Isolation and functional characterization of a glucose-6-phosphate/phosphate translocator (IbG6PPT1) from sweet potato (Ipomoea batatas (L.) Lam.).

Sweet potato (Ipomoea batatas (L.) Lam.) is a good source of carbohydrates, an excellent raw material for starch-based industries, and a strong candidate for biofuel production due to its high starch content. However, the molecular basis of starch biosynthesis and accumulation in sweet potato is still insufficiently understood. Glucose-6-phosphate/phosphate translocators (GPTs) mediate the import of glucose-6-phosphate (Glc6P) into plastids for starch synthesis. Here, we report the isolation of a GPT-encoding gene, IbG6PPT1, from sweet potato and the identification of two additional IbG6PPT1 gene copies in the sweet potato genome. IbG6PPT1 encodes a chloroplast membrane-localized GPT belonging to the GPT1 group and highly expressed in storage root of sweet potato. Heterologous expression of IbG6PPT1 resulted in increased starch content in the leaves, root tips, and seeds and soluble sugar in seeds of Arabidopsis thaliana, but a reduction in soluble sugar in the leaves. These findings suggested that IbG6PPT1 might play a critical role in the distribution of carbon sources in source and sink and the accumulation of carbohydrates in storage tissues and would be a good candidate gene for controlling critical starch properties in sweet potato.

Biochemical and biophysical characterization of a prokaryotic Mg2+ ion channel: Implications for cost-effective purification of membrane proteins.

Although magnesium is the second most abundant cation present in the cell, the transport mechanism of Mg2+ across membranes is poorly understood. Importantly, the prokaryotic MgtE Mg2+ channel is related to mammalian SLC41A1 transporters and, therefore, biochemical and biophysical characterization of MgtE and its orthologs assumes significance. To date, the purification and structure determination of MgtE from Thermus thermophilus has been carried out using the widely used nonionic detergent, n-dodecyl-ß-d-maltopyranoside (DDM). However, DDM is an expensive detergent and alternative methods to produce high-quality proteins in stable and functional form will be practically advantageous to carry out structural studies in a cost-effective manner. In this work, we have utilized 'dual-detergent strategy' to successfully purify MgtE channel in a stable and functional form by employing relatively inexpensive detergents (Triton X-100 and Anzergent 3-14) for membrane solubilization and subsequently changed to DDM during purification. Our results show that Triton X-100 and Anzergent 3-14 extract MgtE well and the quality of purified protein is comparable to DDM-extracted MgtE. Interestingly, addition of high concentration of salt and glycerol during solubilization does not significantly affect the quantity and quality of MgtE. Importantly, limited proteolysis assay, circular dichroism spectroscopy and ensemble tryptophan fluorescence strongly support the use of Triton X-100, in particular, as an inexpensive, alternative detergent for the purification of MgtE without compromising the structural integrity of the channel and Mg2+-induced gating-related conformational dynamics. Overall, these results are relevant for the cost-effective purification of stable and functional membrane proteins in general, and magnesium channels, in particular.

NMR Spectroscopy Approach to Study the Structure, Orientation, and Mechanism of the Multidrug Exporter EmrE.

Multidrug exporters are a class of membrane proteins that remove antibiotics from the cytoplasm of bacteria and in the process confer multidrug resistance to the organism. This chapter outlines the sample preparation and optimization of oriented solid-state NMR experiments applied to the study of structure and dynamics for the model transporter EmrE from the small multidrug resistance (SMR) family.

Accessible Mannitol-Based Amphiphiles (MNAs) for Membrane Protein Solubilisation and Stabilisation.

Integral membrane proteins are amphipathic molecules crucial for all cellular life. The structural study of these macromolecules starts with protein extraction from the native membranes, followed by purification and crystallisation. Detergents are essential tools for these processes, but detergent-solubilised membrane proteins often denature and aggregate, resulting in loss of both structure and function. In this study, a novel class of agents, designated mannitol-based amphiphiles (MNAs), were prepared and characterised for their ability to solubilise and stabilise membrane proteins. Some of MNAs conferred enhanced stability to four membrane proteins including a G protein-coupled receptor (GPCR), the ß2 adrenergic receptor (ß2 AR), compared to both n-dodecyl-d-maltoside (DDM) and the other MNAs. These agents were also better than DDM for electron microscopy analysis of the ß2 AR. The ease of preparation together with the enhanced membrane protein stabilisation efficacy demonstrates the value of these agents for future membrane protein research.

Analysis of endocytosis and ubiquitination of the BOR1 transporter.

Endocytosis and membrane trafficking are the major factors controlling the abundance of plasma membrane proteins, such as transporters and receptors. We have found that Arabidopsis borate transporter BOR1 is polarly localized to the inner (stele-facing) plasma membrane domain of various root cells under boron limitation, and when boron is supplied in excess, BOR1 is rapidly transferred to the vacuole for immediate degradation. The BOR1 polarity and degradation are controlled by membrane trafficking including endocytosis. In this chapter, we describe methods for observation of endocytic trafficking of BOR1, and detection of BOR1 ubiquitination that is required for vacuolar sorting for degradation.

Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/Pi carrier SCaMC1.

SCaMC is an ATP-Mg/Pi carrier protein located at the mitochondrial inner membrane. SCaMC has an unusual N-terminal Ca(2+)-binding domain (NTD) in addition to its characteristic six-helix transmembrane bundle. The NTD of human SCaMC1 (residues 1-193) was expressed and purified in order to study its role in Ca(2+)-regulated ATP-Mg/Pi transport mediated by its transmembrane domain. While Ca(2+)-bound NTD could be crystallized, the apo state resisted extensive crystallization trials. Selenomethionine-labeled Ca(2+)-bound NTD crystals, which belonged to space group P6(2)22 with one molecule per asymmetric unit, diffracted X-rays to 2.9 Šresolution.

Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium.

Anion exchangers are membrane proteins that have been identified in a wide variety of species, where they transport Cl(-) and HCO3(-)across the cell membrane. In this study, we cloned an anion-exchange protein from the genome of the basidiomycete Phanerochaete chrysosporium (PcAEP). PcAEP is a 618-amino acid protein that is homologous to the human anion exchanger (AE1) with 22.9% identity and 40.3% similarity. PcAEP was overexpressed by introducing the PcAEP gene into the genome of Pichia pastoris. As a result, PcAEP localized in the membrane of P. pastoris and was solubilized successfully by n-dodecyl-ß-D-maltoside. His-tagged PcAEP was purified as a single band on SDS-PAGE using immobilized metal affinity chromatography and gel filtration chromatography. Purified PcAEP was found to bind to SITS, an inhibitor of the AE family, suggesting that the purified protein is folded properly. PcAEP expressed and purified using the present system could be useful for biological and structural studies of the anion exchange family of proteins.

Real-time polymerase chain reaction for the quantitative detection of tetA and tetB bacterial tetracycline resistance genes in food.

A new, rapid, sensitive and specific method was developed to directly detect and quantify tetA and tetB in food. Both tet genes are two of the most frequently present tetracycline resistance genes in gram-negative bacteria. A set of primers and Taqman probes was designed for each gene. The standard curves were performed using Escherichia coli BM13 (C600 RifR)/RP4 and E. coli NCTC 50365, which carry tetA and tetB, respectively. Meat and fish samples inoculated with these reference strains were used as a matrix to construct the standard curves for the analysis of 20 samples of chicken meat and 10 samples of hake (Merlucius merlucius). The limits of detection in pure culture were 5 cfu/mL (0.7 log cfu/mL) in the case of tetA, 50 cfu/mL (1.7log cfu/mL) for tetB and 5×10(2)cfu/g (2.7 log cfu/g) for both genes in food samples. The results obtained by real-time quantitative polymerase chain reaction (qPCR) were compared to counts of tetracycline-resistant bacteria obtained by plating extracts of poultry and hake samples in culture media supplemented with 16 mg/L of tetracycline. Counts of tetracycline-resistant bacteria obtained by qPCR showed a positive correlation, especially interesting when compared with microbiological counts of tetracycline-resistant Enterobacteriaceae in poultry meat (r=0.5509) and with tetracycline-resistant mesophilic aerobic bacteria in hake samples (r=0.7146). The obtained results demonstrate that this method could be a useful tool for the direct quantification of the amount of bacterial strains that carry tetA and/or tetB genes in food samples.

The use of isothermal titration calorimetry to study multidrug transport proteins in liposomes.

Biophysical measurements of multidrug transporters in vitro can often be of limited relevance to the natural in vivo behavior. In particular, the properties of transporters when removed from their native bilayer and solubilized in detergents or lipids can differ significantly from their in vivo properties, reducing the value of in vitro measurements for the design of antagonists to the transporters. This problem can be addressed by studying the transport protein in liposomes in which the properties of the liposome bilayer are altered through systematic changes in lipid composition. Isothermal titration calorimetry can be used to determine the properties of the lipid-reconstituted protein in bilayers of different lipid compositions as well as to quantify the percentage recovery of functional protein in different lipids. Both these measurements lead to an accurate analysis of substrate binding activity. The approach is illustrated here for the small multidrug transport protein, EmrE from Escherichia coli. The percentage of functional EmrE successfully reconstituted into liposome depends on lipid composition. Differences in ligand binding and subtle differences in the secondary structure also occur in different lipid compositions.

High yield cell-free production of integral membrane proteins without refolding or detergents.

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

Expression, purification and characterisation of the C-terminal STAS domain of the SLC26 anion transporter prestin.

The membrane protein prestin is the voltage-sensitive molecular motor underlying somatic electromotility of outer hair cells. In order to produce adequate quantities to perform structural and functional studies, we cloned and expressed in bacterial systems three variants of the cytosolic C-terminal STAS domain of prestin from Rattus norvegicus. While the expression level of the longer form of the C-terminal domain (fragment [505-744]) was very low or absent, we succeeded in the overexpression of two shorter fragment of the STAS domain (fragments [529-744], PreCD(L), and [529-720], PreCD(S)). These two polypeptides were purified to homogeneity and characterised by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The two proteins possess a three-dimensional structure and show a great tendency to aggregate. In particular, PreCD(L) is present in solution mainly as dimers and tetramers. These data correlate with that of full-length prestin that forms stable tetramers, suggesting that the C-terminal domain play an important role in modulating the properties of the entire prestin.

Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings.

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added L: -proline (Pro) generates a mitochondrial membrane potential (Delta Psi) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of Delta Psi on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion.

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

Evidence for simultaneous 1Na+:1Mg2+ and ping pong 2Na+:1Mg2+ exchangers in rat thymocyte.

Rat thymocytes showed two Na+/Mg2+ exchangers with high- and low- affinities for external Na+ (Na+o) at physiological internal Mg2+content. The total internal Mg2+ content (Mg2+it) was enhanced by loading with MgCl2 and the ionophore A-23187. Under these conditions, Na+/Mg2+ exchangers were dramatically stimulated by the Mg2+it increase. Na+-induced Mg2+ effluxes were independent of Cl-o or H+. The Na+/Mg2+ exchangers, which we named HANao (high affinity for Na+o) and LANao (low affinity for Na+o), were dissected in Mg2+-loaded thymocytes according to their kinetics and stoichiometries. HANao, which showed an apparent dissociation constant for Na+o (KNa H) = 9.2 +/- 1.6 mmol l(-1) Na+o and a maximal Na+ influx rate (VNa(Na H)max) = 30.5 +/- 6.1 mmol (l cells)(-1) h(-1), was a 1Na+:1Mg2+ simultaneous antiporter insensitive to external magnesium (Mg2+o) whereas that LANao, with KNa L = 65.1 +/- 8.6 mmol l(-1) Na+ and a VNa(Na L)max = 79.5 +/- 14.3 mmol (l cells)(-1) Na+ h(-1), was a 2Na+:1Mg2+ "ping-pong" antiporter which was strongly inhibited by Mg2+o. At physiological concentration of Mg2+o (1 mM), the Na+/Mg2+ exchange through the LANao was inhibited by approximately 50%. Amiloride (10(-4) M) inhibited at similar extent both Na+ and Mg2+ fluxes at high and at low Na+o.

The CFTR associated protein CAP70 interacts with the apical Cl-/HCO3- exchanger DRA in rabbit small intestinal mucosa.

DRA (down regulated in adenoma) is an intestinal anion exchanger, acting in parallel with NHE3 to facilitate ileal and colonic NaCl absorption. Furthermore it is involved in small intestinal bicarbonate secretion. Because DRA has a PDZ interaction motif, which may influence its properties, we searched for DRA-interacting PDZ adapter proteins in the small intestine. Using an overlay assay with the recombinant DRA C-terminus as a ligand, a 70 kDa protein was labeled, which was restricted to the brush border membrane in rabbit duodenal and ileal mucosa and was not detected in the colon. Destruction of the C-terminal PDZ interaction motif abolished this band, suggesting a specific protein-protein interaction. The 70 kDa protein was identified as CAP70 (CFTR associated protein of 70 kDa) by an anti-CAP70 antibody and by two in vitro binding assays after cloning CAP70 from rabbit duodenum and ileum. The interaction was recapitulated in HEK cells transfected with DRA and PDZK1, the human orthologue of CAP70. Corresponding to the overlay assay, no CAP70 mRNA or protein was detected in the colon. In vitro protein-protein interaction studies revealed specific binding of DRA to the 2nd and 3rd PDZ domain, while CFTR is known to interact with PDZ1, PDZ3, and PDZ4. The composition of macromolecular complexes assembled by CAP70 in the distal small bowel is unknown. Its restricted expression shows that it cannot be involved in NaCl absorption in the proximal colon. We suggest that CAP70 mediates regulatory functions specific to the small intestine.

Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand.

Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance to a diverse group of lipophilic cations. To examine the multimeric state(s), size-exclusion HPLC and sedimentation velocity experiments were performed with EmrE solubilized in N-dodecyl-beta-d-maltopyranoside (DM) detergent. EmrE was purified from Escherichia coli membranes using organic extraction with a 3:1 chloroform:methanol solvent followed by LH-20 chromatography and the recovered pure protein was re-solubilized in a buffer containing 2% DM. The purified protein was analyzed by SEC-HPLC to estimate the monodispersity and to determine the amount of bound detergent. The results show that EmrE is homogeneous in DM with a Stokes radius of 3.6nm compatible with that of a monomer. Sedimentation velocity experiments indicated that the EmrE preparation was monodisperse and supports the fact that the organic extracted protein solubilized in DM is monomeric. This monomeric form of the protein analyzed here is also shown to bind substrate in the micromolar range.

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria.

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.

TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution.

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.

A characterization of the lumenal region of human tapasin reveals the presence of two structural domains.

Tapasin is a type I membrane glycoprotein involved with other accessory proteins in the assembly of class I MHC-beta(2)m-peptide complexes in the endoplasmic reticulum. We have probed the three-dimensional structure of the lumenal region of human tapasin (residues 1-392) tagged with a (His)(6) sequence at its C-terminus using biochemical and biophysical techniques. The far-UV circular dichroism spectrum revealed that tapasin possesses well-defined secondary structural elements corresponding predominantly to beta-sheets. A thermal denaturation curve recorded at 216 nm showed a midpoint transition centered at approximately 45 degrees C. Sedimentation analysis showed that tapasin is monomeric in solution with a sedimentation coefficient, S degrees (20,w), of 2.68 S. This value of S degrees (20,w) combined with the value of the molar mass obtained by MALDI mass spectrometry (44.2 kDa) yielded a frictional ratio, f/f(0), of 1.47. Assuming tapasin is a prolate ellipsoid, we calculated an apparent length of 22.5 nm and a diameter of 2.62 nm, consistent with an elongated molecular shape. Controlled proteolysis using various enzymes revealed that a narrow region of tapasin near residue 90 is highly susceptible to digestion, resulting in two fragments that are resistant to further cleavage. The identity of these fragments was determined by amino acid sequencing and MALDI mass spectrometry and revealed a 9 kDa N-terminal fragment and a 34 kDa C-terminal fragment. Collectively, these results suggest that tapasin is comprised of two core domains of different sizes loosely linked by a flexible region.

Optimization of expression and the purification by organic extraction of the integral membrane protein EmrE.

EmrE is a member of the small multidrug resistance family of proteins and functions as a efflux transporter of lipophilic cations. This integral membrane protein is composed of 110 amino acids and is very hydrophobic with small loops exposed to the aqueous environment. This protein has been purified in a variety of ways including extraction with chloroform:methanol mixtures. This study explored culture growth and induction conditions, the parameters of organic solvent extraction, running conditions of a lipophilic column for lipid removal, as well as solubilization conditions. Optimal expression and purification protocols are crucial to the characterization goals of this protein. The experiments presented here led to an improvement in the yield of pure EmrE obtained by organic solvent extraction methods at a level of 0.9+/-0.2mg/L of culture. This is on the order of a 60% improvement over previous reports.