Defining the Mitochondrial Proteomes from Five Rat Organs in a Physiologically Significant Context Using 2D Blue-Native/SDS-PAGE Nicole H. Reifschneider,† Sataro Goto,‡ Hideko Nakamoto,‡ Ryoya Takahashi,‡ Michiru Sugawa,§ Norbert A. Dencher,† and Frank Krause*,† Physical Biochemistry, Department of Chemistry, Darmstadt University of Technology, Petersenstrasse 22, D-64287 Darmstadt, Germany, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba, 274-8510 Japan, and Charite´-Universita¨tsmedizin Berlin, Clinical Neurobiology, Eschenallee 3, D-14050 Berlin, Germany Received December 8, 2005
In accordance with their manifold tasks, various dysfunctions of mitochondria are critically involved in a large number of diseases and the aging process. This has inspired considerable efforts to identify all the mitochondrial proteins by denaturing approaches, notably, the standard gel-based method employing isoelectric focusing. Because a significant part of the mitochondrial proteome is membraneassociated and/or functions as homo- or heterooligomeric protein complexes, there is an urgent need to detect and identify mitochondrial proteins, both membranous and soluble ones, under conditions preserving protein-protein interactions. Here, we investigated mitochondria of five different rat organs (kidney, liver, heart, skeletal muscle, and brain) solubilized with digitonin, enabling the quantitative extraction of the five oxidative phosphorylation (OXPHOS) complexes. The analysis by blue-native (BN)PAGE recovered the OXPHOS complexes to a large extent as supercomplexes and separated many other protein complexes and individual proteins which were resolved by subsequent 2D SDS-PAGE revealing the tissue-diverse mitochondrial proteomes. Using MS peptide mass fingerprinting, we identified in all five organs 92 nonredundant soluble and membrane-embedded non-OXPHOS proteins, among them, many as constituents of known mitochondrial protein complexes as well as novel ones such as the putative “stomatin-like protein 2 complex” with an apparent mass of ca. 1800 kDa. Interestingly, the identification list included 36 proteins known or presumed to be localized to nonmitochondrial compartments, for example, glycolytic enzymes, clathrin heavy chain, valosincontaining protein/p97, VOV1-ATPase, and Na,K-ATPase. We expect that more than 200 distinct nonOXPHOS proteins of digitonin-solubilized rat mitochondria separated by 2D BN/SDS-PAGE, representing a partial “protein interactome” map, can be identified. Keywords: aralar • blue-native electrophoresis • digitonin • metabolon • mitofilin • mitochondria • protein-protein interaction • oxidative phosphorylation • stomatin • supercomplexes
1. Introduction Mitochondria are extremely membrane-rich organelles occurring in almost all eukaryotes. They have a central role in metabolism being the compartment of numerous catabolic and anabolic pathways.1 In addition, the key functions of mitochondria in cellular signaling are now well-recognized, such as intracellular calcium homeostasis2 as well as crucially integrating/modulating signals leading to the programmed cell death (apoptosis), for example, by the release of apoptotic factors to the cytosol.3 * To whom correspondence should be addressed: Dr. Frank Krause, Physical Biochemistry, Department of Chemistry, Darmstadt University of Technology, Petersenstrasse 22, D-64287 Darmstadt, Germany. Phone, +49 (0)6151 165376; fax, +49 (0)6151 164171; e-mail,
[email protected]. † Darmstadt University of Technology. ‡ Toho University. § Charite ´ -Universita¨tsmedizin Berlin. 10.1021/pr0504440 CCC: $33.50
2006 American Chemical Society
The complete set of mitochondrial proteins encompassing a wide range of functions is far from precisely known, so that most proteomic efforts are currently focused to identify as many mitochondrial proteins as possible by the analysis of highly purified mitochondria4,5 using shotgun methods yielding 680 different proteins in human heart mitochondria6,7 and 750 ones in yeast mitochondria8. It is expected that many of the unknown mitochondrial proteins are low-abundant and/or only temporarily localized to mitochondria.5 All mitochondrial proteomic studies, even those having analyzed highly purified organelles, identified a more or less high proportion of proteins which are known or at least presumed to be located in nonmitochondrial compartments (e.g., see refs 6-17). This raises the question which of those proteins are either contaminants due to preparation artifacts, true mitochondria-localized proteins, or mitochondria-associated ones attributable to Journal of Proteome Research 2006, 5, 1117-1132
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research articles physiologically relevant close contacts of mitochondria with other cell compartments. It is well-documented that the properties of mitochondria of different organs from mammals significantly differ from each other which is apparently reflected by differences in size, shape, and number per cell. More importantly, some metabolic processes, both biosynthetic and catabolic pathways, occur in various degrees or are specialized to mitochondria of particular cell types. A recent proteomic survey of mouse brain, heart, kidney, and liver mitochondria combined with RNA expression data revealed a surprisingly large heterogeneity of mitochondrial protein distribution in these tissues, suggesting that only about half of all mitochondrial proteins are ubiquitously present in mammals.14 Significantly, a large proportion of the mitochondrial proteins is membrane-associated and/or functions as homo- or heterooligomeric protein complexes. The most prominent examples are the five OXPHOS complexes, which are embedded in the inner membrane and together comprise ca. 90 different proteins in mammals, as well as the large matrix-located multienzyme complexes catalyzing the decarboxylation of pyruvate, R-ketoglutarate, or branched chain R-ketoacids, respectively. There are even evidences for the structurally and functionally coupled organization of whole metabolic pathways located entirely or partially in mitochondria as “metabolons”, which appear to enable substrate channelling of intermediate metabolites.18-20 In particular, recent studies primarily based on BN-PAGE analysis of digitonin-solubilized mitochondrial membranes have provided strong arguments in favor of the existence of stoichiometric respiratory supercomplexes composed of the complexes III and IV in yeast21-25 and of complexes I, III, and IV in a variety of other eukaryotes21,23,26-34 as well as of the occurrence of dimeric and oligomeric ATP synthases (complex V).21,23,25-30,32,34-36 The elucidation of novel mitochondrial protein-protein interactions and also the reliable detection of the already known “protein interactome” are indispensable tasks for the understanding of mitochondrial (patho)physiology as well as the diagnosis of mitochondrial diseases. Indeed, assembly defects, or affected stability of mitochondrial protein complexes such as the OXPHOS complexes (e.g., see refs 37-39) or others, for example, the heterododecameric (R6β6) propionyl-CoA carboxylase40-42 and the heterooctameric (R4β4) trifunctional protein (TFP)43 in lipid metabolism, are the underlying cause of numerous severe diseases. Here, we approached the mitochondrial proteome of five rat organs by BN-PAGE of mitochondria treated with digitonin enabling the efficient but mild solubilization of mitochondrial membranes. Under these conditions, the quantitatively extracted OXPHOS complexes, which represent a large part of the total mitochondrial protein mass, are preserved to a considerable extent as supercomplexes. In all five organs together, we identified besides 30 subunits of OXPHOS complexes 92 distinct non-OXPHOS proteins, including 36 proteins assigned to nonmitochondrial locations, among them, many as constituents of known protein complexes as well as putative novel ones. The potential of 2D BN/SDS-PAGE of digitoninsolubilized mammalian mitochondria as an invaluable tool to decipher the mitochondrial protein interactome is discussed.
2. Material and Methods Animals. Male rats (F344/DuCrj), 4 weeks of age, were purchased from Charles River Japan, Inc., and maintained 1118
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under specific pathogen-free conditions in the animal facility at Toho University with free access to laboratory chow CE-7 (Clea Japan, Inc.) and water. The ad libitum fed rats had a mean lifespan of 29 months.44 Tissues from brain, liver, kidney, as well as heart and skeletal muscle (hind limbs) of rats between 7 and 30 month of age were frozen in liquid nitrogen and stored at -80 °C upon dissection. Isolation of Crude Mitochondria Fraction. Crude rat mitochondria from liver, kidney, skeletal muscle, and brain were prepared as described28 using slightly modified protocols reported previously.45-47 Crude mitochondria from rat heart were prepared basically using the protocol of Palmer et al.48 The tissue was minced and homogenized in 10 vol of homogenization buffer (5 mM MOPS, pH 7.4, 2 mM EGTA, 220 mM mannitol, 70 mM sucrose, 0.2% bovine serum albumin, and 0.5 mM Pefabloc SC) with a motordriven, loose-fit, Teflon-glass homogenizer (4-5 strokes, 1200 rpm). Subsequently, the homogenate was centrifuged at 500g and 4 °C for 10 min. The supernatant obtained was centrifuged at 3000g and 4 °C for 10 min, and the pellet was washed twice with homogenization buffer. Finally, the mitochondrial pellet was suspended in stock buffer (5 mM MOPS, pH 7.4, 2 mM EGTA, 220 mM mannitol, 70 mM sucrose, and 0.5 mM Pefabloc SC). After isolation, the crude mitochondria were either frozen as aliquots in liquid nitrogen and stored at -80 °C until use or, alternatively, a fraction of them subjected to further purification. Purification of Crude Mitochondria. The purification of the freshly prepared crude mitochondria fractions obtained as described above was conducted according to Sickmann et al.8 Samples were loaded on top of a three-step sucrose gradient (2 mL 60%, 4 mL 32%, 2 mL 23%, 2 mL 15% sucrose in each 10 mM MOPS, pH 7.2, and 1 mM EDTA and centrifuged at 134 000g for 1 h. The mitochondria were collected from the interface between the 32% and 60% sucrose-steps and washed once with 10 vol of buffer (10 mM MOPS, pH 7.2, 250 mM sucrose, 1 mM EDTA, and 0.5 mM Pefabloc SC). Mitochondria were resuspended in respective stock solutions (ref 28, see above), frozen in liquid nitrogen, and stored at -80 °C until use. For comparison, aliquots of frozen crude brain mitochondria analyzed in ref 28 and stored for several months were thawed and subjected to purification as described above. Electrophoresis and Staining. Mitochondria were centrifuged at 20 800g for 10 min, and the pellet was resuspended in solubilization buffer (30 mM HEPES, pH 7.4, 150 mM potassium acetate, and 10% glycerol, containing 0.5 mM Pefabloc SC as serine protease inhibitor, final concentrations). The membranes were solubilized with 8 g digitonin/g protein at a final detergent concentration of 1% by adding a freshly prepared 10% digitonin solution (high purity, Calbiochem Merck) according to the literature.21,28,29,32,35 The samples were incubated for 30 min on ice followed by centrifugation at 20 800g for 10 min. Before the mixtures were loaded onto the gel, the supernatants were mixed with sample buffer (5% Coomassie Blue G-250, 50 mM Bis-Tris, pH 7.0, 500 mM 6-aminocaproic acid) leading to a detergent/dye ratio of 4:1 (g/g). BN-PAGE was performed using the imidazole buffer system,49 and linear 4-13% gradient gels were overlaid with a 3.5% stacking gel as described21,28,29,32,35,49-51 in a Hoefer SE 600 system (18 × 16 × 0.15 cm2, 15 lanes). The digitonin extracts of mitochondria containing approximately 150 µg of protein before solubilization, as determined by the Roti Nano Quant assay, were loaded per lane. Bovine heart mitochondria pre-
Mitochondrial Proteomes from Five Rat Organs
pared from tissue stored for 10 months at -80 °C (4 g digitonin/g protein, 100 µg of protein before solubilization) served as molecular mass standard.32 The BN-PAGE was stopped when the dye front had reached the gel bottom. Upon electrophoresis, gel lanes were excised and stained with Coomassie Blue R-250, or alternatively, for a second dimension Tricine-SDS-PAGE using two 5% stacking gels and a 13% separating gel as described,50-52 the lanes were incubated in a solution of 1% SDS and 1% mercaptoethanol at 20 °C for 1-2 h.50,51 The second dimension gels were silver-stained according to Blum et al.53 and Rabilloud et al.54 which is compatible to digestion by trypsin and analysis by MALDI-TOF-MS. In-Gel Digestion and MALDI-TOF-MS. Proteins from second dimension SDS gels were identified by MALDI-TOF-MS-PMF as described by Rexroth et al.55,56 Briefly, in-gel digestion was accomplished by trypsin (Promega) incubation overnight at 37 °C. Desalination was performed using µ-C18 Zip-Tips (Millipore). Subsequently, peptides were eluted from the tips onto a MALDI sample target using 5 mg/mL 4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid. Peptide samples were analyzed with a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems). Bioinformatics. The freeware “Mascot” at http://www.matrixscience.com was used to match detected masses against the NCBInr database. The search included one possible missing cleavage site as well as possible methionine oxidation and cysteine derivatization by acrylamide (Cys-S-β-propionamide) with a maximal MS error tolerance of 30 ppm. For each PMF search, a probability score was calculated by MASCOT algorithm. PMF correlation was accepted when the probability score was higher than the signal limit, which was set to 58 (p < 0.05) for proteins of Rattus norvegicus. In addition, a minimum of seven matching peptides were required for positive protein identification. Furthermore, the gel position, in particular the apparent mass in the second dimension, of the respective spot had to be in line with the calculated physical data of the protein found in the database and literature search. Thus, some proteins not fulfilling the database search criteria above were tentatively assigned as identified in the case of according apparent masses. Using the Protparam tool (us.expasy.org/tools/protparam.html) from the Swiss Institute of Bioinformatics57 the pI and molecular mass, as well as the grand average of hydropathicity (GRAVY),58 were calculated, whereas the transmembrane area was predicted by the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/).59
3. Results BN-PAGE of Digitonin-Solubilized Crude Mitochondria from Five Rat Organs. The aim to explore native mitochondrial proteins with preserved interactions to other ones is complicated, since a large number of mitochondrial proteins are membrane-associated, which requires to treat mitochondria with detergents in order to efficiently solubilize integral membrane proteins. Using BN-PAGE, we separated the proteins from crude rat mitochondria of kidney, liver, heart, skeletal muscle, and brain each solubilized with 8 g digitonin/g protein, which is above the detergent amount needed to achieve near quantitative extraction of the five OXPHOS complexes, thus, ensuring an optimal extraction yield of membrane proteins. The employed 4-13% polyacrylamide gradient gel allows the separation of protein complexes and individual proteins according to their apparent mass with a good resolution in the range of 3000-100 kDa.21,28,29,32,34,49 Similar to our recent results,
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Figure 1. BN-PAGE of digitonin-solubilized crude rat mitochondria from five tissues as indicated. For mass calibration, digitoninsolubilized bovine heart mitochondria (BHM) were used: individual complexes I-V (130-1000 kDa) and supercomplexes a-e (I1III2IV0-4, 1500-2300 kDa). The characteristic bands of individual OXPHOS complexes and their preserved supercomplexes are recognizable in the five different rat mitochondria, but the overall band pattern differs among the tissues reflecting the distinct protein compositions.
high amounts of preserved respiratory supercomplexes and oligomeric ATP synthases were obtained, albeit partially reduced compared with solubilization using 4 g digitonin/g protein,28 discernible in the first dimension BN gel by a characteristic band pattern (Figure 1). Briefly, the bands of supercomplexes containing complex I such as a (I1III2) and b (I1III2IV1) as well as of dimeric and trimeric ATP synthase (V2 and V3) in the mass range of ca. 1500-2300 kDa appeared above those of the individual complex I (I1). The band of the small supercomplex III2IV1 (∼750 kDa) is visible, too, slightly slower-migrating than the ATP synthase monomer (V1). Other bands represent non-OXPHOS proteins. The overall band pattern of the digitonin-solubilized mitochondria differs among the organs, reflecting the distinct protein contents as well as different detergent stabilities of the OXPHOS supercomplexes which are influenced, for example, by varying lipid compositions/amounts of the respective mitochondrial membranes. Identification of Digitonin-Extracted Proteins from Rat Mitochondria Separated by 2D BN/SDS-PAGE. The high yield of membrane proteins extracted with digitonin from rat mitochondria and the concomitant preservation of OXPHOS supercomplexes separable by subsequent BN-PAGE are excellent features to more comprehensively elucidate proteinprotein interactions of mitochondrial proteins. Hitherto, only a few 2D BN/SDS-PAGE studies identified not more than 21 distinct non-OXPHOS proteins from mitochondria of rat heart and liver60 and human heart,61 as well as rice62 and Arabidopsis63 after solubilization with n-dodecylmaltoside which extracts the individual OXPHOS complexes exclusively by disrupting their supercomplexes. This prompted us to focus on the identification by MALDITOF-MS-PMF of non-OXPHOS proteins which were resolved by a denaturing SDS-PAGE in the second dimension, leading Journal of Proteome Research • Vol. 5, No. 5, 2006 1119
research articles to the migration of single proteins according to their apparent masses in a vertical line below their position in the firstdimension BN-PAGE. Thus, proteins forming a complex, which was preserved and separated essentially according to its mass in the first-dimension BN gel, appeared in a vertical line displaying the same band shape. Overall, 92 non-OXPHOS proteins (without the two succinate dehydrogenase subunits of the TCA cycle being constituents of complex II) were identified in crude and purified (see below) mitochondria of all five tissues. These cover a wide range of molar masses in first (300, 300, 220, 75/∼400, 300, 150, 100, ∼70 ∼70/∼70 ∼70/∼70 ∼70/∼70
detected oligomeric state
homooligomers and heterooligomers with ADP/ATP translocase homodimer homodimer homodimer
∼75/∼70, ∼75/∼75 ∼460/∼750
homotetramer?
850-900/∼1200
VOV1-ATPase
250/∼480
VO-ATPase
∼150-160/∼180
Na,K-ATPase
200/∼180
homotetramer
homodimer
homotetramer
112
52.2/∼52
200/∼180
homotetramer
homotetramer/ homooctamer (ref 49) homodimer (10673439) homodimer (2681790)
63
59.8/∼59
230, 460/∼550
homooctamer
71 72
47.7/∼49 41.0/∼42
∼95/∼120 ∼80/∼120
homodimer homodimer
∼60/∼60
homotetramer
∼360, 90/∼450, ∼90-100 ∼360, 90/∼480
homooctamer/ -dimer homooctamer
∼85/∼110 ∼85/e50
homodimer monomer
∼230/∼260-300
homodimer
homotetramer(12530653) 84 10.8/∼16 Nucleotide metabolism (light blue) homooctamer/-dimer 80 47.0/∼45
53 creatine kinase, mitochondrial 1, ubiquitousl,B 80 47.4/∼45 54 sarcomeric mitochondrial creatine homooctamer/-dimer kinaseM 55 creatine kinase, muscleM homodimer 124 43.0/∼45 56 creatine kinase, brainB homodimer 125 42.7/∼44 Other proteins (light blue) 57 nicotinamide nucleotide homodimer 29 113.9/∼115 transhydrogenaseK,l,H,M heterooligomers (form 4 191.6/∼190 58 clathrin triskelionsK,B clathrin coats) 59 kidney aminopeptidase MK [glycosylated] 60 peripherinM
Mr (complex) reported (calculated)/ apparent [kDa]
homodimer
28
homo- and heterooligomers 98
109.4/∼140
clathrin triskelion clathrin triskelions (∼650-700 kDa) of clathrin coats? /∼2500 kDa ∼220/∼450 homodimer
53.5/∼48
detected: ∼350
homooligomer?
a The protein complexes are listed according to their functions. The known oligomeric state as well as the molecular masses of the protein complexes were deduced using the Swiss-Prot database or the literature as indicated. Only the identified subunits are itemized by the spot numbers used in Figure 2 and Table 1. The apparent masses of the identified subunits and of the detected protein complexes were assigned according to the respective gel positions in the first and second dimension gels as shown in Figure 2. The other calculations and assignments are according to the legend of Table 1.
firmed to be a mitochondria-localized protein.95 While lacking a predictable transmembrane domain, it was identified in a highly enriched inner membrane fraction of mouse liver mitochondria and hypothesized to be involved in regulating ion channels95 like other stomatins.96,97 Interestingly, the related integral plasma membrane protein stomatin is known to form homooligomers,98 additionally strengthening the interpretation of the electrophoretic data that SLP-2 exists as a large homooligomeric complex. Moreover, in liver mitochondria, the Nipsnap1 protein (apparent mass ∼29 kDa) of unknown function was identified as a complex with an apparent mass of ∼100120 kDa, suggesting at least a homotrimeric assembly and/or association with another unidentified protein. The Nipsnap1
protein was also found to be associated with the inner membrane of mouse liver mitochondria.95 Of course, these putative novel protein complexes need further investigation beyond the scope of our study; in particular, the real mass of the SLP-2 complex has to be confirmed by other biochemical means such as gel permeation which solely separates proteins according to their size. Comparative 2D BN/SDS-PAGE Analysis of Crude and Sucrose-Density Gradient-Purified Mitochondria. We have also analyzed mitochondria from each tissue purified by sucrose-density gradient centrifugation which largely enriches proteins that are either truly localized in or tightly associated with mitochondria.13 The purification of crude mitochondria Journal of Proteome Research • Vol. 5, No. 5, 2006 1127
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Table 3. Overview of Putative Novel Protein Complexes Identified in 2D BN/SDS Gelsa
no.
novel protein complex
1 2 3 4 5
6 7 8 9 10 a
identified subunit (spot)
Mr (subunit) calculated/ apparent [kDa]
proposed oligomeric state
Mr (complex) apparent [kDa]
mitochondrial aspartate/glutamate carrier citrinK,L,M mitochondrial aspartate/glutamate carrier aralarH,B,M mitochondrial aspartate/glutamate carrier aralarM dimeric trifunctional protein (TFP)K,L,H building-block of a TCA-urea-cycle metabolon (ketoglutarate dehydrogenase complex, carbamoyl phosphate synthetase 1)K mitofilinK,L,H,M
30 88
74.4/∼70 74.8/∼70
∼180 ∼180
homodimer homodimer
88 20 1 2 3
74.8/∼70 82.7/∼80 164.6/∼165 116.3/∼110 48.9/∼49
g300 ∼700-750 g3000
homotetramer dimer of two heterooctamers (R4β4)2 supercomplex
5
92.3/∼80
large homooligomers
stomatin-like protein 2 (SLP-2) complexL Nipsnap1 proteinL dihydroorotate dehydrogenaseK hypothetical protein MGC94853K
61 83 54 47
38.4/∼40 33.2/∼29 40.3/∼45 62.9/∼60
∼2500, ∼1200, ∼450-600 ∼1800 ∼100-120 ∼80 ∼130
large homooligomer homotrimer homodimer? homodimer
The protein complexes are listed in the order of appearance in the text. The calculations and assignments are according to the legends of Tables 1 and
2.
from rat liver, kidney, heart, and skeletal muscle resulted in hardly collectable or rather small proportions of nonmitochondrial fractions in contrast to that of crude brain mitochondria containing large amounts of myelin and synaptosomal fractions (Figure 3A). Accordingly, the 2D BN/SDS gels of purified liver, heart (not shown), and kidney mitochondria (Figure 3B) were very similar to that of the crude ones, whereas the 2D BN/SDS gels of purified skeletal mitochondria displayed a comparable pattern, but some decrease of glycolytic enzymes and other apparently nonmitochondrial proteins such as the Ca2+-ATPase SERCA1 as the most prominent one (Figure 3C). As expected, striking differences were revealed comparing crude and purified brain mitochondria. Foremost, significant amounts of contaminating proteins occurring in the right lower area of the 2D gels were essentially removed after purification, and truly mitochondrial proteins, especially the OXPHOS complexes, were clearly increased (compare Figure 2 panel E with panel F). The most prominent contaminating proteins (Figure 2E, spots my1-my3, Table 1) were identified as the two predominant protein species of mammalian myelin membranes, the proteolipid protein (PLP, lipophilin) and myelin basic protein, both forming homooligomers in the native membrane and/or detergent-extracts.99,100 If the brain mitochondria were purified from frozen crude mitochondria stored for several months as shown in Figure 2F, the amounts of preserved OXPHOS supercomplexes are significantly reduced, but not or only slightly in the case of mitochondria prepared from fresh crude mitochondria (Figure 3B-D). These results exemplified that the quality of the investigated mitochondria is of great importance to get optimal yields of retained OXPHPOS supercomplexes as earlier noted.28,31,32
4. Discussion 2D BN/SDS-PAGE of Digitonin-Solubilized Rat Mitochondria as a Partial Mitochondrial Protein Interactome Map. The solubilization of mitochondrial membranes with digitonin instead of the most-used detergents n-dodecylmaltoside or Triton X-100 leads to the recovery of the nearly quantitatively extracted OXPHOS complexes predominantly as specific supercomplexes by subsequent BN-PAGE.21-36 This technique separates preserved protein complexes essentially according 1128
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to their molar masses. We took advantage of this favorable performance to explore the mitochondrial proteome in a physiologically meaningful context and started to identify the non-OXPHOS proteins of digitonin-solubilized crude and purified mitochondria from five rat organs separated by 2D BN/ SDS-PAGE. The efficiency of the 4-13% polyacrylamide gradients used for BN-PAGE was sufficient to detect numerous soluble and membrane-associated non-OXPHOS protein complexes, mostly known but also some putative novel ones, and individual proteins of mitochondria and other cell compartments representing a wide range of functions (Tables 1 and 2). The novel mitochondrial protein complexes are either apparent oligomers of smaller units, such as the putatively homooligomeric SLP-2 complex (Table 3, no. 7) and the presumably dimeric TFP heterooctamer (Table 3, no. 4), or contain functionally closely related proteins, such as the presumed building-block (Table 3, no. 5) of a TCA/urea cycle metabolon.18-20 Crucially, the samples applied to BN-PAGE were the supernatants of a one-step extraction with digitonin of mitochondriaenriched fractions containing more or less amounts of nonmitochondrial membranes. Because the detected protein complexes are predominantly known, artificial associations of functionally and spatially related proteins in a one-step detergent extract from a mixture of distinct cell compartments as well as during subsequent BN-PAGE are unlikely. Hence, the simultaneous detection of all these protein complexes and individual proteins together with OXPHOS complexes and supercomplexes in a single gel mutually supports the physiologically relevance of each found protein complex. This is an important argument in favor of the protein complexes not described so far, such as the putative SLP-2 complex, as well as particularly for the OXPHOS supercomplexes whose existence in the inner mitochondrial membrane is still far from being generally accepted.28,34 To date, we could not identify all of the proteins separated by 2D BN/SDS-PAGE using MALDI-TOF-MS-PMF. Besides the ca. 90 subunits of the OXPHOS complexes, we expect that more than 200 distinct non-OXPHOS proteins of digitonin-solubilized crude mammalian mitochondria can be separated by this electrophoresis technique, the more so because the polyacryl-
Mitochondrial Proteomes from Five Rat Organs
research articles stacking gel and a separation gel gradient in the 1D BN-PAGE starting with 3% polyacrylamide allow the entering of protein complexes up to about 10 000 kDa, representing the upper separation limit of the BN-PAGE method. Thus, the pyruvate dehydrogenase complex as the largest known mitochondrial multienzyme complex can be detected49 as well as other species larger than 3000 kDa (N. Ja¨gemann, N. A. Dencher, and F. Krause, unpublished observation) not separated with 4-13% gradient gels used in the present study. Likewise, the extension of the polyacrylamide gradient from 13% up to 18-20% allows a far better separation of small individual proteins and protein complexes with apparent masses below 100 kDa in the first dimension, which otherwise migrate rather crowded near the running front and hence are insufficiently resolved in the subsequent SDS-PAGE. Moreover, the second dimension gels can be adapted for the optimal resolution of more narrow mass ranges from less than 10 kDa to more than 200 kDa. More sophisticated identification methods than employed in our study, such as tandem mass spectrometry,6-8 will contribute to significantly raise the number of identified proteins. Importantly, except the precious information about proteinprotein interactions uniquely provided by 2D BN/SDS-PAGE, and to a similar extent by 2D colorless-native/SDS PAGE (N. H. Reifschneider, S. Goto, N. A. Dencher, and F. Krause, unpublished observation), it has to be noted that more than 300 identified distinct proteins, both OXPHOS61,101 and nonOXPHOS ones, from mitochondria-enriched fractions would surpass the identification numbers of distinct proteins in 2D IEF/SDS-PAGE studies of mitochondria as the hitherto most successful gel-based approaches.9,11,16
Figure 3. Sucrose-density gradient purification of crude rat mitochondria. (A) Sucrose gradients loaded with crude mitochondria after centrifugation. The respective tissues are marked by letters: kidney (K), liver (L), heart (H), skeletal muscle (M), and brain (B). The sucrose concentrations and the purified mitochondria (mito) are indicated on the left side. (B-D) 2D BN/ SDS gels of digitonin-solubilized crude mitochondria (left panels) and the same, freshly prepared mitochondria after purification (right panels) of kidney, skeletal muscle, and brain. The 2D gel of crude kidney mitochondria (B, left panel) is the same shown in Figure 2A. For clarity, only the supercomplexes I1III2IVX (I1III2IV0-4), monomeric (V1), and dimeric (V2) ATP synthases as well as monomeric complexes II and IV are indicated on top of each gel.
amide concentrations in the first and second dimension can be adjusted to focus on special mass ranges. In detail, a 3%
Crude Mitochondria as Physiologically Significant Starting Material for Proteomics. In the last years, much efforts has been spent on the extensive purification of mitochondria as starting material for mitochondrial proteome studies which aimed to identify the entire inventory of truly mitochondrialocalized proteins. Our results demonstrate that the BN-PAGE analysis of digitonin-solubilized crude rat mitochondria leading to the separation of nonmitochondrial proteins and protein complexes from different cell compartments did not significantly affect the detection of mitochondrial proteins and protein complexes. In fact, we stress the suitability and significance to investigate crude mitochondria by BN-PAGE. First, as earlier outlined by us, an extended purification procedure may damage mitochondria, especially those which were already impaired in vivo like aged ones. This may cause the loss of such mitochondria or parts of their proteome for subsequent analysis due to disruption and leaking out45 and/ or may affect the stability of protein-protein interactions during subsequent biochemical analysis32 as shown in the case of OXPHOS supercomplexes of rat brain mitochondria purified from thawed crude ones (Figure 2F). Second, mitochondria of different mammalian cells communicate with other cell compartments such as the nucleus,102 endoplasmic/sarcoplasmic reticulum,103-105 golgi,106 cytosolic proteins,79 and the cytoskeleton107,108 by close physical contacts, which are precisely regulated. Two crucial tasks appear to be the nearby connection of mitochondria as the main ATP generator with ATP-consuming sites of other cell compartments102,109,110 as well as tight coordination of signaling events.2,104-106,111-114 This is exemplified in our study by the occurrence of major nonmitochondrial ATP-consuming enzymes, even in highly purified mammalian mitochondria (e.g., Figure 2F) as in other studies,6,7,10,13-15,17 like Journal of Proteome Research • Vol. 5, No. 5, 2006 1129
research articles Na,K-ATPase (“sodium pump”), V-ATPase, Ca2+-ATPase SERCA1, and valosin-containg protein/p97 (Tables 1 and 2, Figure 2). In conclusion, the 2D BN/SDS-PAGE of digitonin-solubilized crude mitochondria, isolated by the minimum of manipulation needed to recover essentially all of the mitochondria in the tissue sample, provides not only a rather comprehensive, (patho)physiologically highly significant analysis of OXPHOS supercomplexes and other mitochondrial protein-protein interactions but potentially also information about relevant physical contacts between mitochondria and other cell compartments.
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