Structural basis of mitochondrial membrane bending by the I-II-III2-IV2 supercomplex.

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.

The architecture of respiratory supercomplexes.

Mitochondrial electron transport chain complexes are organized into supercomplexes responsible for carrying out cellular respiration. Here we present three architectures of mammalian (ovine) supercomplexes determined by cryo-electron microscopy. We identify two distinct arrangements of supercomplex CICIII2CIV (the respirasome)-a major 'tight' form and a minor 'loose' form (resolved at the resolution of 5.8 Å and 6.7 Å, respectively), which may represent different stages in supercomplex assembly or disassembly. We have also determined an architecture of supercomplex CICIII2 at 7.8 Å resolution. All observed density can be attributed to the known 80 subunits of the individual complexes, including 132 transmembrane helices. The individual complexes form tight interactions that vary between the architectures, with complex IV subunit COX7a switching contact from complex III to complex I. The arrangement of active sites within the supercomplex may help control reactive oxygen species production. To our knowledge, these are the first complete architectures of the dominant, physiologically relevant state of the electron transport chain.

On the role of subunit M in cytochrome cbb3 oxidase.

Cytochrome cbb3 (or C-type) oxidases are a highly divergent group and the least studied members of the heme-copper oxidases (HCOs) superfamily. HCOs couple the reduction of oxygen at the end of the respiratory chain to the active proton translocation across the membrane, contributing to establishment of an electrochemical gradient essential for ATP synthesis. Cbb3 oxidases exhibit unique structural and functional features and have an essential role in the metabolism of many clinically relevant human pathogens. Such characteristics make them a promising therapeutic target. Three subunits, N, O and P, comprise the core cbb3 complex, with N, the catalytic subunit, being highly conserved among all members of the HCO superfamily, including the A-type (aa3, mitochondrial-like) oxidases. An additional fourth subunit containing a single transmembrane (TM) helix was present in the first crystal structure of cbb3. This TM segment was recently proposed to be part of a novel protein CcoM, which was shown to have a putative role in the complex stability and assembly. In this work, we performed large-scale all-atom molecular dynamics simulations of the CcoNOPM complex to further characterize the interactions between subunit M and the core subunits and to determine whether the presence of the fourth subunit influences the water/proton channels previously described for the core complex. The previously proposed putative CcoNOPH complex is also assessed, and the potential functional redundancy of CcoM and CcoQ is discussed.

Interaction of complexes I, III, and IV within the bovine respirasome by single particle cryoelectron tomography.

The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III(2), and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III(2) and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.

From static structure to living protein: computational analysis of cytochrome c oxidase main-chain flexibility.

Crystallographic structure and deuterium accessibility comparisons of CcO in different redox states have suggested conformational changes of mechanistic significance. To predict the intrinsic flexibility and low energy motions in CcO, this work has analyzed available high-resolution crystallographic structures with ProFlex and elNémo computational methods. The results identify flexible regions and potential conformational changes in CcO that correlate well with published structural and biochemical data and provide mechanistic insights. CcO is predicted to undergo rotational motions on the interior and exterior of the membrane, driven by transmembrane helical tilting and bending, coupled with rocking of the ß-sheet domain. Consequently, the proton K-pathway becomes sufficiently flexible for internal water molecules to alternately occupy upper and lower parts of the pathway, associated with conserved Thr-359 and Lys-362 residues. The D-pathway helices are found to be relatively rigid, with a highly flexible entrance region involving the subunit I C-terminus, potentially regulating the uptake of protons. Constriction and dilation of hydrophobic channels in RsCcO suggest regulation of the oxygen supply to the binuclear center. This analysis points to coupled conformational changes in CcO and their potential to influence both proton and oxygen access.

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening.

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ∼60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 µM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.

Allosteric Cooperativity in Proton Energy Conversion in A1-Type Cytochrome c Oxidase.

Cytochrome c oxidase (CcO), the CuA, heme a, heme a3, CuB enzyme of respiratory chain, converts the free energy released by aerobic cytochrome c oxidation into a membrane electrochemical proton gradient (ΔµH+). ΔµH+ derives from the membrane anisotropic arrangement of dioxygen reduction to two water molecules and transmembrane proton pumping from a negative (N) space to a positive (P) space separated by the membrane. Spectroscopic, potentiometric, and X-ray crystallographic analyses characterize allosteric cooperativity of dioxygen binding and reduction with protonmotive conformational states of CcO. These studies show that allosteric cooperativity stabilizes the favorable conformational state for conversion of redox energy into a transmembrane ΔµH+.

Alterations of complex IV in the tissues of a septic mouse model.

OBJECTIVES: To characterize the mitochondrial respiratory chain complex IV(complex IV) activity and protein expression during polymicrobial sepsis. MATERIAL AND METHODS: Polymicrobial peritonitis, a clinically relevant mouse model of sepsis, was generated by cecum ligation and puncture (CLP) in Sprague- Dawley rats. The rats were randomly divided into 3 groups as follows: the sepsis without resuscitation (S), sepsis and fluid resuscitated (R) group, and a control (C) group. Twelve hours after the sepsis model was established, tissue specimens were obtained from the myocardium, liver and skeletal muscle. Mitochondrial respiratory chain complex IV activity of all tissue specimens was detected by spectrophotometry. Western blot was used to measure the liver mitochondrial respiratory chain complex IV protein content. The ultrastructure changes of mitochondria were detected by transmission electron microscopy. RESULTS: In myocardial cells, complex IV activity decreased significantly in the S and R groups as compared to the C group. There were no differences in complex IV activity between groups in skeletal muscle cells while in liver cells, complex IV activity and content was significantly decreased for the S group but no differences were observed between the C and R groups. Increased matrix volume and reduced density with generalized disruption of the normal cristae pattern was most extensive in the liver, followed by cardiac muscle cells with that in skeletal muscle cells been relatively mild in the S group. Mitochondrial fusion/fission and mitochondrial autophagy was also observed in the S group by transmission electron microscopy. Mitochondrial ultrastructure was preserved in the R-group and was similar to that seen in the C-group. CONCLUSIONS: Changes in complex IV activity and mitochondrial ultrastructure, a manifestation of the mitochondrial dysfunction varied depending on cell type. These changes are partly reversed by fluid therapy. Therapies aimed at mitochondrial resuscitation should be explored.

Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases.

The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper-containing oxidases, even though the structures are completely different.

Electron microscopy of cytochrome c oxidase crystals. Monomer-dimer relationship and cytochrome c binding site.

Cytochrome c oxidase was isolated from beef heart mitochondria by detergent extraction yielding two different crystal forms. Extraction with Triton detergents produced vesicular crystals with two-dimensional crystalline arrays of cytochrome c oxidase dimers while extraction with sodium deoxycholate produced crystalline sheets of cytochrome c oxidase monomers. The structures of both crystal forms were determined in two-dimensional projection along an axis normal to the plane of the membrane by cryoelectron microscopy of crystals embedded in vitreous ice (frozen-hydrated). The projection structures of unstained frozen hydrated monomers and of dimers are similar to the structures of the crystals in negative stain. The molecular outline of dimers can be approximated by a parallelogram 44 A by 82 A with an included angle of 80 degrees. Monomers are less regular consisting of two large domains with a smaller domain at one end and a total length of approximately 82 A. Comparison of the two structures reveals the orientation of cytochrome c oxidase monomers within dimers, an orientation which is different from earlier models of monomer-monomer interaction, and suggests a very close interaction between monomers when they associate to form dimers. The crystalline sheets of cytochrome c oxidase monomers bind tightly the small peripheral membrane protein substrate, cytochrome c, and this binding accentuates a tendency of these crystals to stack upon one another. Images of crystals of the cytochrome c oxidase/cytochrome c complex were analyzed by crosscorrelation analysis versus the monomer crystal image. Two types of two-layer crystals have been identified. Both types have one layer rotated by 180 degrees with respect to the other, but they differ in the shifts of origin along crystal axes of the two layers. Difference images formed by subtracting simulated multilayered crystal images (which have no bound cytochrome c) from the complex crystals (cytochrome c oxidase plus cytochrome c) contain one positive difference peak for each cytochrome oxidase monomer within a unit cell. Comparison of the difference peak loci among the different crystal forms is interpreted based upon a consensus cytochrome c binding site in the single layer cytochrome oxidase monomer crystal image.

Properties of the detergent solubilised cytochrome c oxidase (cytochrome cbb(3)) purified from Pseudomonas stutzeri.

Cytochrome cbb(3) is a cytochrome c-oxidising isoenzyme that belongs to the superfamily of respiratory haem/copper oxidases. We have developed a purification method yielding large amounts of pure cbb(3) complex from the soil bacterium Pseudomonas stutzeri. This cytochrome cbb(3) complex consists of three subunits (ccoNOP) in a 1:1:1 stoichiometry and contains two b-type and three c-type haems. The protein complex behaves as a monomer with an overall molecular weight of 114.0+/-8.9 kDa and a s(0)(20,w) value of 8.9+/-0.3 S as determined by analytical ultracentrifugation. Crystals diffracting to 5.0 A resolution have been grown by the vapour diffusion sitting drop method to an average size of 0.1 x 0.1 x 0.3 mm. This is the first crystallisation report of a (cbb(3))-type oxidase.

Brain cytochrome oxidase subunit complementary DNAs: isolation, subcloning, sequencing, light and electron microscopic in situ hybridization of transcripts, and regulation by neuronal activity.

The goal of the present study was to isolate, for the first time, cytochrome oxidase subunit genes from murine brain complementary DNA library and to characterize the expression of these genes from mitochondrial and nuclear sources at both light and electron microscopic levels. Brain subunit III (mitochondrial) shared 100% identity with that of murine L cells. Subunit VIa (nuclear) was known to have tissue-specific isoforms in other species: the ubiquitous liver isoform and the heart/muscle isoform. Our brain subunit VIa shared 93% homology with that of the rat liver and 100% identity with the recently reported murine liver isoform, which is only 62% identical to that of the rat heart isoform. In situ hybridization with riboprobes revealed messenger RNA labelling that was similar, though not identical, to that of cytochrome oxidase histochemistry. Monocular enucleation in adult mice induced a significant down-regulation of both subunit messages in the contralateral lateral geniculate nucleus. However, the decrease in subunit III messenger RNAs surpassed that of subunit VIa at all time periods examined, suggesting that mitochondrial gene expression is more tightly regulated by neuronal activity than that of nuclear ones. At the electron microscopic level, subunit III messenger RNA was localized to the mitochondrial compartment in both cell bodies and processes, while that of nuclear-encoded subunit VIa was present exclusively in the extramitochondrial compartment of somata and not of dendrites or axons. Surprisingly, the message was primarily associated with the rough endoplasmic reticulum, suggesting a novel pathway for its synthesis and trafficking. Our results indicate that the unique properties of neurons impose special requirements for subunits of a single mitochondrial enzyme with dual genomic origins. At sites of high energy demands (such as postsynaptic dendrites and some axon terminals), mitochondrial-encoded cytochrome oxidase subunits can be locally transcribed and translated, and they provide the framework for the subsequent importation and incorporation of nuclear-encoded subunits, which are strictly synthesized in the cell bodies. Dynamic local energy needs are met when subunits from the two genomic sources are assembled to form functional holoenzymes.

Ultrastructural localization of cytochrome oxidase and monoamine oxidase on microcylinders, a Long-Evans rat-specific mitochondrial inclusion.

The presence of a unique inclusion body, the microcylinder, in the intracristal space of mitochondria was previously reported in various types of cells from spotted rats of the Long-Evans strain, but was not found in cells of albino rats. The microcylinder is about 30 nm in diameter and of indefinite length, and is composed of six filamentous subunits surrounding a central one. We performed electron microscopic cytochemical studies on the cells of uriniferous tubules and the corpus striatum in normal spotted rats of the Long-Evans strain and albino rats of Wistar and Sprague-Dawley strains. On the basis of oxidative polymerization of 3, 3'-diaminobenzidine by cytochrome oxidase (CYO) an cupric ferrocyanide deposition by monoamine oxidase (MAO), microcylinders were demonstrated to exhibit activity of these enzymes. Reaction products of other mitochondrial enzymes, such as succinate dehydrogenase and lactate dehydrogenase, were not deposited on microcylinders. We conclude that microcylinders are rat strain-specific mitochondrial inclusions and consist of protein components, particularly containing the mitochondrial enzymes CYO and MAO.

Cytochrome c oxidase: structural studies by electron microscopy of two-dimensional crystals.

Cytochrome c oxidase is a complex integral membrane protein consisting of 13 different polypeptide chains and four metal centers having a total molecular weight of approximately 200,000 daltons. It can be isolated in two 2-dimensional crystalline forms differing in aggregation state of the enzyme. One crystal form consists of cytochrome oxidase dimers (approximately 400,000 daltons) embedded unidirectionally in the lipid bilayer of a collapsed vesicle while the other form consists of crystalline sheets of cytochrome oxidase monomers. Both crystal forms have been studied by electron microscopy during the past two decades, and this paper summarizes the results of early structural studies as well as more recent results applying techniques of cryoelectron microscopy and digital image processing. The structure of frozen-hydrated cytochrome oxidase dimers at 20 A resolution is discussed as well as the packing of monomers within dimers and the site of cytochrome c binding.

Reversed metal replicas of freeze-dried proteins to be visualized with the scanning tunneling microscope.

Scanning tunneling microscopy of metal-coated specimens has become a reliable technique that permits direct three-dimensional visualization of structural details at a level at which individual subunits in protein complexes or even single domains of proteins can be resolved. We describe in this paper a variation of the freeze-drying metal coating procedure that allows us to image with the STM the inner side of the metal replica, previously in contact with the protein molecules. We have tested this new approach with two different well characterized protein systems: freeze-dried two-dimensional crystals of bacteriophage phi 29 connector and the vesicle form of two-dimensional crystals of cytochrome oxidase from beef heart mitochondria. The images obtained have very good contrast and provide direct topographic information of the crystal surface, complementing structural information obtained previously with transmission electron microscopy. The resolution limit is imposed by the size (2-3 nm diameter) and corrugation of the metal grains used to prepare the replica and by the randomness of the metal shadowing.

Chorion laeve trophoblasts of preeclamptic fetal membranes: histochemically detectable enzyme activities do not change at a subcellular level.

We examined the subcellular localization of ADP-degrading activity and cytochrome c oxidase (CCO) activity in chorion laeve trophoblasts from term and near term human fetal membranes, and compared them with those from severe preeclamptic fetal membranes. The methods used for the detection of enzyme activities were the lead nitrate method for ADP-degrading activity and the diaminobenzidine method for CCO. Precipitates indicative of ADP-degrading activity were visible on surface microvillous plasma membranes of chorion laeve trophoblasts both from normal and preeclamptic fetal membranes. The intensity and distribution patterns were the same in the normal and preeclamptic subjects. CCO labeling was visible in almost all laeve trophoblastic mitochondria both in normal and preeclamptic cases. Previously, we demonstrated that in preeclamptic villous trophoblasts there were decreases in ADP-degrading activity and the presence of CCO-negative mitochondria, which were proposed to lead to dysfunction of each villous trophoblast, and finally to placental insufficiency in preeclampsia. Reductions or changes in enzyme intensities/distribution patterns, which are characteristic features of preeclamptic villous trophoblasts, were absent in chorion laeve trophoblasts in preeclampsia. These results suggest that in preeclampsia there are no, or at least less severe, abnormalities in the enzyme activities of chorion laeve trophoblasts, compared with villous trophoblasts, as far as enzyme-histochemically detectable enzymes are concerned.

Electron and proton transfer in the ba(3) oxidase from Thermus thermophilus.

The ba(3)-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa(3)-type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O(2) to water is catalysed at a haem a(3)-Cu(B) catalytic site. The three-dimensional structure of the ba(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa(3) oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba( 3 )-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k congruent with 6.8 x 10(4) s(-1)) and formation of the peroxy (P(R)) intermediate. In the next step a proton was taken up from solution with a rate constant of approximately 1.7 x 10(4) s(-1), associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of approximately 1,100 s(-1), accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was approximately 1.5 protons per enzyme molecule. The results support the earlier proposal that the P(R) and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba(3) oxidases, the proton-uptake reactions occur over the same time scales as in the aa(3)-type oxidases.

Thermostability of proteins: role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus.

The dinuclear copper center (TtCuA) forming the electron entry site in the subunit II of the cytochrome c oxidase in Thermus thermophilus shows high stability toward thermal as well as denaturant-induced unfolding of the protein at ambient pH. We have studied the effect of pH on the stability of the holo-protein as well as of the apo-protein by UV-visible absorption, far-UV, and visible circular dichroism spectroscopy. The results show that the holo-protein both in the native mixed-valence state as well as in the reduced state of the metal ions and the apo-protein of TtCuA were extremely stable toward unfolding by guanidine hydrochloride at ambient pH. The thermal unfolding studies at different values of pH suggested that decreasing pH had almost no effect on the thermal stability of the protein in the absence of the denaturant. However, the stability of the proteins in presence of the denaturant was considerably decreased on lowering the pH. Moreover, the stability of the holo-protein in the reduced state of the metal ion was found to be lower than that in the mixed-valence state at the same pH. The denaturant-induced unfolding of the protein at different values of pH was analyzed using a two-state unfolding model. The values of the free energy of unfolding were found to increase with pH. The holo-protein showed that the variation of the unfolding free energy was associated with a pKa of approximately 5.5. This is consistent with the model that the protonation of a histidine residue may be responsible for the decrease in the stability of the holo-protein at low pH. The results were interpreted in the light of the reported crystal structure of the protein.

Architecture of active mammalian respiratory chain supercomplexes.

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.

Electron microscopy of cytochrome c oxidase-containing proteoliposomes: imaging analysis of protein orientation and monomer-dimer behaviour.

1. Cytochrome c oxidase-containing vesicles were prepared by cholate dialysis using bovine heart cytochrome c oxidase with egg and dioleoylphosphatidylcholine/dioleoylphosphatidylethanolamines (1:1, w/w) at two ratios of phospholipid to protein (25 mg/mg and 10 mg/mg). With each mixture, one or two (FII, FIII) fractions with mostly outward-facing cytochrome aa3 were separated from a fraction (FI) containing mostly inward-facing enzyme and protein-free liposomes by DEAE-Sephacel chromatography. 2. FII and FIII fractions from egg phospholipid mixtures had 60-80% outward-facing enzyme; FII and FIII fractions from dioleoyl phospholipids showed 50-70% outward-facing enzyme. Egg and dioleoyl phospholipid mixtures maintained good respiratory control ratios (8-13) only at the higher lipid/protein ratios. 3. Platinum/carbon replicas of freeze-fractured vesicle surfaces were subjected to image analysis. The results showed two types of membrane projection with average heights of 7.5 nm and 3.5 nm from the fracture plane. The former were more numerous on the convex faces. Calculated areas of the projections indicated the probable presence of both enzyme dimers and higher aggregates. Oxidase dimers may have membrane areas of 70-80 nm2 at the high (7.5 nm) side and 40-50 nm2 on the low (3.5 nm) side. 4. Proteoliposomes prepared with enzyme depleted of subunit III contained predominantly much smaller projecting areas. These probably represent monomers with high side areas of 35-40 nm2 and low side areas of 20-25 nm2. Electron microscopy thus directly confirms the predicted change of aggregation state resulting from subunit depletion. 5. The results are compared with those from two-dimensional crystals. Assuming that the high and low projections are two sides of one family of transmembrane molecules, a total length of 11 nm matches 11-12 nm lengths obtained by crystallography. Our membrane areas match the areas obtained in earlier 'crystal' studies better than the small areas obtained recently by electron cryomicroscopy.