Isolated deficiencies of OXPHOS complexes I and IV are identified accurately and quickly by simple enzyme activity immunocapture assays.

OXPHOS deficits are associated with most reported cases of inherited, degenerative and acquired mitochondrial disease. Traditional methods of measuring OXPHOS activities in patients provide valuable clinical information but require fifty to hundreds of milligrams of biopsy tissue samples in order to isolate mitochondria for analysis. We have worked to develop assays that require less sample and here report novel immunocapture assays (lateral flow dipstick immunoassays) to determine the activities of complexes I and IV, which are far and away the most commonly affected complexes in the class of OXPHOS diseases. These assays are extremely simple to perform, rapid (1-1.5 h) and reproducible with low intra-assay and inter-assay coefficients of variability (CVs) s (<10%). Importantly, there is no need to purify mitochondria as crude extracts of whole cells or tissues are suitable samples. Therefore, the assays allow use of samples obtained non-invasively such as cheek swabs and whole blood, which are not amenable to traditional mitochondrial purification and OXPHOS enzyme analysis. As a first step to assess clinical utility of these novel assays, they were used to screen a panel of cultured fibroblasts derived from patients with isolated deficiencies in complex I or IV caused by identified genetic defects. All patients (5/5) with isolated complex IV deficiencies were identified in this population. Similarly, almost all (22/24) patients with isolated complex I deficiencies were identified. We believe that this assay approach should find widespread utility in initial screening of patients suspected of having mitochondrial disease.

Mitochondrial dysfunction in focal segmental glomerulosclerosis of puromycin aminonucleoside nephrosis.

Focal segmental glomerular sclerosis (FSGS) is a major renal complication of mitochondrial (mt) cytopathies. The present study was designed to investigate the possibility of mtDNA lesion accumulation in podocytes, which are a primary pathogenic site of FSGS, during the development of glomerulopathy in puromycin aminonucleoside nephrosis (PAN). Two renal pathological phases of PAN, nephrosis phase and FSGS phase were studied. We investigated the expression of mt proteins, the copy number of a 4834 base-pair deletion (del-mtDNA), and total mtDNA content by real-time polymerase chain reaction, as well as the mRNA expression levels of the mt transcription factor A (mtTFA) and the nuclear respiratory factor-1 (NRF-1) in glomeruli. The mtDNA encoded cytochrome c oxidase subunit I (COX I) protein level was identical to control in nephrosis phase, however, a 45% reduction was seen in FSGS phase. Intraglomerular del-mtDNA was 16-21 times higher than controls in both phases, but the proportion of this mutation was <1% of total mtDNA. The copy number of total mtDNA at nephrosis phase increased up to 241%, whereas, it decreased to 34% at FSGS phase in glomeruli. The mRNA expression of both mtTFA and NRF-1 was upregulated at nephrosis phase, but mtTFA was downregulated at FSGS phase. A reduction in mtDNA copy number resulted in reduced levels of COX I in glomeruli at FSGS phase, suggesting that mt dysfunction by mtDNA depletion potentially plays a key role in the pathogenesis of FSGS in PAN.

The conformation of the epsilon- and gamma-subunits within the Escherichia coli F(1) ATPase.

F(1) is the water-soluble portion of the ubiquitous F(1)F(0) ATP synthase. Its structure includes three alpha- and three beta-subunits, arranged as a hexameric disc, plus a gamma-subunit that penetrates the center of the disc akin to an axle. Recently Hausrath et al. (Hausrath, A. C., Grüber, G., Matthews, B. W., and Capaldi, R. A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13697-13702) obtained an electron density map of E. coli F(1) at 4.4-A resolution in which the coiled-coil alpha-helices of the gamma-subunit could be seen to extend 45 A from the base of the alpha(3)beta(3) hexamer. Subsequently the structure of a truncated form of the E. coli gamma-subunit in complex with epsilon has been described (Rodgers, A. J. W., and Wilce, M. C. J. (2000) Nat. Struct. Biol. 7, 1051-1054). In the present study the 4.4-A resolution electron density map of E. coli F(1) is re-evaluated in light of the newly available data on the gamma- and epsilon-subunits. It is shown that the map of the F(1) complex is consistent with the structure of the isolated subunits. When E. coli F(1) is compared with that from beef heart, the structures of the E. coli gamma- and epsilon-subunits are seen to be generally similar to their counterparts in the bovine enzyme but to undergo major shifts in position. In particular, the two long, coiled-coil alpha-helices that lie along the axis of F(1) both unwind and rotate. Also the epsilon-subunit rotates around the axis by 81 degrees and undergoes a net translation of about 23 A. It is argued that these large-scale changes in conformation reflect distinct functional states that occur during the rotation of the gamma-subunit within the alpha(3)beta(3) hexamer.

Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme.

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.

A novel subfractionation approach for mitochondrial proteins: a three-dimensional mitochondrial proteome map.

As mitochondria play critical roles in both cell life and cell death, there is great interest in obtaining a human mitochondrial proteome map. Such a map could potentially be useful in diagnosing diseases, identifying targets for drug therapy, and in screening for unwanted drug side effects. In this paper, we present a novel approach to obtaining a human mitochondrial proteome map that combines sucrose gradient centrifugation with standard two-dimensional gel electrophoresis. The resulting three-dimensional separation of proteins allows us to address some of the problems encountered during previous attempts to obtain mitochondrial proteome maps such as resolution of proteins and solubility of hydrophobic proteins during isoelectric focusing. In addition, we show that this new approach provides functional information about protein complexes within the organelle that is not obtained with two-dimensional gel electrophoresis of whole mitochondria.

Cytochrome c oxidase-deficient patients have distinct subunit assembly profiles.

Cytochrome c oxidase (COX) deficiency is the most common respiratory chain defect in childhood and is clinically heterogeneous. We report a study of six patients with COX deficiencies. Two of the patients had as yet undefined defects, three patients had Surf-1 mutations, and one patient had a 15-base pair deletion in the COX III subunit. We show that quantitative measurements of steady-state levels of subunits by monoclonal antibody reactivity, when used in combination with a discontinuous sucrose gradient methods, provide an improved diagnosis of COX deficiencies by distinguishing between kinetic, stability, and assembly defects. The two mutants of undefined etiology had a full complement of subunits with one stable and the other partially unstable to detergent solubilization. Both are likely to carry mutations in nuclear-encoded subunits of the complex. The three Surf-1 mutants and the COX III mutant each had reduced steady-state levels of subunits but variable associations of the residual subunits. This information, as well as aiding in diagnosis, helps in understanding the genotype-phenotype relationships of COX deficiencies and provides insight into the mechanism of assembly of the enzyme complex.

Rotation of the c subunit oligomer in fully functional F1Fo ATP synthase.

The F(1)F(o)-type ATP synthase is the smallest motor enzyme known. Previous studies had established that the central gamma and epsilon subunits of the F(1) part rotate relative to a stator of alpha(3)beta(3) and delta subunits during catalysis. We now show that the ring of c subunits in the F(o) part moves along with the gamma and epsilon subunits. This was demonstrated by linking the three rotor subunits with disulfide bridges between cysteine residues introduced genetically at the interfaces between the gamma, epsilon, and c subunits. Essentially complete cross-linking of the gamma, epsilon, and c subunits was achieved by using CuCl(2) to induce oxidation. This fixing of the three subunits together had no significant effect on ATP hydrolysis, proton translocation, or ATP synthesis, and each of these functions retained inhibitor sensitivity. These results unequivocally place the c subunit oligomer in the rotor part of this molecular machine.

Human complex I defects can be resolved by monoclonal antibody analysis into distinct subunit assembly patterns.

Complex I defects are one of the most frequent causes of mitochondrial respiratory chain disorders. Therefore, it is important to find new approaches for detecting and characterizing Complex I deficiencies. In this paper, we introduce a new set of monoclonal antibodies that react with 39-, 30-, 20-, 18-, 15-, and 8-kDa subunits of Complex I. These antibodies are shown to aid in diagnosis of Complex I deficiencies and add understanding to the genotype-phenotype relationships of different mutations. A total of 11 different patients were examined. Four patients had undefined Complex I defects, whereas the other patients had defects in NDUFV1, NDUFS2 (two patients), NDUFS4 (two patients), NDUFS7, and NDUFS8. We show here that Western blotting with these antibodies, particularly when used in conjunction with sucrose gradient studies and enzymatic activity measurements, helps distinguish catalytic versus assembly defects and further distinguishes between mutations in different subunits. Furthermore, different mutations in the same gene are shown to give very similar subunit profiles, and we show that one of the patients is a good candidate for having a defect in a Complex I assembly factor.

Antibody-based approaches to diagnosis and characterization of oxidative phosphorylation diseases.

Mitochondrial disorders caused by defects in oxidative phosphorylation function are difficult to diagnose. Here we review the emerging use of antibody-based approaches for this diagnosis. Novel methods involving immunohistochemistry and immunocapture of defective enzymes for characterization are described that add to the arsenal of approaches available.

The T9176G mutation of human mtDNA gives a fully assembled but inactive ATP synthase when modeled in Escherichia coli.

A new mutation in human F(1)F(0) ATPase6, T9176G, which changes Leu 217 to an Arg, has been described in two siblings with Leigh syndrome [Carrozzo et al. (2000) Neurology, in press]. This mutation was modeled in Escherichia coli by changing Leu 259 (the equivalent residue) to Arg and the properties of the altered ECF(1)F(0) were compared to those of previously characterized ATPase6 mutants also modeled in the E. coli enzyme. The L259R change produced a fully assembled ECF(1)F(0) which had no significant ATP hydrolysis, ATP synthesis or proton pumping functions. This is very different from previously described human ATPase6 mutations. The presence of Arg at position 259 in subunit a did not make membranes permeable to protons. We conclude that the mutation inhibits functioning by blocking the rotary motor action of the enzyme.

Dynamics of the mitochondrial reticulum in live cells using Fourier imaging correlation spectroscopy and digital video microscopy.

We report detailed studies of the dynamics of the mitochondrial reticulum in live cells using two independent experimental techniques: Fourier imaging correlation spectroscopy and digital video fluorescence microscopy. When both methods are used to study the same system, it is possible to directly compare measurements of preaveraged statistical dynamical quantities with their microscopic counterparts. This approach allows the underlying mechanism of the observed rates to be determined. Our results indicate that the dynamics of the reticulum structure is composed of two independent contributions, each important on very different time and length scales. During short time intervals (1-15 sec), local regions of the reticulum primarily undergo constrained thermally activated motion. During long time intervals (>15 sec), local regions of the reticulum undergo long-range "jump" motions that are associated with the action of cytoskeletal filaments. Although the frequency of the jumps depend on the physiological state of the cells, the average jump distance ( approximately 0.8 microm) is unaffected by metabolic activity. During short time intervals, the dynamics appear to be spatially heterogeneous, whereas the cumulative effect of the infrequent jumps leads to the appearance of diffusive motion in the limit of long time intervals.

The epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases. Structure, arrangement, and role of the epsilon subunit in energy coupling within the complex.

Recent studies show that the epsilon subunit of bacterial and chloroplast F(1)F(0) ATPases is a component of the central stalk that links the F(1) and F(0) parts. This subunit interacts with alpha, beta and gamma subunits of F(1) and the c subunit ring of F(0). Along with the gamma subunit, epsilon is a part of the rotor that couples events at the three catalytic sites sequentially with proton translocation through the F(0) part. Structural data on the epsilon subunit when separated from the complex and in situ are reviewed, and the functioning of this polypeptide in coupling within the ATP synthase is considered.

Mitochondrial DNA depletion causes morphological changes in the mitochondrial reticulum of cultured human cells.

Depletion of mitochondrial DNA (mtDNA) causes defects in respiratory activity and energy production. Recent studies have shown mitochondria to exist primarily as reticular networks, having tubular cristae. Using fluorescence microscopy and transmission electron microscopy, we have examined mitochondrial morphology and interior structure in wildtype and mtDNA-depleted rho0 human fibroblasts and 143B osteosarcoma cell lines. MtDNA depletion results in compromise of the mitochondrial continuum and causes a reduction in amount of cristal membranes, often prompting the remaining cristae to adopt a circular appearance in the mitochondrial interior. These changes emphasize the tight relationship between mitochondrial structure and function.

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact.

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.

Localization of the delta subunit in the Escherichia coli F(1)F(0)-ATPsynthase by immuno electron microscopy: the delta subunit binds on top of the F(1).

The binding site of the delta subunit in the F(1)F(0)-ATPsynthase from Escherichia coli has been determined by electron microscopy of negatively stained, antibody-decorated enzyme molecules. The images show that the antibody is bound at the very top of the F(1) domain indicating that at least part of delta is bound in the dimple formed by the N termini of the alpha and beta subunits. The data may explain why there is only one binding site for delta on the F(1) despite there being three identical alphabeta pairs. The finding also implies that the b subunits of the F(0) have to extend all the way from the membrane surface to the very top of the F(1) domain to make contact with the delta subunit.

A novel mtDNA mutation in the ATPase6 gene studied by E. coli modeling.

This study aimed to understand the pathogenesis of a new mtDNA-related etiology of Leigh syndrome. We identified the T9176G mutation as the molecular basis of Leigh syndrome in a child and looked for alterations in cellular ATP production. We then modeled the new mtDNA mutation in E. coli and analyzed ATP synthesis, hydrolysis, and the ability of the mutated enzyme to pump protons. Our results suggest that the T9176G change results in a novel, fully assembled enzyme which inhibits the holoenzyme probably by blocking the proton pathway.

Structure, functioning, and assembly of the ATP synthase in cells from patients with the T8993G mitochondrial DNA mutation. Comparison with the enzyme in Rho(0) cells completely lacking mtdna.

The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho(0) cells derived from this cell line as controls. ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants. Both activities were highly oligomycin-sensitive. The levels of F(1)F(0) were close to normal, and the enzyme was stable. It is concluded that the loss of ATP synthesis is because of disruption of the proton translocation step within the F(0) part. This is supported by membrane potential measurements using the dye JC-1. Cells with a 91% mutation load grew well and showed only a 25% loss in ATP synthesis. This much reduced effect for only a 9% difference in mutation load mirrors the reduced pathogenicity in patients. F(1)F(0) has been purified for the first time from human cell lines. A partial complex was obtained from Rho(0) cells containing the F(1) subunits associated with several stalk, as well as F(0) subunits, including oligomycin sensitivity conferring protein, b, and c subunits. This partial complex no longer binds inhibitor protein.

Cross-linking and electron microscopy studies of the structure and functioning of the Escherichia coli ATP synthase.

ATP synthase, also called F(1)F(o)-ATPase, catalyzes the synthesis of ATP during oxidative phosphorylation. The enzyme is reversible and is able to use ATP to drive a proton gradient for transport purposes. Our work has focused on the enzyme from Escherichia coli (ECF(1)F(o)). We have used a combination of methods to study this enzyme, including electron microscopy and chemical cross-linking. The utility of these two approaches in particular, and the important insights they give into the structure and mechanism of the ATP synthase, are reviewed.

Structural features of the gamma subunit of the Escherichia coli F(1) ATPase revealed by a 4.4-A resolution map obtained by x-ray crystallography.

The F(1) part of the F(1)F(O) ATP synthase from Escherichia coli has been crystallized and its structure determined to 4.4-A resolution by using molecular replacement based on the structure of the beef-heart mitochondrial enzyme. The bacterial F(1) consists of five subunits with stoichiometry alpha(3), beta(3), gamma, delta, and epsilon. delta was removed before crystallization. In agreement with the structure of the beef-heart mitochondrial enzyme, although not that from rat liver, the present study suggests that the alpha and beta subunits are arranged in a hexagonal barrel but depart from exact 3-fold symmetry. In the structures of both beef heart and rat-liver mitochondrial F(1), less than half of the structure of the gamma subunit was seen because of presumed disorder in the crystals. The present electron-density map includes a number of rod-shaped features which appear to correspond to additional alpha-helical regions within the gamma subunit. These suggest that the gamma subunit traverses the full length of the stalk that links the F(1) and F(O) parts and makes significant contacts with the c subunit ring of F(O).