.2. Isolation of subunits IV and VI by reversed-phase high-performance liquid chromatography. Peaks corresponding to subunits IV and VI as well as cholic acid, heine A, and dithiothreitol ( D T r ) are indicated.

[8]

CYTOCHROME-C OXIDASE FROM S. cerevisiae

109

VI, subunit V, subunit VIIa, and subunit VII (Fig. 3). After the last protein peak is eluted (subunit VII), 1 ml dimethyl sulfoxide is injected to remove denatured material. On completion of the gradient and elution of the dimethyl sulfoxide the column is reequilibrated with solvent A at 2 ml/min prior to the next injection. Step 3: Purification of Subunit H by Gel-Filtration Chromatography. The P2 pellet from step 1 is washed twice, by resuspension and centrifugation (5 rain, 10,000gmax) with 8 M guanidine HC1, 0.05% (v/v) trifluoroacetic acid, and twice with 45% (v/v) n-propanol, 0.05% (v/v) trifluoroacetic acid at 25 °, to remove nonsubunit polypeptide contaminants and to release any residual nuclear-coded subunits that may not have been extracted in step 1. It is then dissociated in 2% (w/v) SDS, 10 mM NaPO4 buffer, pH 7.0, and 1% (v/v) 2-mercaptoethanol for 60 rain at 37 °. The solubilized pellet is filtered through a Millipore HA 0.45-/xm filter and applied to the top of a column (2.5 × 100 cm) of Sephadex G-100 (regular mesh) equilibrated with 50 mM Tris-HC1, pH 7.4, 1 mM Na2-EDTA, 0.5% (w/v) SDS. The column is developed at 25 ° with equilibration buffer at a hydrostatic pressure of 60 cm and a flow rate of 10 to 15 ml/hr, Protein elution is monitored continuously at 280 nm with a UV monitor. Fractions of 1 ml are collected and monitored for their polypeptide composition by SDS-PAGE. Subunit II elutes as the major peak of absorbance just after the excluded volume (Fig. 4).

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FIG. 3. Isolation of subunits V, VII, Vlla, and VIII by reversed-phase high-performance liquid chromatography. Peaks corresponding to subunits V, VII, VIIa, and VIII, as well as residual subunit VI left in the Pa pellet are indicated.

110

A N A L Y S I S O F O X I D A T I V E P H O S P H O R Y L A FION C O M P L F X E S

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F~(;. 4. Purification of subunit If from holocytochrome-coxidase. Sephadex G-100 column chromatography of a crude subunit II preparation extracted from the P2 fraction of holocytochrome-c oxidase. The position of elution of subunit lI is bracketed and the void volume is indicated with an arrow.

Purification ()f Subunits 1 and III Subunits I and IlI are purified from holocytochrome-c oxidase isolated by method 2. Because these subunits are not easily released from the P2 pellet that is used to isolate subunit I1 (as described earlier), we use unprefractionated cytochrome-c oxidase (up to 200 nmol heine A) as starting material. An aliquot (1 to 5 ml) of holoenzyme is dissociated in 10 m M NaPO~, p H 7.0, 5% SDS, 1% 2-mercaptoethanol, 1% glycerol, 0.1% bromphenol blue (as an internal volume marker) by heating at 37 ° for 30 rain and boiling for 2 rain. The dissociated sample is filtered through a 0.45-~m Millipore filter and loaded onto a column (2.5 × 100 cm) of Sephadex G-150, protected at the top with a l-cm layer of Sephadex G-25 (coarse). The column is equilibrated and run with 50 m M Tris-C1, p H 7.4, 1 m M N a 2 - E D T A , 3% SDS. Flow rates should be 10 to 15 ml/hr with a hydrostatic pressure head of approximately 10 cm. Elution is monitored by absorbance at 280 nm and S D S - P A G E . The peaks containing subunits I and II (Fig. 5) are collected, dialyzed against 1000 volumes of 25 m M triethylamine-acetic acid, p H 6.5, overnight and then lyophilized. The pellet is redissociated as above and subjected to rechromatography on Sephadex G-150, as was done earlier. The peaks collected from the rechromatography step are essentially pure. Yields for subunits I and It are approximately 60 to 70%.

CYTO(THROMK-C OXIDASE FROM S. cerevisiae

[8]

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FRACTION NUMBER

FrG. 5. Purilication of subunits 1 and 111 lrom holocytochrome-coxidase. Sephadex G-150 column chromatographyof SDS dissociated holocytochrome-coxidase. The position of elution of subunits I and I11 is brackeled.

Molecular Genetics of Yeast C y t o c h r o m e - c Oxidase A large number of genes are required for the biogenesis and function of yeast cytochrome-c oxidase. These have been identified by classical genetics, via the isolation and characterization of cytochrome-c oxidase deficient mutants, and by reverse genetics, whereby a cloned gene is used to produce a mutant (null or missense) in a wild-type background. Together, these two approaches have revealed the existence of 41 different genes that are specifically required for cytochrome-c oxidase (Table I). These genes fall into three categories: (1) 10 are structural genes for the subunits themselves; (2) 17 are nuclear P E T genes that regulate the expression of the mitochondrial C O X genes; and (3) 13 are nuclear P E T genes that are required for the assembly of the holoenzyme. Methods for the production of missense mutants, and null mutants with cloned genes in vitro, by PCR or other forms of site-directed mutagenesis, and their reintroduction into yeast can be found in other volumes in this series. =5,2~'~ Here, we describe the isolation of cytochrome-c oxidase-deficient mutants by classical genetic methods and also procedures for the biochemical characterization of cytochrome-c oxidase-deficient mutants. -5 (7. Guthrie and O. R. Fmk, this series, Vol. 194. 2
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