The rotor in the membrane of the ATP synthase and relatives.

In recent years, structural information on the F(1) sector of the ATP synthase has provided an insight into the molecular mechanism of ATP catalysis. The structure strongly supports the proposal that the ATP synthase works as a rotary molecular motor. Insights into the membrane domain have just started to emerge but more detailed structural information is needed if the molecular mechanism of proton translocation coupled to ATP synthesis is to be understood. This review will focus mainly on the ion translocating rotor in the membrane domain of the F-type ATPase, and the related vacuolar and archaeal relatives.

The mitochondrial uncoupling protein UCP1: a gated pore.

The uncoupling protein UCP1 is a member of a superfamily of homologous proteins formed by the mitochondrial metabolite transporters. Although they act in vivo as carriers, under specific experimental conditions some of these transporters have been shown to behave as channels. This dual transport operation suggests that these carriers are likely to be formed by two differentiated functional and structural domains. The kinetic model termed "single binding center gated pore" is well suited to understand the behaviour of these carriers. It proposes that in the protein core there must exist a hydrophilic translocation pore whose access is controlled by gates. It is highly likely that the hydrophilic channel is formed by the transmembrane alpha-helices and that loops contribute to the formation of the gates. UCP1 is regulated physiologically by fatty acids and purine nucleotides. Nucleotides maintain the proton conductance inhibited while fatty acids act as cytosolic second messengers of noradrenaline to active UCP1. Based on photoaffinity labeling and mutagenesis data, we propose a structural model for the localization of the binding site. The nucleotide enters through a gate in the cytosolic side and binds deep inside the protein. The three matrix loops contribute to the formation of a hydrophobic binding pocket that would accommodate the purine moiety. Three arginine residues (in helices II, IV, and VI) would interact with the phosphate groups. His214 and Glu190 have been involved in the pH regulation of the nucleotide binding but because they are on the cytosolic side of the protein, we propose that their state of protonation will determine the access of the nucleotide to the binding center.

The rotary mechanism of ATP synthase.

Since the chemiosmotic theory was proposed by Peter Mitchell in the 1960s, a major objective has been to elucidate the mechanism of coupling of the transmembrane proton motive force, created by respiration or photosynthesis, to the synthesis of ATP from ADP and inorganic phosphate. Recently, significant progress has been made towards establishing the complete structure of ATP synthase and revealing its mechanism. The X-ray structure of the F(1) catalytic domain has been completed and an electron density map of the F(1)-c(10) subcomplex has provided a glimpse of the motor in the membrane domain. Direct microscopic observation of rotation has been extended to F(1)-ATPase and F(1)F(o)-ATPase complexes.

Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase.

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F(1)F(o) ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.

Dimerization of bovine F1-ATPase by binding the inhibitor protein, IF1.

In mitochondria, the hydrolytic activity of ATP synthase is regulated by a natural inhibitor protein, IF(1). The binding of IF(1) to ATP synthase depends on pH values, and below neutrality, IF(1) forms a stable complex with the enzyme. Bovine IF(1) has two oligomeric states, dimer and tetramer, depending on pH values. At pH 6.5, where it is active, IF(1) dimerizes by formation of an antiparallel alpha-helical coiled-coil in its C-terminal region. This arrangement places the inhibitory N-terminal regions in opposition, implying that active dimeric IF(1) can bind two F(1) domains simultaneously. Evidence of dimerization of F(1)-ATPase by binding to IF(1) is provided by gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. At present, it is not known whether IF(1) can bring about the dimerization of the F(1)F(0)-ATPase complex.

Activation of the uncoupling protein by fatty acids is modulated by mutations in the C-terminal region of the protein.

The transport properties of the uncoupling protein (UCP) from brown adipose tissue have been studied in mutants where Cys304 has been replaced by either Gly, Ala, Ser, Thr, Ile or Trp. This position is only two residues away from the C-terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane. Mutant proteins have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2-bromopalmitate. Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty acids and nucleotides on the proton permeability and NADH oxidation rate. It is revealed that substitution of Cys304 by non-charged residues alters the response of UCP to fatty acids. The most effective substitution is Cys for Gly since it greatly enhances the sensitivity to palmitate, decreasing threefold the concentration required for half-maximal stimulation of respiration. The opposite extreme is the substitution by Ala which increases twofold the half-maximal concentration. We conclude that the C-terminal region participates in the fatty acid regulation of UCP activity. The observed correlation between yeast growth rates in the presence of bromopalmitate and the calculated activation constants for respiration in isolated mitochondria validates growth analysis as a method to screen the in situ activity of UCP mutants.

A sequence related to a DNA recognition element is essential for the inhibition by nucleotides of proton transport through the mitochondrial uncoupling protein.

The uncoupling protein (UCP) is uniquely expressed in brown adipose tissue, which is a thermogenic organ of mammals. The UCP uncouples mitochondrial respiration from ATP production by introducing a proton conducting pathway through the mitochondrial inner membrane. The activity of the UCP is regulated: nucleotide binding to the UCP inhibits proton conductance whereas free fatty acids increase it. The similarities between the UCP, the ADP/ATP carrier and the DNA recognition element found in the DNA binding domain of the estrogen receptor suggested that these proteins could share common features in their respective interactions with free nucleotides or DNA, and thus defined a putative 'nucleotide recognition element' in the UCP. This article provides demonstration of the validity of this hypothesis. The putative nucleotide recognition element corresponding to the amino acids 261-269 of the UCP was gradually destroyed, and these mutant proteins were expressed in yeast. Flow cytometry, measuring the mitochondrial membrane potential in vivo, showed increased uncoupling activities of these mutant proteins, and was corroborated with studies with isolated mitochondria. The deletion of the three amino acids Phe267, Lys268 and Gly269, resulted in a mutant where proton leak could be activated by fatty acids but not inhibited by nucleotides.

Cysteine residues are not essential for uncoupling protein function.

The uncoupling protein (UCP) of brown adipose tissue is a regulated proton carrier which allows uncoupling of mitochondrial respiration from ATP synthesis and, therefore, dissipation of metabolic energy as heat. In this article we demonstrate that, when UCP is expressed in Saccharomyces cerevisiae, it retains all its functional properties: proton and chloride transport, high-affinity binding of nucleotides and regulation of proton conductance by nucleotides and fatty acids. Site-directed mutagenesis demonstrates that sequential replacement by serine of cysteine residues in the UCP does not affect either its uncoupling activity or its regulation by nucleotides and fatty acids, and therefore establishes that none of the seven cysteine residues present in the wild-type UCP is critical for its activity. These data indicate that transport models involving essential thiol groups can be discounted and that chemical modification data require critical re-evaluation.

Effect of hydrophobic sulphydryl reagents on the uncoupling protein and inner-membrane anion channel of brown-adipose-tissue mitochondria.

The effects of three sulphydryl reagents of differing hydrophobicity (N-ethylmaleimide, N-benzylmaleimide and N,N'-o-phenylenedimaleimide) on ion permeation through the inner membrane of brown-adipose-tissue mitochondria are investigated. GDP-sensitive permeation of chloride and protons (hydroxyl ions) through the uncoupling protein is increased exponentially with time by all three reagents. With increasing hydrophobicity of the reagents, modification is enhanced and an initial inhibited state becomes apparent. Results are interpreted in terms of a two-stage modification via a non-transporting intermediate, which does not bind GDP, to a final highly conducting product. The reagents also react with a hydrophilic sulphydryl group on an independent protein to induce a GDP-insensitive pathway which allows chloride, phosphate and sulphate to cross the membrane. The use of different sulphydryl reagents allows the two pathways to be clearly distinguished.