How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

Import of ß-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the ß-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the ß-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data.

A conserved mitochondrial ATP-binding cassette transporter exports glutathione polysulfide for cytosolic metal cofactor assembly.

An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe(2+) alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.

Peroxisomal ABC transporters: functions and mechanism.

Peroxisomes are arguably the most biochemically versatile of all eukaryotic organelles. Their metabolic functions vary between different organisms, between different tissue types of the same organism and even between different developmental stages or in response to changed environmental conditions. New functions for peroxisomes are still being discovered and their importance is underscored by the severe phenotypes that can arise as a result of peroxisome dysfunction. The ß-oxidation pathway is central to peroxisomal metabolism, but the substrates processed are very diverse, reflecting the diversity of peroxisomes across species. Substrates for ß-oxidation enter peroxisomes via ATP-binding cassette (ABC) transporters of subfamily D; (ABCD) and are activated by specific acyl CoA synthetases for further metabolism. Humans have three peroxisomal ABCD family members, which are half transporters that homodimerize and have distinct but partially overlapping substrate specificity; Saccharomyces cerevisiae has two half transporters that heterodimerize and plants have a single peroxisomal ABC transporter that is a fused heterodimer and which appears to be the single entry point into peroxisomes for a very wide variety of ß-oxidation substrates. Our studies suggest that the Arabidopsis peroxisomal ABC transporter AtABCD1 accepts acyl CoA substrates, cleaves them before or during transport followed by reactivation by peroxisomal synthetases. We propose that this is a general mechanism to provide specificity to this class of transporters and by which amphipathic compounds are moved across peroxisome membranes.

Structures and functions of mitochondrial ABC transporters.

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters.

The multidrug transporter LmrP protein mediates selective calcium efflux.

LmrP is a major facilitator superfamily multidrug transporter from Lactococcus lactis that mediates the efflux of cationic amphiphilic substrates from the cell in a proton-motive force-dependent fashion. Interestingly, motif searches and docking studies suggested the presence of a putative Ca(2+)-binding site close to the interface between the two halves of inward facing LmrP. Binding experiments with radioactive (45)Ca(2+) demonstrated the presence of a high affinity Ca(2+)-binding site in purified LmrP, with an apparent K(d) of 7.2 µm, which is selective for Ca(2+) and Ba(2+) but not for Mn(2+), Mg(2+), or Co(2+). Consistent with our structure model and analogous to crystal structures of EF hand Ca(2+)-binding proteins, two carboxylates (Asp-235 and Glu-327) were found to be critical for (45)Ca(2+) binding. Using (45)Ca(2+) and a fluorescent Ca(2+)-selective probe, calcium transport measurements in intact cells, inside-out membrane vesicles, and proteoliposomes containing functionally reconstituted purified protein provided strong evidence for active efflux of Ca(2+) by LmrP with an apparent K(t) of 8.6 µm via electrogenic exchange with three or more protons. These observations demonstrate for the first time that LmrP mediates selective calcium/proton antiport and raise interesting questions about the functional and physiological links between this reaction and that of multidrug transport.

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling.

The multidrug major facilitator superfamily transporter LmrP from Lactococcus lactis mediates protonmotive-force dependent efflux of amphiphilic ligands from the cell. We compared the role of membrane-embedded carboxylates in transport and binding of divalent cationic propidium and monovalent cationic ethidium. D235N, E327Q, and D142N replacements each resulted in loss of electrogenicity in the propidium efflux reaction, pointing to electrogenic 3H(+)/propidium(2+) antiport. During ethidium efflux, single D142N and D235N replacements resulted in apparent loss of electrogenicity, whereas the E327Q substitution did not affect the energetics, consistent with electrogenic 2H(+)/ethidium(+) antiport. Different roles of carboxylates were also observed in fluorescence anisotropy-based ligand-binding assays. Whereas D235 and E327 were both involved in propidium binding, the loss of one of these carboxylates could be compensated for by the other in ethidium binding. The D142N replacement did not affect the binding of either ligand. These data point to the presence of a dedicated proton binding site containing D142, and a flexible proton/ligand binding site containing D235 and E327, the contributions to proton and ligand binding of which depend on the chemical structure of the bound ligand. Our findings provide the first evidence that multidrug transport by secondary-active transporters can be associated with variable ion coupling.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose.

The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of ß-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed.

Iron cofactor assembly in plants.

Iron is an essential element for all photosynthetic organisms. The biological use of this transition metal is as an enzyme cofactor, predominantly in electron transfer and catalysis. The main forms of iron cofactor are, in order of decreasing abundance, iron-sulfur clusters, heme, and di-iron or mononuclear iron, with a wide functional range. In plants and algae, iron-sulfur cluster assembly pathways of bacterial origin are localized in the mitochondria and plastids, where there is a high demand for these cofactors. A third iron-sulfur cluster assembly pathway is present in the cytosol that depends on the mitochondria but not on plastid assembly proteins. The biosynthesis of heme takes place mainly in the plastids. The importance of iron-sulfur cofactors beyond photosynthesis and respiration has become evident with recent discoveries of novel iron-sulfur proteins involved in epigenetics and DNA metabolism. In addition, increased understanding of intracellular iron trafficking is opening up research into how iron is distributed between iron cofactor assembly pathways and how this distribution is regulated.