Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation.

F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1.

Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria.

The biogenesis of bacterial c-type cytochromes generally involves many gene products--some of which may also have roles in other processes--and their interaction with the disulphide-bond-forming system of the bacterial periplasm. However, in some bacteria a simpler process appears to operate that might be related to the formation of c-type cytochromes in thylakoids of photosynthetic cells. The corresponding process in fungal mitochondria is distinct.

Biological nano motor, ATP synthase F(o)F(1): from catalysis to gammaepsilonc(10-12) subunit assembly rotation.

Proton translocating ATPase (ATP synthase), a chemiosmotic enzyme, synthesizes ATP from ADP and phosphate coupling with the electrochemical ion gradient across the membrane. This enzyme has been studied extensively by combined genetic, biochemical and biophysical approaches. Such studies revealed a unique mechanism which transforms an electrochemical ion gradient into chemical energy through the rotation of a subunit assembly. Thus, this enzyme can be defined as a nano motor capable of coupling a chemical reaction and ion translocation, or more simply, as a protein complex carrying out rotational catalysis. In this article, we briefly discuss our recent work, emphasizing the rotation of subunit assembly (gammaepsilonc(10-12)) which is formed from peripheral and intrinsic membrane subunits.

Synthase (H(+) ATPase): coupling between catalysis, mechanical work, and proton translocation.

Coupling with electrochemical proton gradient, ATP synthase (F(0)F(1)) synthesizes ATP from ADP and phosphate. Mutational studies on high-resolution structure have been useful in understanding this complicated membrane enzyme. We discuss mainly the mechanism of catalysis in the beta subunit of F(1) sector and roles of the gamma subunit in energy coupling. The gamma-subunit rotation during catalysis is also discussed.

Synthesis of holo Paracoccus denitrificans cytochrome c550 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c552 does not. Implications for c-type cytochrome biogenesis.

Expression from a plasmid of the complete gene, including the codons for the N-terminal periplasmic targeting signal, for cytochrome c550 of Paracoccus denitrificans led to the formation of the holo protein in the periplasms of both P. denitrificans and Escherichia coli. Expression of the gene from which the region coding for the targeting signal had been specifically deleted resulted in formation of apo-protein in the cytoplasms of both organisms. These findings are consistent with haem attachment occurring in the periplasm. In contrast, the formation of holo cytochrome c552 from Hydrogenobacter thermophilus following expression of the gene lacking the periplasmic targeting sequence in either P. denitrificans or E. coli is attributed to spontaneous cytoplasmic attachment of haem to the thermostable protein.

Specific thiol compounds complement deficiency in c-type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane-bound disulphide isomerase-like protein.

Escherichia coli JCB606 carries a mutation in the dipZ gene, known to code for a disulphide isomerase-like protein, with the consequence that holo forms of neither exogenous nor endogenous c-type cytochromes are synthesised. This failure has been overcome by adding compounds containing thiol groups to the growth medium. Only L-cysteine and 2-mercaptoethane sulphonic acid were effective, suggesting a (stereo)specific binding site that could be occupied by these compounds in the absence of the catalytic domain of DipZ.

Mutants of Escherichia coli lacking disulphide oxidoreductases DsbA and DsbB cannot synthesise an exogenous monohaem c-type cytochrome except in the presence of disulphide compounds.

Absence through mutation of two proteins involved in periplasmic disulphide bond formation, DsbA and DsbB, results in failure of anaerobically grown Escherichia coli to synthesise the holo forms of either its endogenous c-type cytochrome nitrite reductase or exogenous cytochrome c550 from Paracoccus denitrificans. The synthesis of both cytochromes can be restored to the mutants by inclusion in the growth media of compounds containing disulphide bonds, e.g., the oxidised form of glutathione. The results suggest that the attachment of haem to the CXXCH motif of a periplasmic c-type cytochrome may be preceeded by the formation of one or more intra- or intermolecular disulphide bonds involving the cysteine residues of this motif.

A mutation blocking the formation of membrane or periplasmic endogenous and exogenous c-type cytochromes in Escherichia coli permits the cytoplasmic formation of Hydrogenobacter thermophilus holo cytochrome c552.

A mutant of Escherichia coli, JCB606, shown to be pleiotropically deficient in the formation of endogenous membrane and periplasmic c-type cytochromes, synthesised the apo form of the exogenous cytochrome c550 from Paracoccus denitrificans, but not the holo form. In contrast, a cytoplasmically located holo form of Hydrogenobacter thermophilus cytochrome c552 was found in E. coli JCB606. These findings support the proposition that the formation of the cytoplasmic H. thermophilus cytochrome c552 in E. coli does not involve the physiological pathway of c-type cytochrome biosynthesis in E. coli and that the haem insertion may be catalysed.

Identification of the copper chaperone, CUC-1, in Caenorhabditis elegans: tissue specific co-expression with the copper transporting ATPase, CUA-1.

A cDNA encoding a putative copper chaperone protein, CUC-1, was cloned from Caenorhabditis elegans. CUC-1 had the characteristic motifs of MTCXXC and KKTGK, and showed 49.3 and 39.1% sequence identity with yeast Atx1p and human HAH1, respectively. Expression of CUC-1 cDNA complemented a null atx1 mutant, the yeast copper chaperone gene, thus demonstrating that CUC-1 is a functional copper chaperone. Studies with transgenic worms indicated that cuc-1 and cua-1, which encodes the copper transporting ATPase, are expressed together in intestinal cells of adult and hypodermal cells in the larvae. cua-1 was also expressed in pharyngeal muscle but cuc-1 was not. These results suggest that CUC-1 and CUA-1 constitute a copper trafficking pathway similar to the yeast counterparts in intestinal and hypodermal cells, and CUA-1 may have a different function in pharyngeal muscle.

Analysis of functional domains of Wilson disease protein (ATP7B) in Saccharomyces cerevisiae.

Wilson disease is a genetic disorder of copper metabolism characterized by the toxic accumulation of copper in the liver. The ATP7B gene, which encodes a copper transporting P-type ATPase, is defective in patients with Wilson disease. To investigate the function of ATP7B, wild type or mutated ATP7B cDNA was introduced into a yeast strain lacking the CCC2 gene (delta ccc2), the yeast homologue of ATP7B. Wild type and the H1069Q mutant could rescue delta ccc2, however, the N1270S mutant could not, reflecting phenotypic variability of Wilson disease. In addition, the mutant containing only the sixth copper binding domain could rescue delta ccc2, indicating its functional importance.

A biological molecular motor, proton-translocating ATP synthase: multidisciplinary approach for a unique membrane enzyme.

Proton-translocating ATP synthase (F(o)F(1)) synthesizes ATP from ADP and phosphate, coupled with an electrochemical proton gradient across the biological membrane. It has been established that the rotation of a subunit assembly is an essential feature of the enzyme mechanism and that F(o)F(1) can be regarded as a molecular motor. Thus, experimentally, in the reverse direction (ATP hydrolysis), the chemical reaction drives the rotation of a gammaepsilonc(10-14) subunit assembly followed by proton translocation. We discuss our very recent results regarding subunit rotation in Escherichia coli F(o)F(1) with a combined biophysical and mutational approach.

Caenorhabditis elegans cDNA for a Menkes/Wilson disease gene homologue and its function in a yeast CCC2 gene deletion mutant.

The full-length cDNA coding for a putative copper transporting P-type ATPase (Cu2+-ATPase) was cloned from Caenorhabditis elegans. The putative Cu2+-ATPase is a 1,238-amino acid protein, and highly homologous to the Menkes and Wilson disease gene products mutations of which are responsible for human defects of copper metabolism. The Saccharomyces cerevisiae mutant with a disrupted CCC2 gene (yeast Menkes/Wilson disease gene homologue) was rescued by the cDNA for the C. elegans Cu2+-ATPase but not by the cDNA with an Asp-786 (an invariant phosphorylation site) to Asn mutation, suggesting that the C. elegans Cu2+-ATPase functions as a copper transporter in yeast. The expressed C. elegans protein was detected in yeast vacuolar membranes by immunofluorescence microscopy. The yeast expression system may facilitate further studies on copper transporting P-type ATPases.

Caenorhabditis elegans senses protons through amphid chemosensory neurons: proton signals elicit avoidance behavior.

Acidic pH is known to cause pain sensation through nociceptive neurons as well as taste transduction in mammals. Caenorhabditis elegans avoids an acidic environment (pH lower than approximately 4.0) formed by organic or inorganic acids. This avoidance behavior was dependent on multiple amphid chemosensory neurons, and inhibited by a mutation of capsaicin receptor homologue, and by the addition of amiloride and ruthenium red (inhibitors of proton-gated Na+ channels and capsaicin receptors, respectively). These results indicate that C. elegans recognizes protons as a nociceptive stimulus, through multiple neurons, which elicits avoidance behavior. It is of special interest that a system similar to that of mammalian signal transduction is responsible for this nematode's acid avoidance.

Sensing of cadmium and copper ions by externally exposed ADL, ASE, and ASH neurons elicits avoidance response in Caenorhabditis elegans.

We developed a quantitative assay for Caenorhabditis elegans avoidance behavior. This was then used to demonstrate that the worm moved away from toxic concentrations of Cd2+ and Cu2+, but not Ni2+, all ions that prevented development from larval to adult stages. Mutants that have structural defects in ciliated neurons (che-2 and osm-3) as well as worms with three laser-operated neurons (ADL, ASE, and ASH), showed no avoidance behavior from Cd2+ and Cu2+. These results suggest that the avoidance from Cd2+ and Cu2+ are mediated through multiple neural pathways including ADL, ASE, and ASH neurons. We hypothesize that the three sensing neurons provide increased accuracy of the sensory response and a survival advantage in the natural environment of the worm.

Alteration of haem-attachment and signal-cleavage sites for Paracoccus denitrificans cytochrome C550 probes pathway of c-type cytochrome biogenesis in Escherichia coli.

Paracoccus denitrificans cytochrome C550 is expressed as a periplasmic holo-protein in Escherichia coli; amino acid substitutions of cysteine residues in the haem-binding motif (Cys-X-X-Cys-His), either together or singly, prevented covalent attachment of haem but not polypeptide translocation into the periplasm. When the three alanine residues at positions -3 to -1 in the native signal-cleavage site were deleted, or alanine at -1 was changed to glutamine, signal cleavage was at alternative sites (after only ten residues in the latter case), but haem attachment still occurred. When the same three alanines were changed to Asp-Glu-Asp, a membrane-associated apo product that had retained the complete signal sequence was detected. These and other results presented here indicate that (i) haem attachment is not required for the apo-cytochrome C550 export to the periplasm; (ii) haem cannot attach to apo-cytochrome C550 when attached to the cytoplasmic membrane, suggesting that signal-sequence cleavage precedes periplasmic haem attachment, which can occur at as few as six residues from the mature N-terminus; and (iii) two cysteines are required for haem attachment, possibly because a disulphide bond is an intermediate. The gene for Saccharomyces cerevisiae mitochondrial iso-1-cytochrome c was expressed as a holo-protein in E. coli when fused with the signal sequence plus the first 10 residues of the mature cytochrome C550, indicating that the E. coli cellular apparatus for the c-type cytochrome biogenesis has a broad substrate specificity.

Selected mutations in a mesophilic cytochrome c confer the stability of a thermophilic counterpart.

Mesophilic cytochrome c(551) of Pseudomonas aeruginosa (PA c(551)) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c(552) through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c(551) could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c(552), there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.

Regulation and sequence of the structural gene for cytochrome c552 from Escherichia coli: not a hexahaem but a 50 kDa tetrahaem nitrite reductase.

The structural gene, nrfA, for cytochrome c552, which is the terminal reductase of the formate-dependent pathway for nitrite reduction to ammonia, has been located at co-ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetrahaem c-type cytochrome with a predicted M(r) for the unprocessed product of 53,788. Cleavage of the putative signal peptide at Ala-26 would result in a mature, periplasmic cytochrome of M(r) 50,580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS-polyacrylamide gels for covalently bound haem. This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome c552 by difference spectroscopy. Similar cytochromes were detected in five other E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in-phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In-phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping. Expression from the FNR-dependent nrfA promoter was almost totally repressed during aerobic growth, partially induced during anaerobic growth in the absence of nitrite or in the presence of nitrate, but fully induced only during anaerobic growth in the presence of nitrite. No nitrate repression was detected in a narL mutant, but nitrite induction was unaffected, indicating that the nitrite-sensing mechanism is independent of the NarL protein. Expression from the nrfA promoter was subject to glucose repression but regulation was independent of the CRP-cAMP complex.

Cytochrome c550 expression in Paracoccus denitrificans strongly depends on growth condition: identification of promoter region for cycA by transcription start analysis.

The periplasmic cytochrome c550 content of Paracoccus denitrificans has been shown by immunological detection to be strongly dependent on the mode of growth. Cells grown under anaerobic, denitrifying conditions or methylotrophically in the presence of oxygen contained substantially more cytochrome c550 than cells grown aerobically on multicarbon substrates. A similar pattern was observed when expression of the cycA gene (encoding cytochrome c550), was monitored using an Escherichia coli alkaline phosphatase gene (phoA) fusion as a reporter of cycA promoter activity. The increase in cycA expression observed during growth on C1 substrates was substantially diminished if succinate was also present. These results reveal that expression of cycA is subject to multiple regulatory controls and suggest that cytochrome c550 has a general role in electron transfer to periplasmic reductases required for anaerobic denitrifying growth and from dehydrogenases required for aerobic growth on C1 compounds. Two major transcriptional initiation start points for the cycA gene have been identified.

Essential Cys-Pro-Cys motif of Caenorhabditis elegans copper transport ATPase.

Caenorhabditis elegans putative copper ATPase (CUA-1) had been functionally expressed in a yeast delta ccc2 mutant (copper ATPase gene disruptant). We found that CUA-1 with Cys-Pro-Cys to Cys-Pro-Ala mutation could not rescue the yeast delta ccc2 mutant, suggesting that the carboxyl terminal cysteine residue in the conserved Cys-Pro-Cys motif is essential for copper transport.