A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform.

Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na(+) ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na(+)-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na(+) ion binding site. Our data enhance the understanding of the permeation functions of Bot1.

Cell-free protein synthesis of membrane (1,3)-ß-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs.

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-ß-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.

A Barley xyloglucan xyloglucosyl transferase covalently links xyloglucan, cellulosic substrates, and (1,3;1,4)-beta-D-glucans.

Molecular interactions between wall polysaccharides, which include cellulose and a range of noncellulosic polysaccharides such as xyloglucans and (1,3;1,4)-beta-D-glucans, are fundamental to cell wall properties. These interactions have been assumed to be noncovalent in nature in most cases. Here we show that a highly purified barley xyloglucan xyloglucosyl transferase HvXET5 (EC 2.4.1.207), a member of the GH16 group of glycoside hydrolases, catalyzes the in vitro formation of covalent linkages between xyloglucans and cellulosic substrates and between xyloglucans and (1,3;1,4)-beta-D-glucans. The rate of covalent bond formation catalyzed by HvXET5 with hydroxyethylcellulose (HEC) is comparable with that on tamarind xyloglucan, whereas that with (1,3; 1,4)-beta-D-glucan is significant but slower. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analyses showed that oligosaccharides released from the fluorescent HEC:xyloglucan conjugate by a specific (1,4)-beta-D-glucan endohydrolase consisted of xyloglucan substrate with one, two, or three glucosyl residues attached. Ancillary peaks contained hydroxyethyl substituents (m/z 45) and confirmed that the parent material consisted of HEC covalently linked with xyloglucan. Similarly, partial hydrolysis of the (1,3;1,4)-beta-D-glucan:xyloglucan conjugate by a specific (1,3;1,4)-beta-D-glucan endohydrolase revealed the presence of a series of fluorescent oligosaccharides that consisted of the fluorescent xyloglucan acceptor substrate linked covalently with 2-6 glucosyl residues. These findings raise the possibility that xyloglucan endo-transglucosylases could link different polysaccharides in vivo and hence influence cell wall strength, flexibility, and porosity.