ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry.

Mass spectrometry of membrane protein complexes is still a methodological challenge due to hydrophobic and hydrophilic parts of the species and the fact that all subunits are bound non-covalently together. The present study with the novel laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) reports on the determination of the subunit composition of the F(1)F(o)-ATP synthase from Bacillus pseudofirmus OF4, that of both bovine heart and, for the first time, of human heart mitochondrial F(1)F(o)-ATP synthases. Under selected buffer conditions the mass of the intact F(1)F(o)-ATP synthase of B. pseudofirmus OF4 could be measured, allowing the analysis of complex subunit stoichiometry. The agreement with theoretical masses derived from sequence databases is very good. A comparison of the ATP synthase subunit composition of 5 different ATPases reveals differences in the complexity of eukaryotic and bacterial ATP synthases. However, whereas the overall construction of eukaryotic enzymes is more complex than the bacterial ones, functionally important subunits are conserved among all ATPases.

Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: isolation of putative respiratory strings or patches.

Here, we expand the application of blue native electrophoresis to the separation of mega protein complexes larger than 10 MDa by introducing novel large pore acrylamide gels. We tailored the bis-acrylamide cross-linker amounts relative to the acrylamide monomer to enlarge the pore size of acrylamide gels and to obtain elastic and sufficiently stable gels. The novel gel types were then used to search for suprastructures of mitochondrial respiratory supercomplexes, the hypothetical respiratory strings, or patches. We identified 4-8 MDa assemblies that contain respiratory complexes I, III, and IV and most likely represent dimers, trimers, and tetramers of respiratory supercomplexes. We also isolated multimeric respiratory supercomplexes with apparent masses of 35-45 MDa, the presumed core pieces of respiratory strings or patches. Electron microscopic investigations will be required to clarify whether the isolated assemblies of complexes are ordered and specific, as predicted for respiratory strings and patches in the mitochondrial membrane.

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L.

Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

Laser-induced liquid bead ion desorption-MS of protein complexes from blue-native gels, a sensitive top-down proteomic approach.

We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN-PAGE) and laser-induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent-tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 microg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small-scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.

Mass estimation of native proteins by blue native electrophoresis: principles and practical hints.

Blue native electrophoresis is one of the most popular techniques for mass estimation of native membrane proteins, but the use of non-optimal mass markers and acrylamide gels can compromise accuracy and reliability of the results. We present short protocols taking 10-30 min to prepare optimal sets of membrane protein markers from chicken, rat, mouse, and bovine heart. Especially heart materials from local supermarkets or butcher's shops, e.g. chicken or bovine heart, are ideal sources of high mass membrane protein standards. Considerable discrepancies between the migration behavior of membrane and soluble markers suggest using membrane protein markers for mass estimation of membrane proteins. Soluble standard proteins can be used, with some limitations, when soluble proteins are the focus. Principles and general rules for the determination of mass and oligomeric state of native membrane and soluble proteins are elaborated, and potential pitfalls are discussed.

Native immunoblotting of blue native gels to identify conformation-specific antibodies.

A large repertoire of immunological methods permits monitoring the interaction of antibodies with their specific antigen. However, recognition of a protein by a conformation-specific antibody represents a challenge because native conditions must be kept throughout the assay. Native immunoblotting of blue native gels conserves the native state by using Tween 20 instead of methanol for the obligatory destaining of the blot membrane. We validate the new technique with a set of monoclonal antibodies against respiratory NADH:ubiquinone oxidoreductase.

Native electrophoretic techniques to identify protein-protein interactions.

Permanent protein-protein interactions are commonly identified by co-purification of two or more protein components using techniques like co-immunoprecipitation, tandem affinity purification and native electrophoresis. Here we focus on blue-native electrophoresis, clear-native electrophoresis, high-resolution clear-native electrophoresis and associated techniques to identify stable membrane protein complexes and detergent-labile physiological supercomplexes. Hints for dynamic protein-protein interactions can be obtained using two-hybrid techniques but not from native electrophoresis and other protein isolation techniques except after covalent cross-linking of interacting proteins in vivo prior to protein separation.

Non-classical 2-D electrophoresis.

Classical 2-D electrophoresis (IEF/SDS 2-DE) using isoelectric focusing (IEF) and SDS-PAGE for the second dimension offers very high resolution for the separation of complex protein mixtures, but hydrophobic proteins can aggregate and are considerably under-represented in these 2-D gels. Non-classical 2-DE, as described here, summarizes several heterogeneous techniques, some of which, like BAC/SDS 2-DE and doubled SDS-polyacrylamide gel electrophoresis (dSDS-PAGE), intend to isolate the difficult hydrophobic proteins that are not accessible by classical 2-DE. Other types of non-classical 2-DE start with 1-D separation of native proteins and complexes, like blue-native electrophoresis (BNE), clear-native electrophoresis (CNE), and high-resolution clear-native electrophoresis (hrCNE). These electrophoretic techniques can substitute for chromatographic isolation of protein complexes, and can even isolate supramolecular physiological assemblies. Subsequent resolution in second dimension can be denaturing to resolve the subunits of complexes, as exemplified with BNE/SDS 2-DE, or native like in BNE/BNE 2-DE (the latter using different cathode buffers for 1-D BNE and 2-D BNE). After isolation of highly pure membrane protein complexes by two native electrophoretic separations, the separation protocol may be finished by denaturing 2-DE like BAC/SDS or doubled SDS-PAGE. Thus, a four-dimensional electrophoretic system with minimal loss of protein results that is useful as an efficient micro-scale protein separation protocol, e.g. for mass spectrometric analyses.

Two-dimensional native electrophoretic analysis of respiratory supercomplexes from Yarrowia lipolytica.

Mitochondria of the strictly aerobic yeast Yarrowia lipolytica contain respiratory complex I with close functional and structural similarity to the mammalian enzyme. Unlike mammalian mitochondria, however, Yarrowia mitochondria have been thought not to contain supercomplexes. Here, we identify respiratory supercomplexes composed of complexes I, III and IV also in Y. lipolytica. Evidence for dimeric complex I suggests further association of respiratory supercomplexes into respiratory strings or patches. Similar supercomplex organization in Yarrowia and mammalian mitochondria further makes this aerobic yeast a useful model for the human oxidative phosphorylation system. The analysis of supercomplexes and their constituent complexes was made possible by 2-D native electrophoresis, i.e. by using native electrophoresis for both dimensions. Digitonin and blue-native electrophoresis were generally applied for the initial separation of supercomplexes followed by less mild native electrophoresis variants in the second dimension to release the individual complexes from the supercomplexes. Such 2-D native systems are useful means to identify the constituent proteins and their copy numbers in detergent-labile physiological assemblies, since they can reduce the complexity of supramolecular systems to the level of individual complexes.

Chapter 8 Two-dimensional native electrophoresis for fluorescent and functional assays of mitochondrial complexes.

Supramolecular assemblies of native membrane protein complexes were solubilized from biological membranes by very mild detergents and isolated by native electrophoresis. The complexity of these higher order structures can be reduced for proteomic investigations by applying less mild native electrophoresis variants in the second dimension. Supercomplexes thereby dissociate into the individual complexes. Clear-native and blue-native electrophoresis variants are useful alternatives for the second native dimension, but clear-native electrophoresis is advantageous for the identification of fluorescence-labeled proteins and for in-gel activity assays that are hampered by Coomassie dye. The 2-D native gels comprising two orthogonal native dimensions are useful to determine the stoichiometry of complexes in supercomplexes. Strips from 2-D native gels can also be used for 3-D SDS-PAGE to identify loosely bound accessory subunits of supercomplexes. The subunit composition of supercomplexes and individual complexes is investigated by 4-D gels. The 4-D protocol starts with isolation of highly pure membrane protein complexes by 2-D native electrophoresis, followed by doubled SDS-PAGE to resolve the subunits.

Role of phospholipids in respiratory cytochrome bc(1) complex catalysis and supercomplex formation.

Specific protein-lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc(1) complex and how disruption of a specific cardiolipin binding site in cytochrome c(1) alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc(1) complex was treated with phospholipase A(2). The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from -52 to -82 mV (heme b(L)) and from +113 to -2 mV (heme b(H)). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CL(i)) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc(1) complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein-protein interaction.

Mitochondrial ABC proteins in health and disease.

ABC transporters represent one of the largest families of membrane proteins that are found in all three phyla of life. Mitochondria comprise up to four ABC systems, ABCB7/ATM1, ABCB10/MDL1, ABCB8 and ABCB6. These half-transporters, which assemble into homodimeric complexes, are involved in a number of key cellular processes, e.g. biogenesis of cytosolic iron-sulfur clusters, heme biosynthesis, iron homeostasis, multidrug resistance, and protection against oxidative stress. Here, we summarize recent advances and emerging themes in our understanding of how these ABC systems in the inner and outer mitochondrial membrane fulfill their functions in important (patho) physiological processes, including neurodegenerative and hematological disorders.

Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes.

Mitochondrial ATP synthase is mostly isolated in monomeric form, but in the inner mitochondrial membrane it seems to dimerize and to form higher oligomeric structures from dimeric building blocks. Following a period of electron microscopic single particle analyses that revealed an angular orientation of the membrane parts of monomeric ATP synthases in the dimeric structures, and after extensive studies of the monomer-monomer interface, the focus now shifts to the potentially dynamic state of the oligomeric structures, their potential involvement in metabolic regulation of mitochondria and cells, and to newly identified interactions like physical associations of complexes IV and V. Similarly, larger structures like respiratory strings that have been postulated to form from individual respiratory complexes and their supercomplexes, the respirasomes, come into the focus. Progress by structural investigations is paralleled by insights into the functional roles of respirasomes including substrate channelling and stabilization of individual complexes. Cardiolipin was found to be important for the structural stability of respirasomes which in turn is required to maintain cells and tissues in a healthy state. Defects in cardiolipin remodeling cause devastating diseases like Barth syndrome. Novel species-specific roles of respirasomes for the stability of respiratory complexes have been identified, and potential additional roles may be deduced from newly observed interactions of respirasomes with components of the protein import machinery and with the ADP/ATP translocator.

Features and applications of blue-native and clear-native electrophoresis.

1-D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein-protein interactions can be identified depending on detergent and buffer conditions. 1-D native gels are immediately applicable for in-gel detection of fluorescent-labeled proteins and for in-gel catalytic activity assays. 1-D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1-D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2-D separation of subunits of protein complexes by SDS-PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, and the vast variety of mitochondrial encephalomyopathies.

Structural organization of mitochondrial ATP synthase.

Specific modules and subcomplexes like F(1) and F(0)-parts, F(1)-c subcomplexes, peripheral and central stalks, and the rotor part comprising a ring of c-subunits with attached subunits gamma, delta, and epsilon can be identified in yeast and mammalian ATP synthase. Four subunits, alpha(3)beta(3), OSCP, and h, seem to form a structural entity at the extramembranous rotor/stator interface (gamma/alpha(3)beta(3)) to hold and stabilize the rotor in the holo-enzyme. The intramembranous rotor/stator interface (c-ring/a-subunit) must be dynamic to guarantee unhindered rotation. Unexpectedly, a c(10)a-assembly could be isolated with almost quantitive yield suggesting that an intermediate step in the rotating mechanism was frozen under the conditions used. Isolation of dimeric a-subunit and (c(10))(2)a(2)-complex from dimeric ATP synthase suggested that the a-subunit stabilizes the same monomer-monomer interface that had been shown to involve also subunits e, g, b, i, and h. The natural inhibitor protein Inh1 does not favor oligomerization of yeast ATP synthase. Other candidates for the oligomerization of dimeric ATP synthase building blocks are discussed, e.g. the transporters for inorganic phosphate and ADP/ATP that had been identified as constituents of ATP synthasomes. Independent approaches are presented that support previous reports on the existence of ATP synthasomes in the mitochondrial membrane.

Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I.

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30-37 degrees C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide:oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.

Characterization of domain interfaces in monomeric and dimeric ATP synthase.

We disassembled monomeric and dimeric yeast ATP synthase under mild conditions to identify labile proteins and transiently stable subcomplexes that had not been observed before. Specific removal of subunits alpha, beta, oligomycin sensitivity conferring protein (OSCP), and h disrupted the ATP synthase at the gamma-alpha(3)beta(3) rotor-stator interface. Loss of two F(1)-parts from dimeric ATP synthase led to the isolation of a dimeric subcomplex containing membrane and peripheral stalk proteins thus identifying the membrane/peripheral stalk sectors immediately as the dimerizing parts of ATP synthase. Almost all subunit a was found associated with a ring of 10 c-subunits in two-dimensional blue native/SDS gels. We therefore postulate that c10a1-complex is a stable structure in resting ATP synthase until the entry of protons induces a breaking of interactions and stepwise rotation of the c-ring relative to the a-subunit in the catalytic mechanism. Dimeric subunit a was identified in SDS gels in association with two c10-rings suggesting that a c10a2c10-complex may constitute an important part of the monomer-monomer interface in dimeric ATP synthase that seems to be further tightened by subunits b, i, e, g, and h. In contrast to the monomer-monomer interface, the interface between dimers in higher oligomeric structures remains largely unknown. However, we could show that the natural inhibitor protein Inh1 is not required for oligomerization.

Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.

We introduce high resolution clear native electrophoresis (CNE) as a powerful technique to resolve enzymatically active mitochondrial complexes from cultured human cell lines and skeletal muscle biopsy samples. Quantitative enzymatic assays can be performed using small amounts of cultured cells with low mitochondria content, for example, around 10 mg of sedimented osteosarcoma cells (wet weight) which is equivalent to around 10 million cells. High resolution CNE offers general advantages for in-gel catalytic activity assays compared to blue native electrophoresis. It seems especially suited for assaying mitochondrial ATP synthase and respiratory chain complexes I and II in cell models of human mitochondrial disorders and for detailed analyses of patient cells and tissues with defects in oxidative phosphorylation.

Identification of two proteins associated with mammalian ATP synthase.

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.