Lipid, detergent, and coomassie blue G-250 affect the migration of small membrane proteins in blue native gels:Mitochondrial carriers migrate as monomers not dimers

Background: Mitochondrial carriers were thought to be dimeric based on their migration in blue native gels.  Results: The high molecular mass species observed in blue native gels are composed of protein monomers, detergent, lipid, and Coomassie stain.  Conclusion: The mitochondrial carriers are monomeric not dimeric.  Significance: The apparent mass of small membrane proteins in blue native gels requires significant correction

The molecular features of uncoupling protein 1 support a conventional mitochondrial carrier-like mechanism.

Uncoupling protein 1 (UCP1) is an integral membrane protein found in the mitochondrial inner membrane of brown adipose tissue, and facilitates the process of non-shivering thermogenesis in mammals. Its activation by fatty acids, which overcomes its inhibition by purine nucleotides, leads to an increase in the proton conductance of the inner mitochondrial membrane, short-circuiting the mitochondrion to produce heat rather than ATP. Despite 40 years of intense research, the underlying molecular mechanism of UCP1 is still under debate. The protein belongs to the mitochondrial carrier family of transporters, which have recently been shown to utilise a domain-based alternating-access mechanism, cycling between a cytoplasmic and matrix state to transport metabolites across the inner membrane. Here, we review the protein properties of UCP1 and compare them to those of mitochondrial carriers. UCP1 has the same structural fold as other mitochondrial carriers and, in contrast to past claims, is a monomer, binding one purine nucleotide and three cardiolipin molecules tightly. The protein has a single substrate binding site, which is similar to those of the dicarboxylate and oxoglutarate carriers, but also contains a proton binding site and several hydrophobic residues. As found in other mitochondrial carriers, UCP1 has two conserved salt bridge networks on either side of the central cavity, which regulate access to the substrate binding site in an alternating way. The conserved domain structures and mobile inter-domain interfaces are consistent with an alternating access mechanism too. In conclusion, UCP1 has retained all of the key features of mitochondrial carriers, indicating that it operates by a conventional carrier-like mechanism

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria

Modelling the free energy profile of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier catalyses the equimolar exchange of adenosine di- and tri-phosphates. It operates by an alternating access mechanism in which a single substrate-binding site is made available either to the mitochondrial matrix or the intermembrane space through conformational changes. These changes are prevented in the absence of substrate by a large energy barrier due to the need for sequential disruption and formation of a matrix and cytoplasmic salt bridge network that are located on either side of the central cavity. In analogy to enzyme catalysis, substrate lowers the energy barrier by binding tighter in the intermediate state. Here we provide an in-silico kinetic model that captures the free energy profile of these conformational changes and treats the carrier as a nanomachine moving stochastically from the matrix to cytoplasmic conformation under the influence of thermal energy. The model reproduces the dependency of experimentally determined kcat and KM values on the cytoplasmic network strength with good quantitative accuracy, implying that it captures the transport mechanism and can provide a framework to understand the structure-function relationships of this class of transporter. The results show that maximum transport occurs when the interaction energies of the cytoplasmic network, matrix network and substrate binding are approximately equal such that the energy barrier is minimized. Consequently, the model predicts that there will be other interactions in addition to those of the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier

The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier.

Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT

Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier

Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway

Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans

Supported by Medical Research Council program grant MRC G1001340 (to M Madhani, M Feelisch, and MP Frenneaux). We thank Lesley Cheyne for their contributions to the present study. The authors’ responsibilities were as follows—VSV, M Madhani, JDH, MF, DD, MPF: designed the research; MN, NEKP, KS, BLL, M Minnion, BOF, DV, DC-T, PGC: conducted the research; DV: provided essential materials; MN, NEKP, M Minnion, BOF, DC-T, MF, PGC: analyzed the data; MN, NEKP, PGC, MPF: wrote the paper; MPF: had primary responsibility for the final manuscript; and all authors: read and approved the final manuscript. None of the authors reported a conflict of interest related to the study.Peer reviewedPublisher PD

Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1

The mPTP (mitochondrial permeability transition pore) is a non-specific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT

Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers

Background: Methods for rapid assessment of interactions of small molecules with membrane proteins in detergent are lacking.  Results: Thermostability measurements of mitochondrial transporters display informative trends about detergent, lipid, substrate, and inhibitor interactions.  Conclusion: Mechanistic insights are obtained by studying the thermostability of mitochondrial transporters.  Significance: Information about the nature of compound interactions with membrane proteins can be obtained rapidly