PROTEIN

EXPRESSION

AND PURIFICATION

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One-Step lmmunoaffinity Purification of Comp llex I Subunits from Beef Heart Mitochondria Adrian M. R. Haines,* J. Mark Cooper,* John A. Morgan-Hughes,? John B. Clark,$ and Anthony H. V. Schapira*,t *Department of Neuroscience, Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom; tClinica1 Neurology and SNeurochemistry, Institute of Neurology, London, United Kingdom

Received

December

27, 1991,

and

in revised

form

March

Press,

Inc. .

Mitochondrial complex I (NADH:ubiquinone oxidoreductase, EC 1.6.99.3) is the first oxidoreductase enzyme complex in the electron transport chain. It catalyzes the oxidation of NADH with the subsequent reduction of the lipid-soluble electron carrier, ubiquinone. This, in turn, transfers electrons further along the respiratory chain to the terminal acceptor, oxygen. The transfer of electrons is associated with the vectorial extrusion of protons from the mitochondrial matrix generating a proton motive force across the mitochondrial inner membrane, which is used by complex V (ATP synthase) to generate ATP. Complex I is the largest of the respiratory chain complexes. It comprises at least 26 polypeptides and has a total relative molecular mass of 700-800 kDa. Seven of the complex I polypeptides are encoded by mitochondrial DNA (mtDNA)’ and trans’ Abbreviations buffered saline Tween 20; DMP,

used: DDM, dodecyl maltoside; PBSa, phosphateand azide; PBST, phosphate-buffered saline and dimethyl pimelimidate; BHM, beef heart mitochon-

1046-5928/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

of

23,1992

Polypeptides of beef heart mitochondrial complex I were isolated from 15 mg of solubilized beef heart mitochondria using antibodies immobilized on an agarose chromatography column. The preparation was examined by SDS electrophoresis and Western blotting using affinity-purified antibodies to complex I and compared to beef heart complex I purified according to the conventional method of Hatefi and Rieske. There was a high degree of homology between the two preparations as judged by SDS-polyacrylamide electrophoresis and by immunoblotting with seven affinity-purified antibodies to various complex I subunits. This method could be applied to the preparation of complex I subunits from small samples such as human muscle biopsy specimens. 0 1992 Academic

and Departments

lated on mitochondrial ribosomes (1); the remainder are encoded by nuclear DNA, translated on cytoribosomes, and imported from the cytoplasm. The recent discovery of human diseases associated with specific defects of complex I has focused attention on the molecular structure of this large protein. Abnormalities involving complex I activities and/or abnormalities in complex I subunits have been reported in many patients, with a broad clinical spectrum predominantly involving combinations of opthalmoplegia, myopathy, and encephalopathy and including patients with MELAS (2), Leber’s hereditary optic neuropathy (3), and Parkinson’s disease (4). The molecular abnormalities have involved deficiencies of nuclear encoded complex I subunits (5,6), deletions of mtDNA (7), and point mutations of mtDNA in the tRNALe”(UUR) (8), ND1 (9), and ND4 (10) genes. Identification of the precise molecular basis of these disorders requires the study of complex I polypeptides. Thus far most studies have used immunoblotting with anti-complex I antibodies. This technique is limited by the specificities of the antibodies used and the inability to detect all constituent polypeptides of complex I. We have developed a technique to purify complex I polypeptides from small preparations of mitochondria (15 mg or less). This technique uses a single immunoaffinity chromatography step utilizing immobilized rabbit polyclonal antibodies raised to the iron protein (IP) fraction of complex I. This method may be applied to human biopsy samples and allow direct study of complex I polypeptides. METHODS

Chemicals were obtained from Merck Ltd. unless otherwise stated. Ubiquinone-1 was a kind gift from the Eisai Chemical Co. (Tokyo, Japan). dria; mtDNA, mitochondrial DNA, IP, iron protein fraction; FP, flavoprotein fraction; th, transhydrogenase. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; OA, ovalbumin. 223

Inc. reserved.

224 Preparation of complex I. BHM according to the and flavoprotein fied according to

HAINES

bovine heart mitochondria (BHM) and and beef heart complex I were purified method of Hatefi and Rieske (11). IP (FP) fractions of complex I were purithe method of Galante and Hatefi (12).

Preparation of antibodies. Antibodies were prepared by immunizing New Zealand white rabbits subcutaneously on dorsal sites with primary injections in Freund’s complete adjuvant (Sigma) and booster injections in Freund’s incomplete adjuvant. IP fraction antibodies were prepared by primary immunization with 1 mg of IP fraction and boosted with 0.5 mg. FP fraction antibodies were prepared by primary immunization of 0.3 mg of FP fraction and boosted with 0.15 mg. Antibodies to other complex I subunits were prepared by immunizing with electroeluted protein bands excised from faintly aqueous Coomassie-stained sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels of complex I IP or FP. Affinity purification of antibodies. Complex I was coupled via N-hydroxysuccinimide to agarose beads (Affi-Gel 10 and 15, Bio-Rad Laboratories) using standard procedures. Briefly 30 mg of complex I, purified by the Hatefi method, were solubilized in 5 ml of 0.1 M NaHCO,, pH 8.2, containing 1% Triton X-100 (Sigma). This was mixed with an equal volume of a 1:l mixture of Affi-Gel 10 and Affi-Gel 15 (Bio-Rad Laboratories) for 16 h at 4°C. The slurry was blocked with 1 M ethanolamine-HCl, pH 8 (100 pi/ml gel), transferred to a ClO/ 10 chromatography column (1 X 10 cm; Pharmacia, Milton Keynes, UK), equilibrated with phosphate-buffered saline containing 0.02% sodium azide, pH 7.4 (PBSa), washed with 3 M guanidine-HCI (GuHCl) and re-equilibrated in PBSa. Neat serum, typically l-2 ml, was passed through the column at a flow rate of 0.2 ml/min. Specifically bound IgG was eluted with 3 M GuHCl at the same flow rate, dialyzed once against PBSa and twice against water, snap frozen in liquid nitrogen, and lyophilized. For immunoblotting the lyophilized antibody was made up to the original volume of serum in PBSa containing 1% ovalbumin (Sigma) and stored at 4°C. For the preparation of affinity-purified antibodies for subsequent covalent coupling to protein A, the eluted antibody was dialyzed twice against PBSa, pooled, and concentrated on an Amicon 8400 ultrafiltration cell. Afinity purification of complex I. Complex I was affinity purified using anti-IP fragment antibody covalently coupled via dimethyl pimelimidate (DMP) immobilized to protein A using the Immunopure IgG orientation kit (Pierce Europe BV, Holland). The antibody used was affinity purified as described above. The protein A-anti-IP antibody column was prepared according to the instructions supplied by Pierce. Briefly 4.5 mg of affinity-purified anti-IP fragment antibody in 11.5 ml of

ET AL.

PBSa was diluted with 10 ml of sodium borate wash buffer, pH 8.2 (Pierce), and mixed with the protein A on a rotating wheel at room temperature for 1 h. After washing with borate wash buffer the bound antibody was covalently attached to the protein A by addition of 2 ml of DMP (6.6 mg/ml) in DMP cross-linking buffer (0.2 M triethanolamine, pH 8.2; Pierce) and mixed at room temperature for 1 h. After washing with DMP cross-linking buffer the protein A was blocked with the addition of 2 ml of 0.1 M ethanolamine, pH 8.2, for 10 min at room temperature. The gel slurry was transferred to a ClO/lO column (1 X 10 cm) and equilibrated in PBSa containing 0.2% dodecyl maltoside (Sigma) (PBSa/0.2% DDM), then washed five times with 0.1 M glycine-HCl, pH 2.5, containing 0.2% DDM, and finally re-equilibrated in PBSa/0.2% DDM. For the purification of complex I, 15 mg of BHM were diluted to 1 mg/ml in PBSa/0.2% DDM, allowed to solubilize at room temperature on a rotating wheel for 1 h, and then centrifuged at 43,OOOg,, using an MSE 8 X 50 rotor for 45 min. Fifteen milliliters of the supernatant were passed through the IP antibody-protein A column at a flow rate of 0.2 ml/min, at room temperature. The column was washed with PBSa/0.2% DDM, and specifically bound proteins were eluted with 0.1 M glycineHCl, pH 2.5, containing 0.2% DDM. Peak fractions, as determined by absorbance at 280 nm, were pooled. Detergent was removed by lo-fold dilution in acetone at -20°C overnight, and the resulting precipitate, containing eluted protein, was pelleted by centrifugation. The supernatant was removed and the pellet dried under a stream of nitrogen gas and dissolved in distilled water to three times the original volume of the pooled fractions. This was dialyzed against two 5-liter changes of distilled water at 4°C for 20 h each change to remove residual detergent, frozen in liquid nitrogen, and lyophilized. Gel electrophoresis. SDS gel electrophoresis was carried out under reducing conditions using 20% polyacrylamide Phast gels (Pharmacia). The lyophilized affinity-purified preparation was solubilized in 25 ~1 of SDS reducing sample buffer. Electrophoresis and silver staining were carried out according to the recommendations for the 20% Phast gels, except that electrophoresis was carried out for a total of 120 Vh. Silver-stained gels were scanned using an Ultroscan-enhanced laser densitometer (Pharmacia), and densitometric readings, taken every 20 pm along each lane, were converted into ASCII code using Gelcon computer program (Pharmacia), which allowed importation into Sigmaplot computer graphics program (Sigma) for representation. Western blotting. Proteins were electroblotted from 20% Phast gels onto 0.2-pm-pore nitrocellulose membrane (Schleicher & Schuell) for 30 min at 5.5 mA/cm’ using a semidry blotter (Bio-Rad Laboratories) and a continuous buffer as described by Towbin et al. (13).

IMMUNOAFFINITY

PURIFICATION

Proteins were immunostained with affinity-purified antibodies to complex I polypeptides, using an alkaline phosphatase-recombinant protein G conjugate (Novobiochem, Nottingham, UK). The nitrocellulose was blocked with 5% proprietary low-fat milk powder (Marvel) in PBSa for 30 min and washed once in PBS containing 0.1% Tween 20 (PBST) for 3 min. It was then incubated with affinity-purified antibody diluted in PBST containing 1% ovalbumin (PBST-OA) for 1 h, washed three times in PBST, and incubated with recombinant protein G-alkaline phosphatase conjugate diluted 1:lOOO in PBST-OA for 30 min followed by three washes as before. Visualization was achieved by incubating in the substrate solution, 0.2 mg/ml BCIP (Sigma), 0.4 mg/ml nitro blue tetrazolium (Sigma) in 0.1 M Tris-HCl, pH 9.0, containing 0.1 M NaCl and 50 InM MgCI,, for 5 min. The reaction was stopped by dilution in water, and the blots were thoroughly washed in water and dried. Enzyme assuys. Rotenone sensitive NADH:ubiquinone oxidoreductase assay was carried out according to the method described by Ragan et al. (14), in the presence of 3.2 mg/ml soybean phosphatidylcholine (Type II-S, Sigma). NADH:ferricyanide reductase assays were carried out according to the method of King and Howard (15). Protein was assayed according to the method of Lowry et al. (16). Mitochondria were sonicated at a concentration of 2.8 mg/ml for 6-8 s on ice at a setting of 3 in an MSE 1-73 ultrasonicator fitted with a 3-mm probe to maximize mitochondrial activities. RESULTS

The protein A-IP fragment antibody column was used repeatedly to isolate approximately 100 pg of complex I from 15 mg of beef heart mitochondria (approximately 0.7% of mitochondrial protein). SDS gels of immunoaffinity-prepared complex I and that prepared by the method of Hatefi are shown in Fig. 1. Densitometric scans of these lanes are shown in Fig. 2. The molecular mass nomenclature given for the protein bands is based on that adopted by Cleeter and Ragan (17) and therefore does not necessarily reflect the precise molecular mass of individual protein bands in these gels. There is a high degree of homology in the polypeptide composition between the two preparations. Dinucleotide transhydrogenase (th) is a frequent contaminant of complex I preparations (Fig. 1, lane l), but has been removed by immunoaffinity purification (Fig. 1, lane 2). The main differences are an extra band below the 49kDa subunit and the complete absence of the 42-kDa subunit in the immunoaffinity-purified preparation. Immunoblotting was carried out with four affinitypurified antisera raised to the 75-, 49-, 39-, 30-, and 13kDa subunits (Fig. 1, lanes 3-12), and with two affinitypurified antisera raised to the IP and FP fractions of complex I (Fig. 1, lanes 13-16). The antibody to the IP

OF

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49,

41

3024-

-24

FIG. 1.

Silver-stained SDS-polyacrylamide gel of the Hatefi preparation of complex I (lane 1) and the immunoaffinity preparation of complex I (lane 2). (Lanes 3-16) Immunoblots of the Hatefi preparation of complex I (odd-numbered lanes) and immunoaffinity preparation of complex I (even-numbered lanes). Lanes 3 and 4 were incubated with affinity-purified antibody to the 75kDa subunit (1:200 dilution), lanes 5 and 6 with antibody to the 49-kDa subunit (1:200 dilution), lanes 7 and 8 with antibody to the 39-kDa subunit (1:200 dilution), lanes 9 and 10 with antibody to the 30-kDa subunit (1:lOO dilution), lanes 11 and 12 with antibody to the 13-kDa subunit (1:200 dilution), lanes 13 and 14 with antibody to the IP fraction (1:200 dilution), and lanes 15 and 16 with an antibody to the FP fraction of complex I (1:lOO dilution).

fragment recognizes the 24-kDa protein, which was a minor contaminant (but very antigenic) in the preparation of IP used for immunizing rabbits. The antibody raised to the 39-kDa subunit additionally recognized a protein band of lower molecular mass that may either be a degradation product or a subunit with a similar epitope. With both the Hatefi and the immunoaffinity preparations of complex I, these antibodies showed similar immunoreactivity, with the exception of the antibody raised to the 49-kDa subunit, which also recognized the band just below the 49-kDa subunit. The band observed on SDS-PAGE, below the 49-kDa subunit of complex I, may be the result of proteolytic degradation of the 49kDa subunit since the affinity-purified antibody to this subunit also recognized the lower band in the latter. NADH:ferricyanide reductase and NADH:ubiquinone reductase activities of the complex I purified by the Hatefi method were 147 and 3.8 ~mol/min/mg (92% rotenone sensitive), respectively. However, these activities could not be demonstrated in the immunoaffinityprepared complex I. This was due to the conditions used as both these enzyme activities were completely lost in beef heart mitochondria solubilized in 0.1 M glycineHCl, pH 3.0, containing 0.2% DDM and were decreased by 49 and 86%, respectively, in preparations of bovine heart mitochondria incubated in PBSa with 0.2% DDM. DISCUSSION

The original method of complex I purification from bovine heart mitochondria was first described by Hatefi and co-workers in 1962 (11) and has not been modified appreciably since. This method uses selective solubliza-

226

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B

!

uv 7

8

I-I

9

10

20

tielat~ve

FIG. 2. respectively).

Laser

30

molecular

densitometry Peaks identified

weight

40

50

60

70

80

90

100

(kDo)

scan of lanes 1 and 2 in Fig. 1 (A and B, by immunoblotting are annotated.

tion in the detergents cholate and deoxycholate and precipitation with ammonium sulfate and potassium chloride to yield approximately 1% of mitochondrial protein as complex I. The procedure takes at least 5 days, and large quantities of muscle are required to purify complex I by this method, thus rendering the technique unsuitable for purification from rat or human organs. Cleeter and Ragan (17) immunoprecipitated bovine and human complex I using antibodies raised to the bovine holoenzyme; however, their preparations were contaminated with the heavy and light chains of IgG derived from the immunoprecipitating antibody. The immunoaffinity method described here is a significant improvement on immunoprecipitation since it is not dependent on precipitating antibodies, the preparation is not contaminated with immunoglobulin, the antibody column can be reused, and only small quantities of mitochondria are required. Here 15 mg of BHM was solubilized and this produced 100 /lg of complex I, sufficient to run approximately 50 lanes of Phast gels. The method produces a preparation with a high degree of

ET

AL.

polypeptide composition homology compared to that obtained by the Hatefi method. The immobilized antibody used to isolate the complex I polypeptides was raised to the IP fraction of complex I, but the resultingpreparation showed a pattern representative of the complex I holoenzyme. This indicates that the initial solubilization of beef heart mitochondria in DDM maintains most, if not all, of the integrity of complex I. This integrity is maintained when the complex binds to the antibody immobilized on the column. However, the complete absence of the 42-kDa subunit in the affinity-purified complex I preparation implies that it is loosely associated with the other subunits and is not retained when the complex binds to the immobilized antibody in the presence of 0.2% DDM. The 42-kDa subunit has also been shown to be lost during immunoprecipitation in the presence of the nonionic detergent Triton X-100 (17,lB). Elution at low pH (2.5) interferes with hydrogen bonding and protein-protein interactions and results in release of proteins bound by the antibody. This will also result in splitting of noncovalently linked subunits from each other and this is a likely reason for the complete loss in enzyme activity of the preparation. This is demonstrated by the loss of complex I enzyme activity in beef heart mitochondria incubated in the elution buffer (results not shown). This type of affinity chromatography of complex I should be easily applicable to the isolation of complex I subunits from human muscle and other organ biopsies as the antibody raised to the IP fragment cross reacts with polypeptides of human mitochondria. It can be estimated that it should be possible to purify enough complex I from 1 mg (or less) of mitochondrial protein to run on two or three Phast gels and the possibility of purifying complex I from tissue homogenates is currently being investigated. This will allow the direct study of complex I polypeptide structure in patients with complex I deficiency and will provide valuable new insights into the molecular mechanisms involved in diseases caused by defects of this protein. ACKNOWLEDGMENT Supported Child (UK).

by

a grant

from

Action

Research

for

the

Crippled

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W. D.,

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Parks,

J. K.

(1989)

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in

IMMUNOAFFINITY mitochondrial electron-transport reductase) in Leber’s hereditary Med. 320,1331-1333.

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activity (NADH-coenzyme optic neuropathy. N.

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OF Q J.

4. Schapira, A. H. V., Mann, V. M., Cooper, J. M. Dexter, D., Daniel, S. E., Jenner, P., Clark, J. B., and Marsden, C. D. (1990) Anatomic and disease specificity of NADH CoQ, reductase (complex I) deficiency in Parkinson’s disease. J. Neurochem. 55, 21422145. 5. Schapira, A. H. V., Cooper, J. M., Morgan-Hughes, J. A., Patel, S. D., Cleeter, M. J. W., Ragan, C. I., and Clark, J. B. (1988) Molecular basis of mitochondrial myopathies: Polypeptide analysis in complex I deficiency. Lancet i, 500-503.

8. Goto, Y., Nonako, I., and Horai, S. (1990) A mutation in the tRNAL”“‘“UR’ gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature 348,651-653. 9. Wallace, D. C., Singh, G., Lott, M. T., Hodge, J. A., Schurr, T. G., Lezza. A. M. S.. Elsas. L. J.. and Nikoskelainen. E. K. (1988) Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242,1427-1430. 10. Huoponen, K., Vilkki, J., Aula, P., Nikoskelainen, E. K., and Savontaus, M. (1991) A new mt DNA mutation associated with Leber hereditary optic neuroretinopathy. Am. J. Hum. Genet. 48, 1147-1153.

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Hate& Y., Haavik, A. G., and Griffiths, D. E. (1962) Studies on the electron transfer system. XL. Preparation and properties of mitochondrial DPNH-coenzyme Q reductase. J. Biol. Chem. 237,1676-1680.

12.

Galante, M. Y., and Hate&Y. (1979) Resolution of complex I and isolation of NADH dehydrogenase and an iron-sulfur protein. Arch. Biochem. Biophys. 192,559-568.

13.

Towbin, H., Staehelin, T., and Gordon, transfer of proteins from polyacrylamide sheets: Procedure and some applications. USA 76,4350-4354.

14.

Ragan, C. I., Wilson, M. T., Darley-Usmar, V. M., and Lowe, P. N. (1987) Subfractionation of mitochondria and isolation of the proteins of oxidative phosphorylation, in “Mitochondria: A Practical Approach” (Darley-Usmar, V. M., Rickwood, D., and Wilson, M. T., Eds.), pp. 799112, IRL Press, Oxford.

15.

King, T. E., and Howard, R. L. (1967) Preparation and properties of soluble NADH dehydrogenases from cardiac muscle, in “Methods in Enzymology” (Estabrook, R. W., and Pullman, M. E., Eds.), Vol. 10, pp. 275-294, Academic Press, San Diego, CA.

16.

Lowry, (1951) Chem.

6. Morgan-Hughes, J. A., Schapira, A. H. V., Cooper, J. M., and Clark, J. B. (1988) Molecular defects of NADH ubiquinone oxidoreductase (complex I) in mitochondrial diseases. J. Bioenerg. Biomemb. 20,365-381. 7. Holt, I. J., Harding, A. E., Cooper, J. M., Schapira, A. H. V., Toscano, A., Clark, J. B., and Morgan-Hughes, J. A. (1989) Mitochondrial myopathies: Clinical and biochemical features of 30 patients with major defects of muscle mitochondrial DNA. Ann. Neural. 26,699-708.

COMPLEX

0. H., Rosenbrough, Protein measurement

J. (1979) Electrophoretic gels to nitrocellulose Proc. Natl. Acad. Sci.

N. J., Farr, A. L., and with the folin phenol

Randall, R. J. agent. J. Biol.

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17.

Cleeter, M. W. J., and Ragan, C. I. (1985) The position of the mitochondrial NADH:Ubiquinone plex from several mammalian species. Biochem.

polypeptide comreductase comJ. 230,739-746.

18.

Heron, C., Smith, S., and Ragan, C. I. (1979) An analysis of the polypeptide composition of bovine heart mitochondrial NADH ubiquinone oxidoreductase by two-dimensional polyarylamide gel electrophoresis. Biochem. J. 181, 435-443.