Suppose we want to compute the Delaunay triangulation of a set P whose points are restricted to a collection R of input regions known in advance. Building on recent work by Löffler and Snoeyink, we show how to leverage our knowledge of R for faster Delaunay computation. Our approach needs no fancy machinery and optimally handles a wide variety of inputs, e.g., overlapping disks of different sizes and fat regions. Keywords: Delaunay triangulation - Data imprecision - Quadtree

Kevin Buchin

Maarten Löffler

Pat Morin

Wolfgang Mulzer

Braun, Hans-Peter

Sunderhaus, Stephanie

Boekema, Egbert J.

Kouřil, Roman

University of Groningen

   University of GroningenPurification of the Cytochrome c Reductase/Cytochrome c Oxidase Super Complex of YeastMitochondriaBraun, Hans-Peter; Sunderhaus, Stephanie; Boekema, Egbert J.; Kouřil, RomanPublished in:Methods in EnzymologyDOI:10.1016/S0076-6879(08)04410-8IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2009Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Braun, H-P., Sunderhaus, S., Boekema, E. J., & Kouřil, R. (2009). Purification of the Cytochrome cReductase/Cytochrome c Oxidase Super Complex of Yeast Mitochondria. Methods in Enzymology, 456,183-190. https://doi.org/10.1016/S0076-6879(08)04410-8CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019C H A P T E R T E NMethodsISSN 0* Instit{DepaUnivPurification of the Cytochrome cReductase/Cytochrome c OxidaseSuper Complex of Yeast MitochondriaHans-Peter Braun,* Stephanie Sunderhaus,* Egbert J. Boekema,†and Roman Kourˇil†Contents1. Inin076utertmeersittroductionEnzymology, Volume 456 # 2009-6879, DOI: 10.1016/S0076-6879(08)04410-8 All rigfor Plant Genetics, Faculty of Natural Sciences, Universita¨t Hannover, Hannover, Gent of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Iy of Groningen, Groningen, The NetherlandsElsehtsrmansti1842. Isolation of the III þ IV Supercomplex 1842.1. Cultivation of yeast cells in lactate medium 1842.2. Isolation of mitochondria 1852.3. Solubilization of the mitochondrial membranes 1852.4. Separation of mitochondrial protein complexes by sucrosegradient ultracentrifugation 1852.5. Blue-native PAGE 1863. Characterization of the III þ IV Supercomplex by EM 1874. Perspectives 187Acknowledgments 189References 189AbstractThe protein complexes of the respiratory chain interact by forming large proteinparticles called respiratory supercomplexes or ‘‘respirasomes’’. Biochemicalcharacterization of these particles proved to be difficult because of their insta-bility. Here we describe a strategy to isolate and characterize the cytochrome creductase/cytochrome c oxidase supercomplex of yeast, also termed the III þIV supercomplex, which is based on lactate cultivation of yeast, gentle isolationof mitochondria, membrane solubilization by digitonin, sucrose gradient ultra-centrifugation, and native gel electrophoresis. The procedure yields pure formsof two varieties of the III þ IV supercomplex composed of dimeric complex IIIand one or two copies of monomeric complex IV. Supercomplex preparationscan be used for physiological or structural investigations.vier Inc.reserved.nytute,183184 Hans-Peter Braun et al.1. IntroductionThe cytochrome c reductase and cytochrome c oxidase complexes canbe separately purified from mitochondrial fractions of many organisms. Thisled to the view that the two complexes should be considered as independentstructures (reviewed in Hackenbrock et al., 1986). However, early investi-gations already indicated the occurrence of ordered supramolecular struc-tures that consist of defined interactions of respiratory proteins complexes(Hatefi et al., 1962). These findings were considerably supported by recentinvestigations on the basis of gentle biochemical preparations, native gelelectrophoresis procedures, and electron microscopy (EM) (reviewed inBoekema and Braun, 2007). As a result, several so-called respiratory super-complexes could be defined. One of the best-described respiratorysupercomplexes is the cytochrome c reductase/cytochrome c oxidase super-complex, which also is termed the III þ IV supercomplex. It initially wasdescribed for the bacteria Paracoccus denitrificans, PS3, and Sulfolobus sp.(Berry and Trumpower, 1985; Iwasaki et al., 1985; Sone et al., 1987). Inyeast mitochondria, its existence first was described physiologically byinhibitor titrations (Boumans et al., 1998) and later by biochemical proce-dures (Cruciat et al., 2000; Scha¨gger and Pfeiffer, 2000). Its stability wasfound to depend on the cardiolipin concentration of the inner mitochon-drial membrane (Pfeiffer et al., 2003; Zhang et al., 2002; 2005). Recently, apseudo-atomic structure was presented for the III þ IV supercomplex ofyeast by comparison of medium-resolution 2-D maps from EM and thecrystal structures of the two individual respiratory complexes, which areavailable for higher eukaryotes (Heinemeyer et al., 2007; Nelson and Cox,2008). According to the proposed structure, the cytochrome c bindingpockets of cytochrome c reductase and cytochrome c oxidase are in closeproximity, allowing cytochrome c to efficiently transfer electrons from onecomplex to the other by a simple ping-pong mechanism. Here we describeprocedures to gently purify the III þ IV supercomplex of yeast for itsphysiological and structural characterization.2. Isolation of the III þ IV Supercomplex2.1. Cultivation of yeast cells in lactate mediumBiosynthesis and assembly of respiratory complexes in yeast depends ongrowth conditions during cultivation (Heinemeyer et al., 2007; Scha¨ggerand Pfeiffer, 2000). Lactate media lead to induced biosynthesis of complexIV and also induce formation of the III þ IV supercomplex. For preparingPurification of the III þ IV Super Complex of Yeast 185the III þ IV supercomplex, yeast cells are cultivated in 2.5 L lactatemedium (five 2-L Erlenmeyer vessels with 500 ml medium each composedof 0.3 % [w/v] yeast extract, 5 mM glucose, 7 mM KH2PO4, 20 mMNH4Cl, 4 mM CaCl2, 9 mM NaCl, 3 mM MgCl, 2.2 % [v/v] lactate,0.2MNaOH, pH 5.5) for 24 h at 30 and 120 rpm. Cells are harvested at anoptical density (OD) of 1.2 to 1.3 and directly used for the preparation ofmitochondria.2.2. Isolation of mitochondriaYeast mitochondria are prepared according to standard procedures. Pre-parations should be carried out as fast and gentle as possible. A very suitableprotocol is outlined in Meisinger et al. (2006). For this procedure, the yeastcell wall is digested by zymolyase, cells are disrupted mechanically, andfinally organelles are purified by differential centrifugations and a sucrosegradient ultracentrifugation. Purified mitochondria are resuspended in SEMbuffer (250 mM sucrose, 1 mM EDTA, 10 mMMOPS-KOH, pH 7.2) at aprotein concentration of 20 mg/ml and can be stored at 80. For repro-ducibility of experiments, it is recommended to store stock solutions ofmitochondria at one specific concentration.2.3. Solubilization of the mitochondrial membranesThe solubilization step is most crucial for supercomplex characterization.Digitonin (1-8 g detergent/g protein) nicely allows stabilization of the IIIþIV supercomplex from yeast (Cruciat et al., 2000; Heinemeyer et al., 2007;Scha¨gger and Pfeiffer, 2000). Routinely, 1 ml resuspended organelles(corresponding to 20 mg mitochondrial protein) are sedimented by centri-fugation for 10 min at 14,000g. The pellet is directly resolved in 1 mlsolubilization buffer (5% [w/v] digitonin, 30 mM HEPES, 150 mM potas-sium acetate, pH 7.4) and incubated for 20 min on ice. Insoluble material isremoved by centrifugation for 10 min at 18,000g.2.4. Separation of mitochondrial protein complexes bysucrose gradient ultracentrifugationThe solubilized mitochondrial protein complexes are directly transferredonto a linear sucrose gradient (volume: 12 ml; 0.3 to 1.5 M sucrose in 15mM TRIS base, pH 7.0, 20 mM KCl, 0.2% digitonin) and centrifuged for20 h at 150,000g and 4. Afterwards, the gradient is fractionated intotwenty-six 0.5-ml fractions from bottom to top, which most easily isachieved by use of a peristaltic pump linked to a needle, which gently istransferred into the tube.186 Hans-Peter Braun et al.2.5. Blue-native PAGESmall aliquots of the sucrose gradient fractions (50 ml) can be used foranalysis of the protein complex composition of the fractions by blue-nativePAGE (Wittig et al., 2006). Because 5% digitonin specifically destabilizesthe dimeric ATP synthase supercomplex from yeast, the two largest bandsvisible on the gels represent III þ IV supercomplexes (Fig. 10.1). Asrevealed by densitometric measurements, the largest band represents asupercomplex composed of dimeric complex III and two copies of mono-meric complex IV, the second largest band a supercomplex of dimericcomplex III and one copy of complex IV (Heinemeyer et al., 2007;Scha¨gger and Pfeiffer, 2000). Overall purity of the two supercomplexes is>90% in fraction 4 and >80% in fraction 5 of the gradient (Fig. 10.1)as estimated by silver staining of blue-native gels (not shown). Thesefractions can be directly taken for physiological measurements or structural1 2III2+ IV2III2+ IV1III2V3 4 5 6 7Sucrose gradient ultracentrifucation8 9 10 111D blue native PAGE12 13Figure 10.1 Purification of III þ IV supercomplexes from yeast mitochondria. Mito-chondrial membranes are solubilized bydigitonin, and protein complexes are separatedby sucrose gradient ultracentrifugation (horizontal separation dimension). Aliquots of13 fractions of the gradient are subsequently separated by1-D blue-native PAGE (verti-cal separation dimension). Identities of the four largest protein complexes are given tothe left of the gel: III2þ IV1 and III2þ IV2, supercomplexes composed of dimeric com-plex III and one or two copies of monomeric complex IV;V, ATP synthase complex; III2,dimeric complex III.Purification of the III þ IV Super Complex of Yeast 187analysis with EM (see below). Activity of the purified supercomplexes alsocan be directly monitored by in-gel activity assays for complex IV andcomplex III (Wittig et al., 2007; Zerbetto et al., 1997). For an even higherpurification of the yeast IIIþ IV supercomplexes, which might be necessaryfor cryo-EM or x-ray crystallography, the bands representing these super-complexes can be directly electroeluted from a 1-D Blue-native gel accord-ing to standard procedures (Wittig et al., 2006).3. Characterization of the III þ IVSupercomplex by EMPurified supercomplex fractions can be used for structural analysis withnegative stain single particle EM (Heinemeyer et al., 2007). EM can recordthe signal of single molecules, and EM images can typically contain theprojection maps of dozens to hundreds of proteins or other macromole-cules. These images, however, are very noisy, and to retrieve the signal ofmolecules thousands of single particle projections need to be summed.Because molecules may have freedom to attach to the support film of theEM specimens, extensive image processing is necessary. Single particleimage analysis is a well-developed method to analyze EM projections. Inshort, projections are compared by statistical methods and subsequentlysorted into homogeneous groups (‘‘classes’’), which represent specific angu-lar projections of studied proteins. Before they can be summed into final2-D maps, the projections also need to be aligned, which means thatrotational and translational shifts are calculated and imposed to bring themin optimal equivalent positions. The 2-D maps disclose many features of thestudied macromolecules. In favorable cases, where the sample is homoge-neous and randomly oriented molecules can be recorded, projections canbecome merged into 3-D structures. Recent reviews give an overview ofthe methods of single particle EM (Frank, 2002; van Heel et al., 2000).On single particle analysis, the obtained 2-D maps of the yeast III2IV2supercomplex show that it has a symmetric shape with dimeric complex IIIin the center and two laterally attached complex IV monomers at oppositepositions (Fig. 10.2).4. PerspectivesWith lactate medium for yeast cultivation, a gentle method for thepreparation of yeast mitochondria, membrane solubilization by digitoninand sucrose gradient ultracentrifugation, supercomplexes composed ofcytochrome c reductase and cytochrome c oxidase can be efficientlyFigure 10.2 Structure of the III2IV2 supercomplex from yeast as obtained by singleparticle electron microscopy. Upper part, 2-D map of 832 aligned projections in aposition vertical to the membrane plane. Lower part, pseudo-atomic structure of theIII2þ IV2 supercomplex derived by fitting the crystal structures for the III2 complex (inred) of yeast (Lange and Hunte, 2002) and complex IV (in green) of beef(Tsukihara et al.,1996) into the projectionmap.Modified fromHeinemeyer et al. (2007).188 Hans-Peter Braun et al.purified. Blue-native PAGE is a helpful tool to monitor the purificationsteps. Furthermore, blue-native PAGE can be used as the starting point foranother purification step, which is based on electroelution and which leadsto highly pure supercomplexes suitable for x-ray crystallography. However,despite considerable success in supercomplex purification, procedures stillhave to be further developed to characterize even larger protein structures,which are assumed to occur under in vivo conditions. Laboratory strains ofSaccharomyces cerevisiae lack the NADH dehydrogenase complex (complex I),which in other organisms additionally is associated to complexes III and IV.Further protein complexes of the inner mitochondrial membrane possiblyassociated with IIIþ IV supercomplexes are the ADP/ATP translocase or thepreprotein translocase of the inner mitochondrial membrane, the so-calledTIM complex (Dienhart and Stuart, 2008; Saddar et al., 2008). Developmentof novel procedures might allow unraveling the megacomplex organization ofthe inner mitochondrial membrane.Purification of the III þ IV Super Complex of Yeast 189ACKNOWLEDGMENTSResearch in our laboratories was supported by grants of the Deutsche Forschungsge-meinschaft (Br 1829-7/3 and Br 1829-8/1) and the Dutch Science Foundation NWO-CW.REFERENCESBerry, E. A., and Trumpower, B. L. (1985). Isolation of ubiquinol oxidase from Paracoccusdenitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes. J. Biol.Chem. 262, 2458–2467.Boekema, E. J., and Braun, H. P. (2007). Supramolecular structure of the mitochondrialoxidative phosphorylation system. J. Biol. Chem. 282, 1–4.Boumans, H., Grivell, L. A., and Berden, J. A. (1998). The respiratory chain in yeast behavesas a single functional unit. J. Biol. Chem. 273, 4872–4877.Cruciat, C. M., Brunner, S., Baumann, F., Neupert, W., and Stuart, R. A. (2000). Thecytochrome bc1 and cytochrome c oxidase complexes associate to form a single supra-complex in yeast mitochondria. J. Biol. Chem. 275, 18093–18098.Dienhart, M. K., and Stuart, R. A. (2008). The yeast Aac2 protein exists in physicalassociation with the cytochrome bc1-COX supercomplex and the TIM23 machinery.Mol. Biol. Cell 19, 3934–3943.Frank, J. (2002). Single-particle imaging of macromolecules by cryo-electron microscopy.Annu. Rev. Biophys. Biomol. Struct. 31, 303–319.Hatefi, Y., Haavik, A. G., Fowler, L. R., and Griffiths, D. E. (1962). Studies on the electrontransfer system. Reconstitution of the electron transfer system. J. Biol. Chem. 237,2661–2669.Hackenbrock, C. R., Chazotte, B., and Gupta, S. S. (1986). The random collision modeland a critical assessment of the diffusion and collision in mitochondrial electron transport.J. Bioenerg. Biomembr. 18, 331–368.Heinemeyer, J., Braun, H. P., Boekema, E. J., and Kouril, R. (2007). A structural model ofthe cytochrome c reductase/oxidase supercomplex from yeast mitochondria. J. Biol.Chem. 282, 12240–12248.Iwasaki, T., Matsuura, K., and Oshima, T. (1995). Resolution of the aerobic respiratorysystem of the thermoacidophilic archaeon, Sulfolobus sp. Strain 7. I. The archael terminaloxidase supercomplex is a functional fusion of respiratory complexes III and IV with noc-type cytochromes. J. Biol. Chem. 270, 30881–30892.Lange, C., and Hunte, C. (2002). Crystal structure of the yeast cytochrome bc1 complex withits bound substrate cytochrome c. Proc Natl. Acad. Sci. USA 99, 2800–2805.Meisinger, C., Pfanner, N., and Truscott, K. N. (2006). Isolation of yeast mitochondria.Methods Mol. Biol. 313, 33–39.Nelson, D. L., and Cox, M. M. (2008). In ‘‘Lehninger—Principles of Biochemistry’’ 5th Ed.,pp. 718–719. Freeman, New York.Pfeiffer, K., Gohil, V., Stuart, R. A., Hunte, C., Brandt, U., Greenberg, M. L., andScha¨gger, H. (2003). Cardiolipin stabilizes respiratory chain supercomplexes. J. Biol.Chem. 278, 52873–52880.Saddar, S., Dienhart, M. K., and Stuart, R. A. (2008). The F1F0-ATP synthase complexinfluences the assembly state of the cytochrome bc1-cytochrome oxidase supercomplexand its association with the TIM23 machinery. J. Biol. Chem. 283, 6677–6686.Scha¨gger, H., and Pfeiffer, K. (2000). Supercomplexes in the respiratory chains of yeast andmammalian mitochondria. EMBO J. 19, 1777–1783.190 Hans-Peter Braun et al.Sone, N., Sekimachi, M., and Kutoh, E. (1987). Identification and properties of a quinoloxidase supercomplex composed of a bc1 complex and cytochrome oxidase in thethermophilic bacterium PS3. J. Biol. Chem. 262, 15386–15391.Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., and Yoshikawa, S. (1996). The whole structureof the 13-subunit oxidized cytochrome c oxidase at 2.8 A˚. Science 272, 1136–1144.van Heel, M., Gowen, B., Matadeen, R., Orlova, E. V., Finn, R., Pape, T., Cohen, D.,Stark, H., Schmidt, R., Schatz, M., and Patwardhan, A. (2000). Single-particle electroncryo-microscopy: Towards atomic resolution. Q. Rev. Biophys. 33, 307–369.Wittig, I., Braun, H. P., and Scha¨gger, H. (2006). Blue-Native PAGE.NATURE Protocols 1,418–428.Wittig, I., Karas, M., and Scha¨gger, H. (2007). High resolution clear native electrophoresisfor in-gel functional assays and fluorescence studies of membrane protein complexes.Mol. Cell. Proteomics 6, 1215–1225.Zerbetto, E., Vergani, L., and Dabbeni-Sala, F. (1997). Quantitation of muscle mitochon-drial oxidative phosphorylation enzymes via histochemical staining of blue native poly-acrylamide gels. Electrophoresis 18, 2059–2064.Zhang, M., Mileykovskaya, E., and Dowhan, W. (2002). Gluing the respiratory chaintogether. J. Biol. Chem. 277, 43553–43556.Zhang, M., Mileykovskaya, E., and Dowhan, W. (2005). Cardiolipin is essential for organi-zation of complexes III and IV into a supercomplex in intact yeast mitochondria. J. Biol.Chem. 280, 29403–29408.

Purification of the Cytochrome c Reductase/Cytochrome c Oxidase Super Complex of Yeast Mitochondria

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University of Groningen Research Database

  
 University of Groningen
Purification of the Cytochrome c Reductase/Cytochrome c Oxidase Super Complex of Yeast
Mitochondria
Braun, Hans-Peter; Sunderhaus, Stephanie; Boekema, Egbert J.; Kouřil, Roman
Published in:
Methods in Enzymology
DOI:
10.1016/S0076-6879(08)04410-8
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2009
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Braun, H-P., Sunderhaus, S., Boekema, E. J., & Kouřil, R. (2009). Purification of the Cytochrome c
Reductase/Cytochrome c Oxidase Super Complex of Yeast Mitochondria. Methods in Enzymology, 456,
183-190. DOI: 10.1016/S0076-6879(08)04410-8
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
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Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
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Download date: 11-02-2018
C H A P T E R T E N
Purification of the Cytochrome c
Reductase/Cytochrome c Oxidase
Super Complex of Yeast Mitochondria
Hans-Peter Braun,* Stephanie Sunderhaus,* Egbert J. Boekema,†
and Roman Kourˇil†
Contents
1. Introduction 184
2. Isolation of the III þ IV Supercomplex 184
2.1. Cultivation of yeast cells in lactate medium 184
2.2. Isolation of mitochondria 185
2.3. Solubilization of the mitochondrial membranes 185
2.4. Separation of mitochondrial protein complexes by sucrose
gradient ultracentrifugation 185
2.5. Blue-native PAGE 186
3. Characterization of the III þ IV Supercomplex by EM 187
4. Perspectives 187
Acknowledgments 189
References 189
Abstract
The protein complexes of the respiratory chain interact by forming large protein
particles called respiratory supercomplexes or ‘‘respirasomes’’. Biochemical
characterization of these particles proved to be difficult because of their insta-
bility. Here we describe a strategy to isolate and characterize the cytochrome c
reductase/cytochrome c oxidase supercomplex of yeast, also termed the III þ
IV supercomplex, which is based on lactate cultivation of yeast, gentle isolation
of mitochondria, membrane solubilization by digitonin, sucrose gradient ultra-
centrifugation, and native gel electrophoresis. The procedure yields pure forms
of two varieties of the III þ IV supercomplex composed of dimeric complex III
and one or two copies of monomeric complex IV. Supercomplex preparations
can be used for physiological or structural investigations.
Methods in Enzymology, Volume 456 # 2009 Elsevier Inc.
ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)04410-8 All rights reserved.
* Institute for Plant Genetics, Faculty of Natural Sciences, Universita¨t Hannover, Hannover, Germany
{
Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute,
University of Groningen, Groningen, The Netherlands
183
1. Introduction
The cytochrome c reductase and cytochrome c oxidase complexes can
be separately purified from mitochondrial fractions of many organisms. This
led to the view that the two complexes should be considered as independent
structures (reviewed in Hackenbrock et al., 1986). However, early investi-
gations already indicated the occurrence of ordered supramolecular struc-
tures that consist of defined interactions of respiratory proteins complexes
(Hatefi et al., 1962). These findings were considerably supported by recent
investigations on the basis of gentle biochemical preparations, native gel
electrophoresis procedures, and electron microscopy (EM) (reviewed in
Boekema and Braun, 2007). As a result, several so-called respiratory super-
complexes could be defined. One of the best-described respiratory
supercomplexes is the cytochrome c reductase/cytochrome c oxidase super-
complex, which also is termed the III þ IV supercomplex. It initially was
described for the bacteria Paracoccus denitrificans, PS3, and Sulfolobus sp.
(Berry and Trumpower, 1985; Iwasaki et al., 1985; Sone et al., 1987). In
yeast mitochondria, its existence first was described physiologically by
inhibitor titrations (Boumans et al., 1998) and later by biochemical proce-
dures (Cruciat et al., 2000; Scha¨gger and Pfeiffer, 2000). Its stability was
found to depend on the cardiolipin concentration of the inner mitochon-
drial membrane (Pfeiffer et al., 2003; Zhang et al., 2002; 2005). Recently, a
pseudo-atomic structure was presented for the III þ IV supercomplex of
yeast by comparison of medium-resolution 2-D maps from EM and the
crystal structures of the two individual respiratory complexes, which are
available for higher eukaryotes (Heinemeyer et al., 2007; Nelson and Cox,
2008). According to the proposed structure, the cytochrome c binding
pockets of cytochrome c reductase and cytochrome c oxidase are in close
proximity, allowing cytochrome c to efficiently transfer electrons from one
complex to the other by a simple ping-pong mechanism. Here we describe
procedures to gently purify the III þ IV supercomplex of yeast for its
physiological and structural characterization.
2. Isolation of the III þ IV Supercomplex
2.1. Cultivation of yeast cells in lactate medium
Biosynthesis and assembly of respiratory complexes in yeast depends on
growth conditions during cultivation (Heinemeyer et al., 2007; Scha¨gger
and Pfeiffer, 2000). Lactate media lead to induced biosynthesis of complex
IV and also induce formation of the III þ IV supercomplex. For preparing
184 Hans-Peter Braun et al.
the III þ IV supercomplex, yeast cells are cultivated in 2.5 L lactate
medium (five 2-L Erlenmeyer vessels with 500 ml medium each composed
of 0.3 % [w/v] yeast extract, 5 mM glucose, 7 mM KH2PO4, 20 mM
NH4Cl, 4 mM CaCl2, 9 mM NaCl, 3 mM MgCl, 2.2 % [v/v] lactate,
0.2MNaOH, pH 5.5) for 24 h at 30 and 120 rpm. Cells are harvested at an
optical density (OD) of 1.2 to 1.3 and directly used for the preparation of
mitochondria.
2.2. Isolation of mitochondria
Yeast mitochondria are prepared according to standard procedures. Pre-
parations should be carried out as fast and gentle as possible. A very suitable
protocol is outlined in Meisinger et al. (2006). For this procedure, the yeast
cell wall is digested by zymolyase, cells are disrupted mechanically, and
finally organelles are purified by differential centrifugations and a sucrose
gradient ultracentrifugation. Purified mitochondria are resuspended in SEM
buffer (250 mM sucrose, 1 mM EDTA, 10 mMMOPS-KOH, pH 7.2) at a
protein concentration of 20 mg/ml and can be stored at 80. For repro-
ducibility of experiments, it is recommended to store stock solutions of
mitochondria at one specific concentration.
2.3. Solubilization of the mitochondrial membranes
The solubilization step is most crucial for supercomplex characterization.
Digitonin (1-8 g detergent/g protein) nicely allows stabilization of the IIIþ
IV supercomplex from yeast (Cruciat et al., 2000; Heinemeyer et al., 2007;
Scha¨gger and Pfeiffer, 2000). Routinely, 1 ml resuspended organelles
(corresponding to 20 mg mitochondrial protein) are sedimented by centri-
fugation for 10 min at 14,000g. The pellet is directly resolved in 1 ml
solubilization buffer (5% [w/v] digitonin, 30 mM HEPES, 150 mM potas-
sium acetate, pH 7.4) and incubated for 20 min on ice. Insoluble material is
removed by centrifugation for 10 min at 18,000g.
2.4. Separation of mitochondrial protein complexes by
sucrose gradient ultracentrifugation
The solubilized mitochondrial protein complexes are directly transferred
onto a linear sucrose gradient (volume: 12 ml; 0.3 to 1.5 M sucrose in 15
mM TRIS base, pH 7.0, 20 mM KCl, 0.2% digitonin) and centrifuged for
20 h at 150,000g and 4. Afterwards, the gradient is fractionated into
twenty-six 0.5-ml fractions from bottom to top, which most easily is
achieved by use of a peristaltic pump linked to a needle, which gently is
transferred into the tube.
Purification of the III þ IV Super Complex of Yeast 185
2.5. Blue-native PAGE
Small aliquots of the sucrose gradient fractions (50 ml) can be used for
analysis of the protein complex composition of the fractions by blue-native
PAGE (Wittig et al., 2006). Because 5% digitonin specifically destabilizes
the dimeric ATP synthase supercomplex from yeast, the two largest bands
visible on the gels represent III þ IV supercomplexes (Fig. 10.1). As
revealed by densitometric measurements, the largest band represents a
supercomplex composed of dimeric complex III and two copies of mono-
meric complex IV, the second largest band a supercomplex of dimeric
complex III and one copy of complex IV (Heinemeyer et al., 2007;
Scha¨gger and Pfeiffer, 2000). Overall purity of the two supercomplexes is
>90% in fraction 4 and >80% in fraction 5 of the gradient (Fig. 10.1)
as estimated by silver staining of blue-native gels (not shown). These
fractions can be directly taken for physiological measurements or structural
1 2
III2+ IV2
III2+ IV1
III2
V
3 4 5 6 7
Sucrose gradient ultracentrifucation
8 9 10 11
1D
 b
lu
e 
na
ti
ve
 P
A
G
E
12 13
Figure 10.1 Purification of III þ IV supercomplexes from yeast mitochondria. Mito-
chondrial membranes are solubilized bydigitonin, and protein complexes are separated
by sucrose gradient ultracentrifugation (horizontal separation dimension). Aliquots of
13 fractions of the gradient are subsequently separated by1-D blue-native PAGE (verti-
cal separation dimension). Identities of the four largest protein complexes are given to
the left of the gel: III2þ IV1 and III2þ IV2, supercomplexes composed of dimeric com-
plex III and one or two copies of monomeric complex IV;V, ATP synthase complex; III2,
dimeric complex III.
186 Hans-Peter Braun et al.
analysis with EM (see below). Activity of the purified supercomplexes also
can be directly monitored by in-gel activity assays for complex IV and
complex III (Wittig et al., 2007; Zerbetto et al., 1997). For an even higher
purification of the yeast IIIþ IV supercomplexes, which might be necessary
for cryo-EM or x-ray crystallography, the bands representing these super-
complexes can be directly electroeluted from a 1-D Blue-native gel accord-
ing to standard procedures (Wittig et al., 2006).
3. Characterization of the III þ IV
Supercomplex by EM
Purified supercomplex fractions can be used for structural analysis with
negative stain single particle EM (Heinemeyer et al., 2007). EM can record
the signal of single molecules, and EM images can typically contain the
projection maps of dozens to hundreds of proteins or other macromole-
cules. These images, however, are very noisy, and to retrieve the signal of
molecules thousands of single particle projections need to be summed.
Because molecules may have freedom to attach to the support film of the
EM specimens, extensive image processing is necessary. Single particle
image analysis is a well-developed method to analyze EM projections. In
short, projections are compared by statistical methods and subsequently
sorted into homogeneous groups (‘‘classes’’), which represent specific angu-
lar projections of studied proteins. Before they can be summed into final
2-D maps, the projections also need to be aligned, which means that
rotational and translational shifts are calculated and imposed to bring them
in optimal equivalent positions. The 2-D maps disclose many features of the
studied macromolecules. In favorable cases, where the sample is homoge-
neous and randomly oriented molecules can be recorded, projections can
become merged into 3-D structures. Recent reviews give an overview of
the methods of single particle EM (Frank, 2002; van Heel et al., 2000).
On single particle analysis, the obtained 2-D maps of the yeast III2IV2
supercomplex show that it has a symmetric shape with dimeric complex III
in the center and two laterally attached complex IV monomers at opposite
positions (Fig. 10.2).
4. Perspectives
With lactate medium for yeast cultivation, a gentle method for the
preparation of yeast mitochondria, membrane solubilization by digitonin
and sucrose gradient ultracentrifugation, supercomplexes composed of
cytochrome c reductase and cytochrome c oxidase can be efficiently
Purification of the III þ IV Super Complex of Yeast 187
purified. Blue-native PAGE is a helpful tool to monitor the purification
steps. Furthermore, blue-native PAGE can be used as the starting point for
another purification step, which is based on electroelution and which leads
to highly pure supercomplexes suitable for x-ray crystallography. However,
despite considerable success in supercomplex purification, procedures still
have to be further developed to characterize even larger protein structures,
which are assumed to occur under in vivo conditions. Laboratory strains of
Saccharomyces cerevisiae lack the NADH dehydrogenase complex (complex I),
which in other organisms additionally is associated to complexes III and IV.
Further protein complexes of the inner mitochondrial membrane possibly
associated with IIIþ IV supercomplexes are the ADP/ATP translocase or the
preprotein translocase of the inner mitochondrial membrane, the so-called
TIM complex (Dienhart and Stuart, 2008; Saddar et al., 2008). Development
of novel procedures might allow unraveling the megacomplex organization of
the inner mitochondrial membrane.
Figure 10.2 Structure of the III2IV2 supercomplex from yeast as obtained by single
particle electron microscopy. Upper part, 2-D map of 832 aligned projections in a
position vertical to the membrane plane. Lower part, pseudo-atomic structure of the
III2þ IV2 supercomplex derived by fitting the crystal structures for the III2 complex (in
red) of yeast (Lange and Hunte, 2002) and complex IV (in green) of beef
(Tsukihara et al.,1996) into the projectionmap.Modified fromHeinemeyer et al. (2007).
188 Hans-Peter Braun et al.
ACKNOWLEDGMENTS
Research in our laboratories was supported by grants of the Deutsche Forschungsge-
meinschaft (Br 1829-7/3 and Br 1829-8/1) and the Dutch Science Foundation NWO-CW.
REFERENCES
Berry, E. A., and Trumpower, B. L. (1985). Isolation of ubiquinol oxidase from Paracoccus
denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes. J. Biol.
Chem. 262, 2458–2467.
Boekema, E. J., and Braun, H. P. (2007). Supramolecular structure of the mitochondrial
oxidative phosphorylation system. J. Biol. Chem. 282, 1–4.
Boumans, H., Grivell, L. A., and Berden, J. A. (1998). The respiratory chain in yeast behaves
as a single functional unit. J. Biol. Chem. 273, 4872–4877.
Cruciat, C. M., Brunner, S., Baumann, F., Neupert, W., and Stuart, R. A. (2000). The
cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supra-
complex in yeast mitochondria. J. Biol. Chem. 275, 18093–18098.
Dienhart, M. K., and Stuart, R. A. (2008). The yeast Aac2 protein exists in physical
association with the cytochrome bc1-COX supercomplex and the TIM23 machinery.
Mol. Biol. Cell 19, 3934–3943.
Frank, J. (2002). Single-particle imaging of macromolecules by cryo-electron microscopy.
Annu. Rev. Biophys. Biomol. Struct. 31, 303–319.
Hatefi, Y., Haavik, A. G., Fowler, L. R., and Griffiths, D. E. (1962). Studies on the electron
transfer system. Reconstitution of the electron transfer system. J. Biol. Chem. 237,
2661–2669.
Hackenbrock, C. R., Chazotte, B., and Gupta, S. S. (1986). The random collision model
and a critical assessment of the diffusion and collision in mitochondrial electron transport.
J. Bioenerg. Biomembr. 18, 331–368.
Heinemeyer, J., Braun, H. P., Boekema, E. J., and Kouril, R. (2007). A structural model of
the cytochrome c reductase/oxidase supercomplex from yeast mitochondria. J. Biol.
Chem. 282, 12240–12248.
Iwasaki, T., Matsuura, K., and Oshima, T. (1995). Resolution of the aerobic respiratory
system of the thermoacidophilic archaeon, Sulfolobus sp. Strain 7. I. The archael terminal
oxidase supercomplex is a functional fusion of respiratory complexes III and IV with no
c-type cytochromes. J. Biol. Chem. 270, 30881–30892.
Lange, C., and Hunte, C. (2002). Crystal structure of the yeast cytochrome bc1 complex with
its bound substrate cytochrome c. Proc Natl. Acad. Sci. USA 99, 2800–2805.
Meisinger, C., Pfanner, N., and Truscott, K. N. (2006). Isolation of yeast mitochondria.
Methods Mol. Biol. 313, 33–39.
Nelson, D. L., and Cox, M. M. (2008). In ‘‘Lehninger—Principles of Biochemistry’’ 5th Ed.,
pp. 718–719. Freeman, New York.
Pfeiffer, K., Gohil, V., Stuart, R. A., Hunte, C., Brandt, U., Greenberg, M. L., and
Scha¨gger, H. (2003). Cardiolipin stabilizes respiratory chain supercomplexes. J. Biol.
Chem. 278, 52873–52880.
Saddar, S., Dienhart, M. K., and Stuart, R. A. (2008). The F1F0-ATP synthase complex
influences the assembly state of the cytochrome bc1-cytochrome oxidase supercomplex
and its association with the TIM23 machinery. J. Biol. Chem. 283, 6677–6686.
Scha¨gger, H., and Pfeiffer, K. (2000). Supercomplexes in the respiratory chains of yeast and
mammalian mitochondria. EMBO J. 19, 1777–1783.
Purification of the III þ IV Super Complex of Yeast 189
Sone, N., Sekimachi, M., and Kutoh, E. (1987). Identification and properties of a quinol
oxidase supercomplex composed of a bc1 complex and cytochrome oxidase in the
thermophilic bacterium PS3. J. Biol. Chem. 262, 15386–15391.
Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-
Itoh, K., Nakashima, R., Yaono, R., and Yoshikawa, S. (1996). The whole structure
of the 13-subunit oxidized cytochrome c oxidase at 2.8 A˚. Science 272, 1136–1144.
van Heel, M., Gowen, B., Matadeen, R., Orlova, E. V., Finn, R., Pape, T., Cohen, D.,
Stark, H., Schmidt, R., Schatz, M., and Patwardhan, A. (2000). Single-particle electron
cryo-microscopy: Towards atomic resolution. Q. Rev. Biophys. 33, 307–369.
Wittig, I., Braun, H. P., and Scha¨gger, H. (2006). Blue-Native PAGE.NATURE Protocols 1,
418–428.
Wittig, I., Karas, M., and Scha¨gger, H. (2007). High resolution clear native electrophoresis
for in-gel functional assays and fluorescence studies of membrane protein complexes.
Mol. Cell. Proteomics 6, 1215–1225.
Zerbetto, E., Vergani, L., and Dabbeni-Sala, F. (1997). Quantitation of muscle mitochon-
drial oxidative phosphorylation enzymes via histochemical staining of blue native poly-
acrylamide gels. Electrophoresis 18, 2059–2064.
Zhang, M., Mileykovskaya, E., and Dowhan, W. (2002). Gluing the respiratory chain
together. J. Biol. Chem. 277, 43553–43556.
Zhang, M., Mileykovskaya, E., and Dowhan, W. (2005). Cardiolipin is essential for organi-
zation of complexes III and IV into a supercomplex in intact yeast mitochondria. J. Biol.
Chem. 280, 29403–29408.
190 Hans-Peter Braun et al.


The protein complexes of the respiratory chain interact by forming large protein particles called respiratory supercomplexes or ‘‘respirasomes’’. Biochemical characterization of these particles proved to be difficult because of their instability. Here we describe a strategy to isolate and characterize the cytochrome c reductase/cytochrome c oxidase supercomplex of yeast, also termed the III + IV supercomplex, which is based on lactate cultivation of yeast, gentle isolation of mitochondria, membrane solubilization by digitonin, sucrose gradient ultracentrifugation, and native gel electrophoresis. The procedure yields pure forms of two varieties of the III + IV supercomplex composed of dimeric complex III and one or two copies of monomeric complex IV. Supercomplex preparations can be used for physiological or structural investigations

Dissertations of the University of Groningen

   University of GroningenPurification of the Cytochrome c Reductase/Cytochrome c Oxidase Super Complex of YeastMitochondriaBraun, Hans-Peter; Sunderhaus, Stephanie; Boekema, Egbert J.; Kouřil, RomanPublished in:Methods in EnzymologyDOI:10.1016/S0076-6879(08)04410-8IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2009Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Braun, H-P., Sunderhaus, S., Boekema, E. J., & Kouřil, R. (2009). Purification of the Cytochrome cReductase/Cytochrome c Oxidase Super Complex of Yeast Mitochondria. Methods in Enzymology, 456,183-190. https://doi.org/10.1016/S0076-6879(08)04410-8CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.C H A P T E R T E NMethodsISSN 0* Instit{DepaUnivPurification of the Cytochrome cReductase/Cytochrome c OxidaseSuper Complex of Yeast MitochondriaHans-Peter Braun,* Stephanie Sunderhaus,* Egbert J. Boekema,†and Roman Kouřil†Contents1. Inin076utertmeersittroductionEnzymology, Volume 456 # 2009-6879, DOI: 10.1016/S0076-6879(08)04410-8 All rigfor Plant Genetics, Faculty of Natural Sciences, Universität Hannover, Hannover, Gent of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Iy of Groningen, Groningen, The NetherlandsElsehtsrmansti1842. Isolation of the III þ IV Supercomplex 1842.1. Cultivation of yeast cells in lactate medium 1842.2. Isolation of mitochondria 1852.3. Solubilization of the mitochondrial membranes 1852.4. Separation of mitochondrial protein complexes by sucrosegradient ultracentrifugation 1852.5. Blue-native PAGE 1863. Characterization of the III þ IV Supercomplex by EM 1874. Perspectives 187Acknowledgments 189References 189AbstractThe protein complexes of the respiratory chain interact by forming large proteinparticles called respiratory supercomplexes or ‘‘respirasomes’’. Biochemicalcharacterization of these particles proved to be difficult because of their insta-bility. Here we describe a strategy to isolate and characterize the cytochrome creductase/cytochrome c oxidase supercomplex of yeast, also termed the III þIV supercomplex, which is based on lactate cultivation of yeast, gentle isolationof mitochondria, membrane solubilization by digitonin, sucrose gradient ultra-centrifugation, and native gel electrophoresis. The procedure yields pure formsof two varieties of the III þ IV supercomplex composed of dimeric complex IIIand one or two copies of monomeric complex IV. Supercomplex preparationscan be used for physiological or structural investigations.vier Inc.reserved.nytute,183184 Hans-Peter Braun et al.1. IntroductionThe cytochrome c reductase and cytochrome c oxidase complexes canbe separately purified from mitochondrial fractions of many organisms. Thisled to the view that the two complexes should be considered as independentstructures (reviewed in Hackenbrock et al., 1986). However, early investi-gations already indicated the occurrence of ordered supramolecular struc-tures that consist of defined interactions of respiratory proteins complexes(Hatefi et al., 1962). These findings were considerably supported by recentinvestigations on the basis of gentle biochemical preparations, native gelelectrophoresis procedures, and electron microscopy (EM) (reviewed inBoekema and Braun, 2007). As a result, several so-called respiratory super-complexes could be defined. One of the best-described respiratorysupercomplexes is the cytochrome c reductase/cytochrome c oxidase super-complex, which also is termed the III þ IV supercomplex. It initially wasdescribed for the bacteria Paracoccus denitrificans, PS3, and Sulfolobus sp.(Berry and Trumpower, 1985; Iwasaki et al., 1985; Sone et al., 1987). Inyeast mitochondria, its existence first was described physiologically byinhibitor titrations (Boumans et al., 1998) and later by biochemical proce-dures (Cruciat et al., 2000; Schägger and Pfeiffer, 2000). Its stability wasfound to depend on the cardiolipin concentration of the inner mitochon-drial membrane (Pfeiffer et al., 2003; Zhang et al., 2002; 2005). Recently, apseudo-atomic structure was presented for the III þ IV supercomplex ofyeast by comparison of medium-resolution 2-D maps from EM and thecrystal structures of the two individual respiratory complexes, which areavailable for higher eukaryotes (Heinemeyer et al., 2007; Nelson and Cox,2008). According to the proposed structure, the cytochrome c bindingpockets of cytochrome c reductase and cytochrome c oxidase are in closeproximity, allowing cytochrome c to efficiently transfer electrons from onecomplex to the other by a simple ping-pong mechanism. Here we describeprocedures to gently purify the III þ IV supercomplex of yeast for itsphysiological and structural characterization.2. Isolation of the III þ IV Supercomplex2.1. Cultivation of yeast cells in lactate mediumBiosynthesis and assembly of respiratory complexes in yeast depends ongrowth conditions during cultivation (Heinemeyer et al., 2007; Schäggerand Pfeiffer, 2000). Lactate media lead to induced biosynthesis of complexIV and also induce formation of the III þ IV supercomplex. For preparingPurification of the III þ IV Super Complex of Yeast 185the III þ IV supercomplex, yeast cells are cultivated in 2.5 L lactatemedium (five 2-L Erlenmeyer vessels with 500 ml medium each composedof 0.3 % [w/v] yeast extract, 5 mM glucose, 7 mM KH2PO4, 20 mMNH4Cl, 4 mM CaCl2, 9 mM NaCl, 3 mM MgCl, 2.2 % [v/v] lactate,0.2MNaOH, pH 5.5) for 24 h at 30 and 120 rpm. Cells are harvested at anoptical density (OD) of 1.2 to 1.3 and directly used for the preparation ofmitochondria.2.2. Isolation of mitochondriaYeast mitochondria are prepared according to standard procedures. Pre-parations should be carried out as fast and gentle as possible. A very suitableprotocol is outlined in Meisinger et al. (2006). For this procedure, the yeastcell wall is digested by zymolyase, cells are disrupted mechanically, andfinally organelles are purified by differential centrifugations and a sucrosegradient ultracentrifugation. Purified mitochondria are resuspended in SEMbuffer (250 mM sucrose, 1 mM EDTA, 10 mMMOPS-KOH, pH 7.2) at aprotein concentration of 20 mg/ml and can be stored at 80. For repro-ducibility of experiments, it is recommended to store stock solutions ofmitochondria at one specific concentration.2.3. Solubilization of the mitochondrial membranesThe solubilization step is most crucial for supercomplex characterization.Digitonin (1-8 g detergent/g protein) nicely allows stabilization of the IIIþIV supercomplex from yeast (Cruciat et al., 2000; Heinemeyer et al., 2007;Schägger and Pfeiffer, 2000). Routinely, 1 ml resuspended organelles(corresponding to 20 mg mitochondrial protein) are sedimented by centri-fugation for 10 min at 14,000g. The pellet is directly resolved in 1 mlsolubilization buffer (5% [w/v] digitonin, 30 mM HEPES, 150 mM potas-sium acetate, pH 7.4) and incubated for 20 min on ice. Insoluble material isremoved by centrifugation for 10 min at 18,000g.2.4. Separation of mitochondrial protein complexes bysucrose gradient ultracentrifugationThe solubilized mitochondrial protein complexes are directly transferredonto a linear sucrose gradient (volume: 12 ml; 0.3 to 1.5 M sucrose in 15mM TRIS base, pH 7.0, 20 mM KCl, 0.2% digitonin) and centrifuged for20 h at 150,000g and 4. Afterwards, the gradient is fractionated intotwenty-six 0.5-ml fractions from bottom to top, which most easily isachieved by use of a peristaltic pump linked to a needle, which gently istransferred into the tube.186 Hans-Peter Braun et al.2.5. Blue-native PAGESmall aliquots of the sucrose gradient fractions (50 ml) can be used foranalysis of the protein complex composition of the fractions by blue-nativePAGE (Wittig et al., 2006). Because 5% digitonin specifically destabilizesthe dimeric ATP synthase supercomplex from yeast, the two largest bandsvisible on the gels represent III þ IV supercomplexes (Fig. 10.1). Asrevealed by densitometric measurements, the largest band represents asupercomplex composed of dimeric complex III and two copies of mono-meric complex IV, the second largest band a supercomplex of dimericcomplex III and one copy of complex IV (Heinemeyer et al., 2007;Schägger and Pfeiffer, 2000). Overall purity of the two supercomplexes is>90% in fraction 4 and >80% in fraction 5 of the gradient (Fig. 10.1)as estimated by silver staining of blue-native gels (not shown). Thesefractions can be directly taken for physiological measurements or structural1 2III2+ IV2III2+ IV1III2V3 4 5 6 7Sucrose gradient ultracentrifucation8 9 10 111D blue native PAGE12 13Figure 10.1 Purification of III þ IV supercomplexes from yeast mitochondria. Mito-chondrial membranes are solubilized bydigitonin, and protein complexes are separatedby sucrose gradient ultracentrifugation (horizontal separation dimension). Aliquots of13 fractions of the gradient are subsequently separated by1-D blue-native PAGE (verti-cal separation dimension). Identities of the four largest protein complexes are given tothe left of the gel: III2 þ IV1 and III2 þ IV2, supercomplexes composed of dimeric com-plex III and one or two copies of monomeric complex IV;V, ATP synthase complex; III2,dimeric complex III.Purification of the III þ IV Super Complex of Yeast 187analysis with EM (see below). Activity of the purified supercomplexes alsocan be directly monitored by in-gel activity assays for complex IV andcomplex III (Wittig et al., 2007; Zerbetto et al., 1997). For an even higherpurification of the yeast IIIþ IV supercomplexes, which might be necessaryfor cryo-EM or x-ray crystallography, the bands representing these super-complexes can be directly electroeluted from a 1-D Blue-native gel accord-ing to standard procedures (Wittig et al., 2006).3. Characterization of the III þ IVSupercomplex by EMPurified supercomplex fractions can be used for structural analysis withnegative stain single particle EM (Heinemeyer et al., 2007). EM can recordthe signal of single molecules, and EM images can typically contain theprojection maps of dozens to hundreds of proteins or other macromole-cules. These images, however, are very noisy, and to retrieve the signal ofmolecules thousands of single particle projections need to be summed.Because molecules may have freedom to attach to the support film of theEM specimens, extensive image processing is necessary. Single particleimage analysis is a well-developed method to analyze EM projections. Inshort, projections are compared by statistical methods and subsequentlysorted into homogeneous groups (‘‘classes’’), which represent specific angu-lar projections of studied proteins. Before they can be summed into final2-D maps, the projections also need to be aligned, which means thatrotational and translational shifts are calculated and imposed to bring themin optimal equivalent positions. The 2-D maps disclose many features of thestudied macromolecules. In favorable cases, where the sample is homoge-neous and randomly oriented molecules can be recorded, projections canbecome merged into 3-D structures. Recent reviews give an overview ofthe methods of single particle EM (Frank, 2002; van Heel et al., 2000).On single particle analysis, the obtained 2-D maps of the yeast III2IV2supercomplex show that it has a symmetric shape with dimeric complex IIIin the center and two laterally attached complex IV monomers at oppositepositions (Fig. 10.2).4. PerspectivesWith lactate medium for yeast cultivation, a gentle method for thepreparation of yeast mitochondria, membrane solubilization by digitoninand sucrose gradient ultracentrifugation, supercomplexes composed ofcytochrome c reductase and cytochrome c oxidase can be efficientlyFigure 10.2 Structure of the III2IV2 supercomplex from yeast as obtained by singleparticle electron microscopy. Upper part, 2-D map of 832 aligned projections in aposition vertical to the membrane plane. Lower part, pseudo-atomic structure of theIII2þ IV2 supercomplex derived by fitting the crystal structures for the III2 complex (inred) of yeast (Lange and Hunte, 2002) and complex IV (in green) of beef(Tsukihara et al.,1996) into the projectionmap.Modified fromHeinemeyer et al. (2007).188 Hans-Peter Braun et al.purified. Blue-native PAGE is a helpful tool to monitor the purificationsteps. Furthermore, blue-native PAGE can be used as the starting point foranother purification step, which is based on electroelution and which leadsto highly pure supercomplexes suitable for x-ray crystallography. However,despite considerable success in supercomplex purification, procedures stillhave to be further developed to characterize even larger protein structures,which are assumed to occur under in vivo conditions. Laboratory strains ofSaccharomyces cerevisiae lack the NADH dehydrogenase complex (complex I),which in other organisms additionally is associated to complexes III and IV.Further protein complexes of the inner mitochondrial membrane possiblyassociated with IIIþ IV supercomplexes are the ADP/ATP translocase or thepreprotein translocase of the inner mitochondrial membrane, the so-calledTIM complex (Dienhart and Stuart, 2008; Saddar et al., 2008). Developmentof novel procedures might allow unraveling the megacomplex organization ofthe inner mitochondrial membrane.Purification of the III þ IV Super Complex of Yeast 189ACKNOWLEDGMENTSResearch in our laboratories was supported by grants of the Deutsche Forschungsge-meinschaft (Br 1829-7/3 and Br 1829-8/1) and the Dutch Science Foundation NWO-CW.REFERENCESBerry, E. A., and Trumpower, B. L. (1985). Isolation of ubiquinol oxidase from Paracoccusdenitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes. J. Biol.Chem. 262, 2458–2467.Boekema, E. J., and Braun, H. P. (2007). Supramolecular structure of the mitochondrialoxidative phosphorylation system. J. Biol. Chem. 282, 1–4.Boumans, H., Grivell, L. A., and Berden, J. A. (1998). The respiratory chain in yeast behavesas a single functional unit. J. Biol. Chem. 273, 4872–4877.Cruciat, C. M., Brunner, S., Baumann, F., Neupert, W., and Stuart, R. A. (2000). Thecytochrome bc1 and cytochrome c oxidase complexes associate to form a single supra-complex in yeast mitochondria. J. Biol. Chem. 275, 18093–18098.Dienhart, M. K., and Stuart, R. A. (2008). The yeast Aac2 protein exists in physicalassociation with the cytochrome bc1-COX supercomplex and the TIM23 machinery.Mol. Biol. Cell 19, 3934–3943.Frank, J. (2002). Single-particle imaging of macromolecules by cryo-electron microscopy.Annu. Rev. Biophys. Biomol. Struct. 31, 303–319.Hatefi, Y., Haavik, A. G., Fowler, L. R., and Griffiths, D. E. (1962). Studies on the electrontransfer system. Reconstitution of the electron transfer system. J. Biol. Chem. 237,2661–2669.Hackenbrock, C. R., Chazotte, B., and Gupta, S. S. (1986). The random collision modeland a critical assessment of the diffusion and collision in mitochondrial electron transport.J. Bioenerg. Biomembr. 18, 331–368.Heinemeyer, J., Braun, H. P., Boekema, E. J., and Kouril, R. (2007). A structural model ofthe cytochrome c reductase/oxidase supercomplex from yeast mitochondria. J. Biol.Chem. 282, 12240–12248.Iwasaki, T., Matsuura, K., and Oshima, T. (1995). Resolution of the aerobic respiratorysystem of the thermoacidophilic archaeon, Sulfolobus sp. Strain 7. I. The archael terminaloxidase supercomplex is a functional fusion of respiratory complexes III and IV with noc-type cytochromes. J. Biol. Chem. 270, 30881–30892.Lange, C., and Hunte, C. (2002). Crystal structure of the yeast cytochrome bc1 complex withits bound substrate cytochrome c. Proc Natl. Acad. Sci. USA 99, 2800–2805.Meisinger, C., Pfanner, N., and Truscott, K. N. (2006). Isolation of yeast mitochondria.Methods Mol. Biol. 313, 33–39.Nelson, D. L., and Cox, M. M. (2008). In ‘‘Lehninger—Principles of Biochemistry’’ 5th Ed.,pp. 718–719. Freeman, New York.Pfeiffer, K., Gohil, V., Stuart, R. A., Hunte, C., Brandt, U., Greenberg, M. L., andSchägger, H. (2003). Cardiolipin stabilizes respiratory chain supercomplexes. J. Biol.Chem. 278, 52873–52880.Saddar, S., Dienhart, M. K., and Stuart, R. A. (2008). The F1F0-ATP synthase complexinfluences the assembly state of the cytochrome bc1-cytochrome oxidase supercomplexand its association with the TIM23 machinery. J. Biol. Chem. 283, 6677–6686.Schägger, H., and Pfeiffer, K. (2000). Supercomplexes in the respiratory chains of yeast andmammalian mitochondria. EMBO J. 19, 1777–1783.190 Hans-Peter Braun et al.Sone, N., Sekimachi, M., and Kutoh, E. (1987). Identification and properties of a quinoloxidase supercomplex composed of a bc1 complex and cytochrome oxidase in thethermophilic bacterium PS3. J. Biol. Chem. 262, 15386–15391.Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., and Yoshikawa, S. (1996). The whole structureof the 13-subunit oxidized cytochrome c oxidase at 2.8 Å. Science 272, 1136–1144.van Heel, M., Gowen, B., Matadeen, R., Orlova, E. V., Finn, R., Pape, T., Cohen, D.,Stark, H., Schmidt, R., Schatz, M., and Patwardhan, A. (2000). Single-particle electroncryo-microscopy: Towards atomic resolution. Q. Rev. Biophys. 33, 307–369.Wittig, I., Braun, H. P., and Schägger, H. (2006). Blue-Native PAGE.NATURE Protocols 1,418–428.Wittig, I., Karas, M., and Schägger, H. (2007). High resolution clear native electrophoresisfor in-gel functional assays and fluorescence studies of membrane protein complexes.Mol. Cell. Proteomics 6, 1215–1225.Zerbetto, E., Vergani, L., and Dabbeni-Sala, F. (1997). Quantitation of muscle mitochon-drial oxidative phosphorylation enzymes via histochemical staining of blue native poly-acrylamide gels. Electrophoresis 18, 2059–2064.Zhang, M., Mileykovskaya, E., and Dowhan, W. (2002). Gluing the respiratory chaintogether. J. Biol. Chem. 277, 43553–43556.Zhang, M., Mileykovskaya, E., and Dowhan, W. (2005). Cardiolipin is essential for organi-zation of complexes III and IV into a supercomplex in intact yeast mitochondria. J. Biol.Chem. 280, 29403–29408.

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