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Significance Flowering plants exhibit exceptional diversity of form and stature. Their ability to achieve great heights is supported in part by their water-conducting specialized cells, the vessel elements, which have gradually evolved and resulted in increased hydraulic efficiency. Here we report the discovery, functional, and evolutionary analysis of a previously uncharacterized gene, ENLARGED VESSEL ELEMENT (EVE), that contributes to vessel element dimensions in the perennial woody species Populus. EVE’s appearance among the streptophyte algae and ultimate expansion in flowering plants may represent an important addition to the genetic toolkit required for plant vascular development. Surprisingly, EVE homologs are also detected in algae-infecting prasinoviruses, suggesting that it has been horizontally transferred., The radiation of angiosperms led to the emergence of the vast majority of today’s plant species and all our major food crops. Their extraordinary diversification occurred in conjunction with the evolution of a more efficient vascular system for the transport of water, composed of vessel elements. The physical dimensions of these water-conducting specialized cells have played a critical role in angiosperm evolution; they determine resistance to water flow, influence photosynthesis rate, and contribute to plant stature. However, the genetic factors that determine their dimensions are unclear. Here we show that a previously uncharacterized gene, ENLARGED VESSEL ELEMENT (EVE), contributes to the dimensions of vessel elements in Populus, impacting hydraulic conductivity. Our data suggest that EVE is localized in the plasma membrane and is involved in potassium uptake of differentiating xylem cells during vessel development. In plants, EVE first emerged in streptophyte algae, but expanded dramatically among vessel-containing angiosperms. The phylogeny, structure and composition of EVE indicates that it may have been involved in an ancient horizontal gene-transfer event.

About 50% of the world’s arable land is strongly acidic (soil pH < 5). The low pH of these soils solubilizes root-toxic ionic aluminium (Al3+) species from clay minerals, driving the evolution of various counteractive adaptations in cultivated crops. The food crop Sorghum bicolor, for example, upregulates the membrane-embedded transporter protein SbMATE in its roots. SbMATE mediates efflux of the anionic form of the organic acid, citrate, into the soil rhizosphere, chelating Al3+ ions and thereby imparting Al-resistance based on excluding Al+3 from the growing root tip. Here, we use electrophysiological, radiolabeled, and fluorescence-based transport assays in two heterologous expression systems to establish a broad substrate recognition profile of SbMATE, showing the transport of 14C- citrate anion, as well as the organic monovalent cation, ethidium, but not the divalent ethidium-derivative, propidium. The transport cycle is proton and/or sodium-driven, and shares certain molecular mechanisms with bacterial MATE-family transporters. We further complement our transport assays by directly measuring substrate binding to detergent-purified SbMATE protein. Finally, we use the functionally-folded, purified membrane protein as an antigen to discover high-affinity, native conformation-binding and transport function-altering nanobodies using an animal-free, mRNA/cDNA display technology. Our results demonstrate the utility of using Pichia pastoris as an efficient eukaryotic host to express large quantities of functional plant transporter proteins for in vitro characterization. The nanobody discovery approach is applicable to other low immunogenic plant proteins.

EmrD is a multidrug transporter from the Major Facilitator Superfamily that expels amphipathic compounds across the inner membrane of Escherichia coli . Here, we report the x-ray structure of EmrD determined to a resolution of 3.5 angstroms. The structure reveals an interior that is composed mostly of hydrophobic residues, which is consistent with its role transporting amphipathic molecules. Two long loops extend into the inner leaflet side of the cell membrane. This region can serve to recognize and bind substrate directly from the lipid bilayer. We propose that multisubstrate specificity, binding, and transport are facilitated by these loop regions and the internal cavity.

Co-crystal structures of P-glycoprotein with a series of engineered ligands reveal multiple ligand-binding modes, a ligand-binding site on the outer surface of the transporter and a conformational change that may couple to ATP hydrolysis., P-glycoprotein (P-gp) is a transporter of great clinical and pharmacological significance. Several structural studies of P-gp and its homologs have provided insights into its transport cycle, but questions remain regarding how P-gp recognizes diverse substrates and how substrate binding is coupled to ATP hydrolysis. Here, four new P-gp co-crystal structures with a series of rationally designed ligands are presented. It is observed that the binding of certain ligands, including an ATP-hydrolysis stimulator, produces a large conformational change in the fourth transmembrane helix, which is positioned to potentially transmit a signal to the nucleotide-binding domains. A new ligand-binding site on the surface of P-gp facing the inner leaflet of the membrane is also described, providing vital insights regarding the entry mechanism of hydrophobic drugs and lipids into P-gp. These results represent significant advances in the understanding of how P-gp and related transporters bind and export a plethora of metabolites, antibiotics and clinically approved and pipeline drugs.

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATP-Binding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse P-gp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.

Transporter proteins from the MATE (multidrug and toxic compound extrusion) family are involved in metabolite transport in plants and in multidrug resistance in bacteria and mammals. MATE transporters were the only remaining class of multidrug resistance transporter whose structure was not known. The X-ray structure of a prototypical MATE protein, NorM from Vibrio cholerae, has now been determined to 3.65 A resolution, revealing a protein topology distinct from other membrane-protein structures solved to date. The structure is in an 'outward-facing' conformation, with a cation-binding site in close proximity to residues previously deemed critical for transport. Transporter proteins from the MATE (multidrug and toxic compound extrusion) family are involved in metabolite transport in plants, and in multiple-drug resistance in bacteria and mammals. Here, the X-ray crystal structure of a MATE transporter from Vibrio cholerae is reported. The structure is in an outward-facing conformation, and reveals a cation-binding site near to residues previously deemed essential for transport. Transporter proteins from the MATE (multidrug and toxic compound extrusion)1 family are vital in metabolite transport in plants2,3, directly affecting crop yields worldwide4. MATE transporters also mediate multiple-drug resistance (MDR) in bacteria and mammals5, modulating the efficacy of many pharmaceutical drugs used in the treatment of a variety of diseases6,7,8,9. MATE transporters couple substrate transport to electrochemical gradients and are the only remaining class of MDR transporters whose structure has not been determined10. Here we report the X-ray structure of the MATE transporter NorM from Vibrio cholerae determined to 3.65 A, revealing an outward-facing conformation with two portals open to the outer leaflet of the membrane and a unique topology of the predicted 12 transmembrane helices distinct from any other known MDR transporter. We also report a cation-binding site in close proximity to residues previously deemed critical for transport11. This conformation probably represents a stage of the transport cycle with high affinity for monovalent cations and low affinity for substrates.

• Fluorophotometry was used to evaluate the blood-ocular barrier in rats following streptozocin-induced diabetes, experimental systemic hypertension, sodium iodate treatment, diet-induced galactosemia, and aldose reductase inhibitors. After administration of intravenous (IV) fluorescein sodium, diabetes, hypertension, or sodium iodate treatment resulted in an increased vitreous accumulation of IV fluorescein. Accumulation of dextran-labeled fluorescein (3,000 and 19,000 molecular weight [mol wt]) was not increased in diabetic or sodium iodate-treated animals. However, 3,000-mol wt dextran-labeled dye accumulated in the vitreous of hypertensive rats. The disappearance of fluorescein injected into the vitreous was significantly delayed in diabetic and sodium iodate-treated rats, whereas this rate was normal in hypertensive animals. Galactosemia did not alter vitreous fluorophotometric measurements. Pretreatment for systemic effects with aldose reductase inhibitors did not correct the vitreous fluorophotometric measurements of diabetic rats.