Lateral roots originate deep within the parental root from a small number of founder cells at the periphery of vascular tissues and must emerge through intervening layers of tissues. We describe how the hormone auxin, which originates from the developing lateral root, acts as a local inductive signal which re-programmes adjacent cells. Auxin induces the expression of a previously uncharacterized auxin influx carrier LAX3 in cortical and epidermal cells directly overlaying new primordia. Increased LAX3 activity reinforces the auxin-dependent induction of a selection of cell-wall-remodelling enzymes, which are likely to promote cell separation in advance of developing lateral root primordia.

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

This paper is dedicated to the memory of our friend and colleague Andreas Schmidlin P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an~10 A Ê resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding af®nity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P i . Biochemical data suggest that these rearrangements may involve rotation of transmembrane a-helices. A mechanism for transport is suggested.

Abstract:ABC (ATP Binding Cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilises detergents to solubilise the protein from the membrane, effectively removing it from its native lipid environment. Subsequently lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. We present here the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead using a styrene maleic acid co-polymer (SMA). SMA inserts in a bilayer and assembles into discrete particles, essentially solubilising the membrane into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). We show that this polymer can extract several eukaryotic ABC transporters; P-glycoprotein (ABCB1), MRP1 (ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (ABCC7), from a range of different expression systems. The SMALP encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably to those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared to detergent solubilisation. This study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function. Summary statement:A styrene maleic acid copolymer can be effectively used to extract and purify large eukaryotic transmembrane proteins in the absence of detergents, forming small bilayer discs encapsulating the protein, which have great potential for future structure & function studies.

The M2 protein of influenza virus forms ion channels activated by low pH which are proton permeable and play a key role in the life cycle of the virus. M2 is a 97-residue integral membrane protein containing a single transmembrane (TM) helix. M2 is present as disulfide-linked homotetramers. The TM domain of M2 has been modeled as a bundle of four parallel M2 helices. The helix bundle forms a left-handed supercoil surrounding a central pore. Residue H37 has been implicated in the mechanism of low-pH activation of the channel. Models generated with H37 in a fully deprotonated state exhibit a pore occluded by a ring of H37 side chains oriented toward the lumen of the pore. Models with H37 in a fully protonated state no longer exhibit such occlusion of the pore, as the H37 side chains adopt a more interfacial location. Extended molecular dynamics simulations with water molecules within and at the mouths of the pores support this distinction between the H37-deprotonated and H37-protonated models. These simulations suggest that only in the H37-protonated model is there a continuous column of water extending the entire length of the central pore. A mechanism for activation of M2 by low pH is presented in which the H37-deprotonated model corresponds to the "closed" form of the channel, while the H37-protonated model corresponds to the "open" form. A switch from the closed to the open form of the channel occurs if H37 is protonated midway through a simulation. The open channel is suggested to contain a wire of H-bonded water molecules which enables proton permeability.

In most organisms, ABC transporters constitute one of the largest families of membrane proteins. In humans, their functions are diverse and underpin numerous key physiological processes, as well as being causative factors in a number of clinically relevant pathologies. Advances in our understanding of these diseases have come about through combinations of genetic and protein biochemical investigations of these transporters and the power of in vitro and in vivo investigations is helping to develop genotype–phenotype understanding. However, the importance of ABC transporter research goes far beyond human biology; microbial ABC transporters are of great interest in terms of understanding virulence and drug resistance and industrial biotechnology researchers are exploring the potential of prokaryotic ABC exporters to increase the capacity of synthetic biology systems. Plant ABC transporters play important roles in transport of hormones, xenobiotics, metals and secondary metabolites, pathogen responses and numerous aspects of development, all of which are important in the global food security area. For 3 days in Chester, this Biochemical Society Focused Meeting brought together researchers with diverse experimental approaches and with different fundamental questions, all of which are linked by the commonality of ABC transporters.

Peroxisomes are organelles that perform diverse metabolic functions in different organisms, but a common function is β-oxidation of a variety of long chain aliphatic, branched, and aromatic carboxylic acids. Import of substrates into peroxisomes for β-oxidation is mediated by ATP binding cassette (ABC) transporter proteins of subfamily D, which includes the human adrenoleukodystropy protein (ALDP) defective in X-linked adrenoleukodystrophy (X-ALD). Whether substrates are transported as CoA esters or free acids has been a matter of debate. Using COMATOSE (CTS), a plant representative of the ABCD family, we demonstrate that there is a functional and physical interaction between the ABC transporter and the peroxisomal long chain acyl-CoA synthetases (LACS)6 and -7. We expressed recombinant CTS in insect cells and showed that membranes from infected cells possess fatty acyl-CoA thioesterase activity, which is stimulated by ATP. A mutant, in which Serine 810 is replaced by asparagine (S810N) is defective in fatty acid degradation in vivo, retains ATPase activity but has strongly reduced thioesterase activity, providing strong evidence for the biological relevance of this activity. Thus, CTS, and most likely the other ABCD family members, represent rare examples of polytopic membrane proteins with an intrinsic additional enzymatic function that may regulate the entry of substrates into the β-oxidation pathway. The cleavage of CoA raises questions about the side of the membrane where this occurs and this is discussed in the context of the peroxisomal coenzyme A (CoA) budget. T he peroxisome is the sole site of β-oxidation of fatty acids and related molecules in plants and fungi and is essential in metabolism of very long chain fatty acids and bioactive lipid molecules in mammals. ATP binding cassette (ABC) proteins of subfamily D are required for the transport of these substrates across the peroxisome membrane (1). These are peroxisomal ABC transporter 1 and 2 (Pxa1p/Pxa2p) in yeast, adrenoleukodystrophy protein (ALDP/ABCD1), adrenoleukodystrophy related protein (ALDPR/ ABCD2), and the 70 kDa peroxisome membrane protein (PMP70/ ABCD3) in mammals, and Comatose (CTS; also known as PED3, PXA1, and AtABCD1) in plants. Defects in ALDP result in Xlinked adrenoleukodystrophy, a neurological disorder in which very long chain fatty acids accumulate (2). Similarly, cts mutants are defective in germination and mobilization of stored triacylglycerol (3).Activation by formation of a CoA thioester is a prerequisite for entry of substrates into β-oxidation (4) but whether ABCD proteins accept free fatty acids or acyl-CoAs has been contentious. Arabidopsis mutants lacking CTS accumulate acyl-CoAs (5) and the basal ATPase activity of the protein is stimulated by acyl-CoAs rather than free fatty acids (6). Studies with yeast cells and trypanosomes suggest that Pxa1p/Pxa2p and the trypanosome ABCD protein GAT1 transport acyl-CoAs (7, 8) and human ALDP, ALDR, and Arabidopsis CTS are all able to complement the Saccharomyces cerevisiae pxa1...

Auxin signaling is central to plant growth and development, yet hardly anything is known about its evolutionary origin. While the presence of key players in auxin signaling has been analyzed in various land plant species, similar analyses in the green algal lineages are lacking. Here, we survey the key players in auxin biology in the available genomes of Chlorophyta species. We found that the genetic potential for auxin biosynthesis and AUXIN1 (AUX1)/LIKE AUX1-and P-GLYCOPROTEIN/ATP-BINDING CASSETTE subfamily B-dependent transport is already present in several single-celled and colony-forming Chlorophyta species. In addition, our analysis of expressed sequence tag libraries from Coleochaete orbicularis and Spirogyra pratensis, green algae of the Streptophyta clade that are evolutionarily closer to the land plants than those of the Chlorophyta clade, revealed the presence of partial AUXIN RESPONSE FACTORs and/or AUXIN/INDOLE-3-ACETIC ACID proteins (the key factors in auxin signaling) and PIN-FORMED-like proteins (the best-characterized auxin-efflux carriers). While the identification of these possible AUXIN RESPONSE FACTOR-and AUXIN/INDOLE-3-ACETIC ACID precursors and putative PIN-FORMED orthologs calls for a deeper investigation of their evolution after sequencing more intermediate genomes, it emphasizes that the canonical auxin response machinery and auxin transport mechanisms were, at least in part, already present before plants "moved" to land habitats.