Low Transport Activity Research Articles

Abstract 4903: Levels of folate transporters impact the compartmentalization of one-carbon metabolism in the mitochondria vs cytosol providing a unique vulnerability to SHMT inhibition

Abstract These studies characterize the critical role of the Reduced Folate Carrier (RFC) and the Proton Coupled Folate Transporter (PCFT) as determinants of cancer cell reliance on mitochondrial vs cytosolic one carbon (C1) metabolism, to identify a unique susceptibility towards SHMT1 and SHMT2 inhibition in cancer cells. C1 metabolism is frequently reprogrammed in cancer cells to provide nucleotides, amino acids, and glutathione, and to maintain redox homeostasis for proliferation. The mitochondrial C1 converting enzymes serine hydroxymethyltransferase (SHMT) 2 and methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top overexpressed metabolic enzymes in human cancers, suggesting these are important cancer-specific targets. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates (with AGF347 being the lead) that potently inhibit mitochondrial SHMT2, as well as SHMT1. Folates are essential cofactors for C1 converting reactions; folates and antifolates primarily rely on the major facilitative folate transporters RFC and PCFT for internalization by tumor cells. In these studies, we explore the effects of folate transporter expression on the compartmentalization of C1 metabolism in the mitochondria and cytosol and the potential impact on SHMT1 and SHMT2 inhibition. We used a tetracycline inducible system for RFC expression in RFC/PCFT-null HeLa cells, with and without constitutive PCFT, and characterized the impact on transport and accumulation of folates and AGF347 in response to folate transporter expression. Increased accumulation of folates was observed with increasing RFC in the absence of PCFT. In the presence of PCFT, baseline folate accumulation was substantial and independent of RFC expression, indicating a major role of PCFT as a facilitative folate transporter. SHMT2 catalyzes the conversion of serine and THF to glycine and 5, 10-methylene THF in the mitochondria, while SHMT1 catalyzes the reverse reaction in the cytosol, producing THF and serine. To determine how folate transporter expression affects the C1 flux through SHMT1 vs SHMT2, [2,3,3-2H]serine tracer experiments were performed; increased mitochondrial C1 flux through SHMT2 was observed with increasing RFC in the absence of PCFT, whereas the mitochondrial flux through SHMT2 predominated in the presence of PCFT. By cell proliferation assays it was discovered that cells with lower RFC expression exhibit a hypersensitive phenotype towards AGF347 in the absence of PCFT, indicating a unique therapeutic opportunity for targeting SHMT forms in cancer cells with low folate transport activity. Here we identify RFC and PCFT as key determinants for the compartmentalization of C1 flux in the mitochondria vs the cytosol, in turn affecting the sensitivity of cancer cells towards SHMT inhibition. Citation Format: Mathew Joseph Schneider, Carrie O'connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry H. Matherly. Levels of folate transporters impact the compartmentalization of one-carbon metabolism in the mitochondria vs cytosol providing a unique vulnerability to SHMT inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4903.

Influence of lung cancer model characteristics on tumor targeting behavior of nanodrugs.

Evidence is mounting that there is a significant gap between the antitumor efficacy of nanodrugs in preclinical mouse tumor models and in clinical human tumors, and that differences in tumor models are likely to be responsible for this gap. Herein, we investigated the enhanced permeability and retention (EPR) effect in mouse lung cancer models with different tumor growth rates, volumes and locations, and analyzed the nanodrug tumor targeting behaviors limited by tumor vascular pathophysiological characteristics in various tumor models. The results showed that the fast-growing tumors were characterized by lower vascular tight junctions, leading to higher vascular paracellular transport activity and nanodrug tumor accumulation. The paracellular transport activity increased with the growth of tumor, but the vascular density and transcellular transport activity decreased, and as a result, the average tumor accumulation of passive targeting nanodrugs decreased. Orthotopic tumors were rich in blood vessels, but had low vascular transcellular and paracellular transport activities, making it difficult for nanodrug accumulation in orthotopic tumors via passive targeting strategies. The antitumor efficacy of passive targeting nanodrugs in various lung cancer-bearing mice validated the aforementioned nanodrug accumulation behavior, and nanodrugs based on the angiogenesis-tumor sequential targeting strategy achieved obviously improved efficacy in orthotopic lung cancer-bearing mice. These results suggest that the EPR effect varies in different tumor models and should not be used as a universal targeting strategy for antitumor nanodrugs. Besides, attention should be paid to the animal tumor models in the evaluation of nanodrugs so as to avoid exaggerating the antitumor efficacy.

Transporter-interfering chemicals inhibit P-glycoprotein of yellowfin tuna (Thunnus albacares)

Marine pollutants bioaccumulate at high trophic levels of marine food webs and are transferred to humans through consumption of apex species. Yellowfin tuna (Thunnus albacares) are marine predators, and one of largest commercial fisheries in the world. Previous studies have shown that yellowfin tuna can accumulate high levels of persistent organic pollutants, including Transporter Interfering Chemicals (TICs), which are chemicals shown to bind to mammalian xenobiotic transporters and interfere with their function. Here, we examined the extent to which these same compounds might interfere with the activity of the yellowfin tuna (Thunnus albacares) ortholog of this transporter. To accomplish this goal we identified, expressed, and functionally assayed tuna ABCB1. The results demonstrated a common mode of vertebrate ABCB1 interaction with TICs that predicts effects across these species, based on high conservation of specific interacting residues. Importantly several TICs showed potent inhibition of Ta-ABCB1, such as the organochlorine pesticides Endrin (EC50 = 1.2 ± 0.2 μM) and Mirex (EC50 = 2.3 ± 0.9 μM). However, unlike the effects observed on mouse ABCB1, low concentrations of the organochlorine pesticide TICs p,p’-DDT and its metabolite p,p’-DDD co-stimulated verapamil-induced Ta-ABCB1 ATPase activity possibly suggesting a low transport activity for these ligands in tuna. These results provide a mechanistic basis for understanding the potential vulnerability of tuna to these ubquitous pollutants.

Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform.

ABSTRACTThe capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.

Exploring the Autoinhibitory Domain (AID) of the Electrogenic Na/HCO3 Transporter NBCe1‐B, and Binding Motifs for the Cytosolic Protein IRBIT

The electrogenic Na/HCO3 cotransporter NBCe1 is expressed widely throughout the body, and plays crucial roles in trans‐epithelial HCO3− transport (e.g., renal proximal tubule, PT) and regulation of intracellular pH (e.g., cells of central nervous system, CNS). The five known NBCe1 variants are: NBCe1‐A variant, mainly in PT; B, ubiquitous; C, CNS; and D and E, murine reproductive organs. For each variant, the extreme NH2‐terminus (Nt) markedly affects transport activity. For the A and D variants, the initial 41 amino acids (aa) are unique and, at least in A, confer high transport activity. For the B, C, and E variants, the initial 85 aa are likewise unique and, at least in B and C, confer low transport activity. Among these variants, at least B interacts with IRBIT [inositol trisphosphate (IP3)‐receptor binding protein released with IP3], which binds to the unique Nt and markedly enhances transport activity. In a previous study, we examined NBCe1‐B (e1B) residues 28–62, inclusive, and found that the cationic cluster (residues 40–48, inclusive) is an essential element of the AID that confers low activity to e1B. The purpose of the present study is to: (a) determine whether other portions of the unique Nt of e1B (i.e., residues 2–27 and 63–85, inclusive) contribute to the AID, (b) better characterize the cationic cluster, and (c) refine the structural requirements of the IRBIT‐e1B interaction under physiological conditions. Our approach is to co‐express in Xenopus oocytes one of several e1B constructs ± super‐IRBIT (sIRBIT, which lacks the PP1 binding site), followed by two‐electrode voltage‐clamping, extracellular surface biotinylation, and NeutrAvidin pull‐down of plasma‐membrane proteins. As assayed by western blot, we find that sIRBIT co‐purifies with biotinylated e1B protein, but only for e1B constructs with an intact AID (i.e., low‐activity constructs that sIRBIT strongly stimulates). For example, sIRBIT does not copurify with the Δ940‐48 e1B construct, which lacks the cationic cluster (i.e., AID disrupted). We find that residues 63–85, inclusive, are unnecessary for both the AID and for sIRBIT binding. Currently, we are using alanine scanning to explore residues 2–27, inclusive. When we replace the normal cation cluster—mostly made of arginine (R) residues—with increasing numbers (N) of lysine residues (KN) or histidine residues (HN), we observe that (a) the KN and HN constructs also produce autoinhibition, (b) IRBIT relieves the autoinhibition, and (c) sIRBIT co‐purifies with the KN and HN constructs, and (d) the amount of co‐purified sIRBIT gradually increases with increasing N for both KN and HN constructs, as is the case with RN constructs. Thus, the cationic cluster—whether made up of RN, KN, or HN—is essential both for autoinhibition and sIRBIT binding.Support or Funding InformationThe Office of Naval Research, N00014‐15‐1‐2060,N00014‐16‐1‐2535

Self-Assembled Columnar Triazole Quartets: An Example of Synergistic Hydrogen-Bonding/Anion-π Interactions.

The self-assembly of triazole amphiphiles was examined in solution, the solid state, and in bilayer membranes. Single-crystal X-ray diffraction experiments show that stacked protonated triazole quartets (T4 ) are stabilized by multiple strong interactions with two anions. Hydrogen bonding/ion pairing of the anions are combined with anion-π recognition to produce columnar architectures. In bilayer membranes, low transport activity is observed when the T4 channels are operated as H+ /X- translocators, but higher transport activity is observed for X- in the presence of the K+ -carrier valinomycin. These self-assembled superstructures, presenting intriguing structural behaviors such as directionality, and strong anion encapsulation by hydrogen bonding supported by vicinal anion-π interactions can serve as artificial supramolecular channels for transporting anions across lipid bilayer membranes.

Self‐Assembled Columnar Triazole Quartets: An Example of Synergistic Hydrogen‐Bonding/Anion–π Interactions

AbstractThe self‐assembly of triazole amphiphiles was examined in solution, the solid state, and in bilayer membranes. Single‐crystal X‐ray diffraction experiments show that stacked protonated triazole quartets (T4) are stabilized by multiple strong interactions with two anions. Hydrogen bonding/ion pairing of the anions are combined with anion–π recognition to produce columnar architectures. In bilayer membranes, low transport activity is observed when the T4 channels are operated as H+/X− translocators, but higher transport activity is observed for X− in the presence of the K+‐carrier valinomycin. These self‐assembled superstructures, presenting intriguing structural behaviors such as directionality, and strong anion encapsulation by hydrogen bonding supported by vicinal anion–π interactions can serve as artificial supramolecular channels for transporting anions across lipid bilayer membranes.

Evaluating Physiological Interactions between the Electrogenic Na/HCO3 Transporter NBCe1‐B and its Cytosolic Binding Partner IRBIT

The electrogenic Na/HCO3 transporter NBCe1 is expressed in numerous organs, where it plays many important roles, including trans‐epithelial HCO3− transport (e.g., pancreatic ducts) and maintenance of intracellular pH (e.g., cells in CNS). Presently, five NBCe1 variants are known: the A variant, mainly in kidney; B, ubiquitous; C, CNS; and D and E, murine reproductive organs. The extreme NH2‐terminus (Nt) of each variant is a strong determinant of transport activity. A and D have a unique Nt of 41 amino acids (aa) that, at least in A, confers high transport activity. B, C, and E have a unique Nt of 85 aa that, at least in B and C, confers low transport activity. However, B interacts with IRBIT [inositol trisphosphate (IP3)‐receptor binding protein released with IP3], which binds to the unique Nt of NBCe1‐B, and markedly enhances the transport activity of NBCe1‐B. Previously, others examined the interaction by fusing a fragment of the unique Nt of NBCe1‐B/C/E to maltose‐binding protein (MBP) for use in a MBP pull‐down assay, and found that NBCe1‐B (e1B) residues 1–62 are necessary for the interaction. In a recent study, we showed that the cationic cluster (residues 40–48 inclusive) is an essential element of the autoinhibitory domain (AID) that confers low activity on e1B. The purpose of the present study is to refine the structural requirements of the IRBIT‐e1B interaction under physiological conditions. Here, we co‐express 7 constructs of e1B (one at a time) together with super‐IRBIT (sIRBIT, which lacks the PP1 binding site) in Xenopus oocytes, followed by two‐electrode voltage‐clamp, extracellular‐surface‐biotinylation, and NeutrAvidin pull‐down of plasma‐membrane proteins. We find that sIRBIT copurifies, as assayed by western blot, with biotinylated e1B protein, but only for low‐activity e1B constructs (i.e., AID intact) for which sIRBIT enhances transport activity. sIRBIT does not copurify with the Δ940–48 e1B construct, which lacks the cationic cluster (i.e., AID disrupted). Thus, the cationic cluster is not only essential for autoinhibition, but also for sIRBIT binding. Our approach will be useful for elucidating other motifs that may be necessary for the interaction of IRBIT with NBCe1‐B, ‐C, or ‐E.Support or Funding InformationSupported by grants from the Office of Naval Research, N00014‐15‐1‐2060, N00014‐16‐1‐2535This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cysteine Scanning Mutagenesis of TM4b-4c Loop of Glutamate Transporter EAAT1 Reveals Three Conformationally Sensitive Residues.

Glutamatergic synaptic transmitters are cleared from the synaptic cleft through excitatory amino acid transporters (EAATs) that are responsible for recycling glutamate and transporting it into neurons and glial cells. To probe the structural role of the TM4b-4c loop of EAAT1 (Rattus norvegicus), each of the 57 amino acid residues was mutated to cysteine. Thirteen of the single mutants have very low transport activity. Aqueous accessibility of the introduced cysteines from the remaining mutants was then explored by membrane-permeant and membrane-impermeant sulfhydryl reagents in different conditions. F190C, V238C, and A243C were affected by MTSET, whereas Q189C, F190C, V238C, A243C, and L244C were sensitive to MTSEA. Q189C and L244C transport activity was diminished in the presence of potassium, which is expected to favor the inward-facing conformation of the transporter. Inversely, L244C was protected by glutamate. The modification of A243C by MTSEA was enhanced by either potassium and glutamate or dl-threo-β-benzyloxyaspartate. From these results, we suggest that residues F190C, V238C, and A243C may be located near the extracellular surface, and the TM4b-4c loop forms multiple reentrant membrane loops on the cell surface. Alternatively, F190C, V238C, and A243C may function in the transport pathway, which is exposed to MTSET. In addition, Q189C, A243C, and L244C are conformationally sensitive and may play a role in the transport cycle.

Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ΔITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ΔITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ΔITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ΔITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ΔITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ΔITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

Na+ /H+ exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells.

Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+/H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1‐dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1‐dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady‐state pHi and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.

Silicon fertilization of potato: expression of putative transporters and tuber skin quality.

A silicon transporter homolog was upregulated by Si fertilization and drought in potato roots and leaves. High Si in tuber skin resulted in anatomical and compositional changes suggesting delayed skin maturation. Silicon (Si) fertilization has beneficial effects on plant resistance to biotic and abiotic stresses. Potatoes, low Si accumulators, are susceptible to yield loss due to suboptimal growth conditions; thus Si fertilization may contribute to crop improvement. The effect of Si fertilization on transcript levels of putative transporters, Si uptake and tuber quality was studied in potatoes grown in a glasshouse and fertilized with sodium silicate, under normal and drought-stress conditions. Anatomical studies and Raman spectroscopic analyses of tuber skin were conducted. A putative transporter, StLsi1, with conserved amino acid domains for Si transport, was isolated. The StLsi1 transcript was detected in roots and leaves and its level increased twofold following Si fertilization, and about fivefold in leaves upon Si × drought interaction. Nevertheless, increased Si accumulation was detected only in tuber peel of Si-fertilized plants--probably due to passive movement of Si from the soil solution--where it modified skin cell morphology and cell-wall composition. Compared to controls, skin cell area was greater, suberin biosynthetic genes were upregulated and skin cell walls were enriched with oxidized aromatic moieties suggesting enhanced lignification and suberization. The accumulating data suggest delayed tuber skin maturation following Si fertilization. Despite StLsi1 upregulation, low accumulation of Si in roots and leaves may result from low transport activity. Study of Si metabolism in potato, a major staple food, would contribute to the improvement of other low Si crops to ensure food security under changing climate.

Engineering persister-specific antibiotics with synergistic antimicrobial functions.

Most antibiotics target growth processes and are ineffective against persister bacterial cells, which tolerate antibiotics due to their reduced metabolic activity. These persisters act as a genetic reservoir for resistant mutants and constitute a root cause of antibiotic resistance, a worldwide problem in human health. We re-engineer antibiotics specifically for persisters using tobramycin, an aminoglycoside antibiotic that targets bacterial ribosomes but is ineffective against persisters with low metabolic and cellular transport activity. By giving tobramycin the ability to induce nanoscopic negative Gaussian membrane curvature via addition of 12 amino acids, we transform tobramycin itself into a transporter sequence. The resulting molecule spontaneously permeates membranes, retains the high antibiotic activity of aminoglycosides, kills E. coli and S. aureus persisters 4–6 logs better than tobramycin, but remains noncytotoxic to eukaryotes. These results suggest a promising paradigm to renovate traditional antibiotics.

Rational use of plasma protein and tissue binding data in drug design.

It is a commonly accepted assumption that only unbound drug molecules are available to interact with their targets. Therefore, one of the objectives in drug design is to optimize the compound structure to increase in vivo unbound drug concentration. In this review, theoretical analyses and experimental observations are presented to illustrate that low plasma protein binding does not necessarily lead to high in vivo unbound plasma concentration. Similarly, low brain tissue binding does not lead to high in vivo unbound brain tissue concentration. Instead, low intrinsic clearance leads to high in vivo unbound plasma concentration, and low efflux transport activity at the blood-brain barrier leads to high unbound brain concentration. Plasma protein and brain tissue binding are very important parameters in understanding pharmacokinetics, pharmacodynamics, and toxicities of drugs, but these parameters should not be targeted for optimization in drug design.

Transport of Kynurenic Acid by Rat Organic Anion Transporters rOAT1 and rOAT3: Species Difference between Human and Rat in OAT1

A tryptophan catabolite, kynurenic acid, is involved in schizophrenia and uremia; there is little information on the mechanism of its disposition. Recently, our laboratory showed that kynurenic acid is a good substrate of human organic anion transporters hOAT1 and hOAT3. In this study, we performed uptake experiment using Xenopus laevis oocytes to characterize the transport of kynurenic acid by rat homologs of the transporters, rOAT1, and rOAT3. These transporters stimulated the uptake of kynurenic acid into oocytes, and transport by rOAT3 was marked. The Km values of the transport were estimated to be 8.46 μM for rOAT1 and 4.81 μM for rOAT3, and these values are comparable to their human homologs. The transport activity of kynurenic acid by rOAT1 was about one quarter of that of p-aminohippurate, although they were at the similar levels in hOAT1. A comparative experiment with hOAT1 was added in this study, showing that uptake amounts of kynurenic acid by hOAT1-expressing oocytes were 4 times greater than rOAT1-expressing oocytes. rOAT3 transported kynurenic acid as efficiently as estrone sulfate; this phenomenon was also observed in hOAT3. In conclusion, transport of kynurenic acid by rOAT1 and rOAT3 was shown. The characteristics of rOAT3 were similar to hOAT3, but low transport activity of kynurenic acid by rOAT1 was exhibited compared with hOAT1.

Effect of Multiple Cysteine Substitutions on the Functionality of Human Multidrug Resistance Protein 1 Expressed in Human Embryonic Kidney 293 Cells: Identification of Residues Essential for Function

Multidrug resistance protein 1 (MRP1) is a broad-specificity membrane transporter belonging to the C branch of the ATP binding cassette (ABC) superfamily. MRP1 confers resistance to various chemotherapeutic drugs and transports a wide range of conjugated organic anions. Several ABCC proteins, including MRP1, are unusual among ABC transporters in having a third membrane-spanning domain (MSD), MSD0, at their N termini. MRP1 lacking this additional MSD (ΔMRP1) is able to traffic to the plasma membrane of mammalian cells and to transport a number of well characterized substrates. A cysteineless (cysless) ΔMRP1 has been expressed in yeast and reported to be functional. However, we found that trafficking of such a construct in human cells was severely compromised, and, even when expressed in insect Sf21 cells, the protein had extremely low transport activity. Therefore, we have systematically examined the effects of substituting cysteines in the four domains of ΔMRP1, initially with alanine. These studies allowed us to identify five cysteines that cannot be replaced with alanine without inactivating the protein. Substitution of two of these residues with alternative amino acids has allowed us to produce an almost cysless form of ΔMRP1 that traffics to the plasma membrane and transports leukotriene C(4), 17β-estradiol 17-β-D-glucuronide, and estrone-3-sulfate with kinetic characteristics similar to those of the wild-type protein. The distribution of the remaining Cys residues is such that the protein will provide a useful template for a variety of cysteine based mutagenesis studies.

Arabidopsis thalianaNIP7;1: An Anther-Specific Boric Acid Transporter of the Aquaporin Superfamily Regulated by an Unusual Tyrosine in Helix 2 of the Transport Pore

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1 forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9-11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte development.

Expression of a chloroplast ATP/ADP transporter in E. coli membranes: Behind the Mistic strategy

Eukaryotic membrane protein expression is still a major bottleneck for structural studies. Production in E. coli often leads to low expression level and/or aggregated proteins. In the last decade, strategies relying on new fusion protein expression revealed promising results. Fusion with the amphipatic Mistic protein has been described to favor expression in E. coli membranes. Although, this approach has already been reported for a few membrane proteins, little is known about the activity of the fused proteins. We used this strategy and obtained high expression levels of a chloroplast ATP/ADP transporter from A. thaliana (NTT1) and characterized its transport properties. NTT1 fused to Mistic has a very low transport activity which can be recovered after in vivo Mistic fusion cleavage. Moreover, detailed molecular characterization of purified NTT1 mature form, NTT1 fused to Mistic or NTT1 cleaved-off from this fusion highlights the correct fold of the latter one. Therefore, considering the higher quantity of purified NTT1 mature form obtained via the Mistic fusion approach, this is a valuable strategy for obtaining quantities of pure and active proteins that are adequate for structural studies.

A Glutamine Residue Conserved in the Neurotransmitter:Sodium:Symporters Is Essential for the Interaction of Chloride with the GABA Transporter GAT-1

Neurotransmitter:sodium symporters are crucial for efficient synaptic transmission. The transporter GAT-1 mediates electrogenic cotransport of GABA, sodium, and chloride. The presence of chloride enables the transporter to couple the transport of the neurotransmitter to multiple sodium ions, thereby enabling its accumulation against steep concentration gradients. Here we study the functional impact of mutations of the putative chloride-binding residues on transport by GAT-1, with the emphasis on a conserved glutamine residue. In contrast to another putative chloride coordinating residue, Ser-331, where mutation to glutamate led to chloride-independent GABA transport, the Q291E mutant was devoid of any transport activity, despite substantial expression at the plasma membrane. Low but significant transport activity was observed with substitution mutants with small side chains such as Q291S/A/G. Remarkably, when these mutations were combined with the S331E mutation, transport was increased significantly, even though the activity of the S331E single mutant was only ∼25% of that of wild type GAT-1. Transport by these double mutants was largely chloride-independent. Like mutants of other putative chloride coordinating residues, the apparent affinity of the active Gln-291 single mutants for chloride was markedly reduced along with a change their anion selectivity. In addition to the interaction of the transporter with chloride, Gln-291 is also required at an additional step during transport. Electrophysiological analysis of the Q291N and Q291S mutants, expressed in Xenopus laevis oocytes, is consistent with the idea that this additional step is associated with the gating of the transporter.

Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function

Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.