Transport Mutants Research Articles

Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli.

Fluorescent tracers have been used to measure solute transport, but transport kinetics have not been evaluated by comparison of radiolabeled tracers. Using Streptococcus equinus JB1 and other bacteria, the objective of this study was to determine if a fluorescent analogue of glucose (2-NBDG) would be transported with the same kinetics and transporters as [(14)C]glucose. We uniquely modified a technique for measuring transport of radiolabeled tracers so that transport of a fluorescent tracer (2-NBDG) could also be measured. Deploying this technique for S. equinus JB1, we could detect 2-NDBG transport quantitatively and within 2 s. We found the Vmax of 2-NBDG transport was 2.9-fold lower than that for [(14)C]glucose, and the Km was 9.9-fold lower. Experiments with transport mutants suggested a mannose phosphotransferase system (PTS) was responsible for 2-NBDG transport in S. equinus JB1 as well as Escherichia coli. Upon examination of strains from 12 species of rumen bacteria, only the five that possessed a mannose PTS were shown to transport 2-NBDG. Those five uniformly transported [(14)C]mannose and [(14)C]deoxyglucose (other glucose analogues at the C-2 position) at high velocities. Species that did not transport 2-NBDG at detectable velocities did not possess a mannose PTS, though they collectively possessed several other glucose transporters. These results, along with retrospective genomic analyses of previous 2-NBDG studies, suggest that only a few bacterial transporters may display high activity toward 2-NBDG. Fluorescent tracers have the potential to measure solute transport qualitatively, but their bulky fluorescent groups may restrict (i) activity of many transporters and (ii) use for quantitative measurement.

Mechanisms of Passive Inward Transport in Hyperaldosteronism Mutants and Normal Na/K Pumps

Hyperaldosteronism (HA)‐inducing mutations in the α1 subunit of adrenal Na/K pumps (NKAs) have been found to induce a passive Na influx through the mutated NKAs under near‐physiological conditions (> 100 mM Nao;4.5 mM Ko). Under such conditions, wild type (WT) NKA catalyzes the uphill transport of 3 Na out of the cell in exchange for 2 K using the energy of one ATP molecule, a process that induces an outward current. However, under non‐physiological conditions, WT NKAs passively import H when the [Na]o and [K]o are not enough to saturate externally facing ion bindingsites. Here we use electrophysiology(two electrode voltage clamp and patch clamp) in oocytes expressing mutant NKAs to test the characteristics of the ion pathways through WT and mutant NKAs and whether the inward currents (IIN) induced by HA mutations are large enough to significantly depolarize the adrenal cells, or whether the observed depolarization (~30 mV) is secondary to the distorted ion homeostasis.WT H IIN occurs through a mechanism where the ions traverse the ion binding site that exclusively binds Na (site III). To test the contribution to IIN of ion binding sites I and II (which bind Na or K during the normal cycle), we simultaneously introduced 3 mutations(E327D, N776T, E779D) to disrupt ion binding there by increasing the size of the ion‐binding cavity. Resembling the HA mutations, the triple mutant presented a large IIN carried by Na under physiological conditions. Evaluation of the single mutants attributed this IIN to E327D in trans membrane segment (TM) 4. It has been proposed that the Na ions transported through IIN in two HA mutations, L97R (in TM1) and V325G (in TM4), involves a pathway for Na ions distinct from the H pathway in WT NKAs. To test this proposal, we concomitantly introduced the site III mutation D926N, known to abolish H permeation in WT pumps, with L97R, V325G, and E327D templates. Large IIN was observed in both L97R/D926N and V325G/D926N, while the E327D/D926N lacked IIN. This suggests that the pathway for Na current through these two HA mutants is different than the pathway for H through WT NKA. Additional studies will further clarify whether the effect of D926N on E327D is due to lack of expression, lack of a required partial reaction, or due to a pathway distinct from the HA mutants. We are also using giant inside‐out patches to compare the intracellular NKA ligand (ATP, Na, K, etc.) requirements for Na current through WT and mutant NKAs.To evaluate if the IIN observed in mutant NKAs is enough to significantly depolarize the adrenal cells, we are measuring IIN while counting the total number of NKAs present in the membrane. If the total IIN/NKA molecule is similar to the total IP/NKA molecule in WT pump, it is unlikely that the depolarization is a direct reflection of charge influx through the mutated pumps in the cell (half of the total pumps).Support or Funding InformationFunded by NSF1515434

The Function of the Iron–Sulfur Cluster Assembly Machinery is involved in Electron Transport Chain Activity and Oxidative Stress in Saccharomyces cerevisiae

For their function, many proteins depend on cofactor binding to specific sequence motifs. Fe‐S clusters are conserved and versatile inorganic cofactors that participate in a variety of processes and functions. In eukaryotes, biogenesis and recycling of Fe‐S clusters play important roles in the mechanisms of iron homeostasis involved in mitochondrial function. These Fe‐S clusters are assembled into apoproteins by the iron–sulfur cluster machinery (ISC). The present study aimed to determine the effects of mutations in the ISC genes on mitochondrial functionality and iron homeostasis under oxidative stress in S. cerevisiae BY4741 and its KanMX4 interruption ISC genes mutants: ssq1Δ, grx5Δ and isa1Δ. Intracellular ROS, Fe2+ and mitochondrial membrane potential in yeast cultures were determined using oxidant‐sensitive, cell‐permeant fluorescent probes (DHE, PGFL and Rho123). Fluorescence was quantified by flow cytometry, viewed and colocalized by confocal microscopy. Mitochondria were isolated and permeabilized for spectrophotometric determination of respiratory complexes activities. Oxygen consumption rate (OCR) was measured with a Clark‐type oxygen electrode coupled to a biological oxygen monitor. Electron transport chain (ETC) supercomplexes were identified by BN‐PAGE and Raman spectroscopy was performed to detect mitochondrial Fe‐S content. The results indicate that ROS generation caused by oxidizing agents like menadione is increased by ISC system dysfunction, due to loss of iron homeostasis (i.e. augmented free Fe2+), suggesting that ROS and Fe2+ from Fe‐S cluster proteins react creating a strong oxidative status in mitochondria. This was associated to an impairment on the activity of the complexes II, III and IV from the ETC in the ISC mutants. Indeed, increased oxygen generation instead consumption was observed in SSQ1 and ISA1 mutants suggesting a massive generation of ROS and its detoxification by superoxide dismutase and catalase. In mitochondria from SSQ1 and ISA1 mutants, the content of [Fe–S] centers was decreased along with III2IV2 respiratory supercomplex formation, but not in the iron‐deficient ATX1 (cytosolic copper metallochaperone) and MRS4 (mitochondrial iron transporter) mutants, used as controls. In conclusion these results indicate that the ISC system is important in iron‐homeostasis, ROS stress and in the assembly of the supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

Neurofibromin Loss of Function Drives Excessive Grooming in Drosophila.

Neurofibromatosis I is a common genetic disorder that results in tumor formation, and predisposes individuals to a range of cognitive/behavioral symptoms, including deficits in attention, visuospatial skills, learning, language development, and sleep, and autism spectrum disorder-like traits. The nf1-encoded neurofibromin protein (Nf1) exhibits high conservation, from the common fruit fly, Drosophila melanogaster, to humans. Drosophila provides a powerful platform to investigate the signaling cascades upstream and downstream of Nf1, and the fly model exhibits similar behavioral phenotypes to mammalian models. In order to understand how loss of Nf1 affects motor behavior in flies, we combined traditional activity monitoring with video analysis of grooming behavior. In nf1 mutants, spontaneous grooming was increased up to 7x. This increase in activity was distinct from previously described dopamine-dependent hyperactivity, as dopamine transporter mutants exhibited slightly decreased grooming. Finally, we found that relative grooming frequencies can be compared in standard activity monitors that measure infrared beam breaks, enabling the use of activity monitors as an automated method to screen for grooming phenotypes. Overall, these data suggest that loss of nf1 produces excessive activity that is manifested as increased grooming, providing a platform to dissect the molecular genetics of neurofibromin signaling across neuronal circuits.

A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell.

Tissue-specific root ion profiling reveals essential roles of the CAX and ACA calcium transport systems in response to hypoxia in Arabidopsis.

Waterlogging is a major abiotic stress that limits the growth of plants. The crucial role of Ca(2+) as a second messenger in response to abiotic and biotic stimuli has been widely recognized in plants. However, the physiological and molecular mechanisms of Ca(2+) distribution within specific cell types in different root zones under hypoxia is poorly understood. In this work, whole-plant physiological and tissue-specific Ca(2+) changes were studied using several ACA (Ca(2+)-ATPase) and CAX (Ca(2+)/proton exchanger) knock-out Arabidopsis mutants subjected to waterlogging treatment. In the wild-type (WT) plants, several days of hypoxia decreased the expression of ACA8, CAX4, and CAX11 by 33% and 50% compared with the control. The hypoxic treatment also resulted in an up to 11-fold tissue-dependent increase in Ca(2+) accumulation in root tissues as revealed by confocal microscopy. The increase was much higher in stelar cells in the mature zone of Arabidopsis mutants with loss of function for ACA8, ACA11, CAX4, and CAX11 In addition, a significantly increased Ca(2+) concentration was found in the cytosol of stelar cells in the mature zone after hypoxic treatment. Three weeks of waterlogging resulted in dramatic loss of shoot biomass in cax11 plants (67% loss in shoot dry weight), while in the WT and other transport mutants this decline was only 14-22%. These results were also consistent with a decline in leaf chlorophyll fluorescence (F v/F m). It is suggested that CAX11 plays a key role in maintaining cytosolic Ca(2+) homeostasis and/or signalling in root cells under hypoxic conditions.

Functional Studies of Nucleotide Sugar Transporters

Nucleotide sugar transporters (NSTs) are eukaryotic membrane proteins of the SLC35 family of Solute Carrier Proteins. They transport the nucleotide sugars from the cytosol to the Endoplasmic reticulum (ER) and the Golgi apparatus, thus, forming the link between sugar transport and glycosylation. The crucial role of the NSTs have been highlighted by a variety of transporter mutants that lead to severe congenital diseases in humans (LAD IIC, Schenkenbecken Dysplasia) and decreased virulence of parasites and yeast pathogens.It is well established that NSTs function as antiporters, transporting nucleotide sugars from the cytosol to the lumen of the ER and Golgi in exchange for nucleoside monophosphate (NMP). Currently, biochemical characterization has primarily been achieved by complementation assays or through uptake assays performed using isolated yeast Golgi/ER vesicles. The main aim of the study is to understand molecular recognition, hence specificity and mechanism of transport used by this class of proteins from pure detergent-solubilized protein separated from all other cellular components. We will present the protein expression, purification and biophysical characterization of several NST transporters using Isothermal Calorimetry and Microscale thermophoresis.

Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1.

The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3(-)), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To explore the effects and mode of action of NO3(-) on Lpr, we used an extended set of NO3(-) transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1 and nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3(-) conditions. First, a strong positive relationship between Lpr and NO3(-) accumulation, in shoots rather than in roots, was revealed. Secondly, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role for the high-affinity NO3(-) transporter NRT2.1 in increasing Lpr These results indicate that NO3(-)signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative real-time reverse transcription-PCR and enzyme-linked immunosorbent assays (ELISAs) were used to investigate the effects of NO3(-) availability on plasma membrane aquaporin (plasma membrane intrinsic protein; PIP) expression. Whereas PIP regulation mostly occurs at the post-translational level in wild-type plants, a regulation of PIPs at both the transcriptional and translational levels was uncovered in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3(-) involves novel shoot to root signaling and NRT2.1-dependent functions.

Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant.

Fungal infections are of major relevance due to the increased numbers of immunocompromised patients, frequently delayed diagnosis, and limited therapeutics. To date, the growth and nutritional requirements of fungi during infection, which are relevant for invasion of the host, are poorly understood. This is particularly true for invasive pulmonary aspergillosis, as so far, sources of (macro)elements that are exploited during infection have been identified to only a limited extent. Here, we have investigated sulfur (S) utilization by the human-pathogenic mold Aspergillus fumigatus during invasive growth. Our data reveal that inorganic S compounds or taurine is unlikely to serve as an S source during invasive pulmonary aspergillosis since a sulfate transporter mutant strain and a sulfite reductase mutant strain are fully virulent. In contrast, the S-containing amino acid cysteine is limiting for fungal growth, as proven by the reduced virulence of a cysteine auxotroph. Moreover, phenotypic characterization of this strain further revealed the robustness of the subordinate glutathione redox system. Interestingly, we demonstrate that methionine synthase is essential for A. fumigatus virulence, defining the biosynthetic route of this proteinogenic amino acid as a potential antifungal target. In conclusion, we provide novel insights into the nutritional requirements ofA. fumigatus during pathogenesis, a prerequisite to understanding and fighting infection.

A Western Blot Protocol for Detection of Proteins Heterologously Expressed in Xenopus laevis Oocytes.

Oocytes of the African clawed frog, Xenopus laevis, are often used for expression and biochemical characterization of transporter proteins as the oocytes are particularly suitable for uptake assays and electrophysiological recordings. Assessment of the expression level of expressed transporters at the individual oocyte level is often desirable when comparing properties of wild type and mutant transporters. However, a large content of yolk platelets in the oocyte cytoplasm makes this a challenging task. Here we report a method for fast and easy, semiquantitative Western blot analysis of proteins heterologously expressed in Xenopus oocytes.

Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii

Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite's carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.

Mutations in the Na(+)/citrate cotransporter NaCT (SLC13A5) in pediatric patients with epilepsy and developmental delay.

Mutations in the SLC13A5 gene that codes for the Na(+)/citrate cotransporter, NaCT, are associated with early onset epilepsy, developmental delay and tooth dysplasia in children. In the present study we identify additional SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and treatment strategies. There are currently no effective treatments, but some anti-epileptic drugs targeting the GABA system reduce seizure frequency. Acetazolamide, a carbonic anhydrase inhibitor and atypical anti-seizure medication decreases seizures in 4 patients. In contrast to previous reports, the ketogenic diet and fasting produce worsening of symptoms. The effects of the mutations on NaCT transport function and protein expression were examined by transient transfections of COS-7 cells. There was no transport activity from any of the mutant transporters, although some of the mutant transporter proteins were present on the plasma membrane. The structural model of NaCT suggests that these mutations can affect helix packing or substrate binding. We tested various treatments, including chemical chaperones and low temperatures, but none improve transport function in the NaCT mutants. Interestingly, coexpression of NaCT and the mutants results in decreased protein expression and activity of the wild-type transporter, indicating functional interaction. In conclusion, our study has identified additional SLC13A5 mutations in patients with chronic epilepsy starting in the neonatal period, with the mutations producing inactive Na(+)/citrate transporters.

Root and bacterial secretions regulate the interaction between plants and PGPR leading to distinct plant growth promotion effects

Plant growth-promoting rhizobacteria (PGPR) have garnered interest in agriculture due to their ability to influence the growth and production of host plants. ATP-binding cassette (ABC) transporters play important roles in plant-microbe interactions by modulating plant root exudation. The present study aimed to provide a more precise understanding of the mechanism and specificity of the interaction between PGPR and host plants. In the present study, the effects of interactions between a PGPR strain, Bacillus cereus AR156, and Arabidopsis thaliana wild type (Col-0) or its ABC transporter mutants on plant growth have been studied. B. cereus AR156 promoted the shoot growth of Col-0 and Atabcg30 but repressed the growth of Atabcc5. Bacterial volatiles and secretion promoted the shoot growth of Col-0 and Atabcg30 but had no effect on Atabcc5. We also found that root exudates of Col-0 induced the expression of B. cereus AR156 genes related to siderophore and chitinase production; while root exudates of Atabcc5 inhibited the expression level of those genes. Further analysis of root exudates revealed that amino acids, organic acids, and sugars were significantly less abundant in Atabcc5 when compared to Col-0. Our findings highlight that both host plant and PGPR play active roles in the outcome of the plant-microbe interaction.

Feeding on Leaves of the Glucosinolate Transporter Mutant gtr1gtr2 Reduces Fitness of Myzus persicae.

As aphids are a pest on various crops worldwide, a better understanding of the interaction between aphids and plant host defenses is required. The green peach aphid (Myzus persicae) feeds on a variety of plant species, including the model plant Arabidopsis thaliana (Arabidopsis), in which glucosinolates function as a major part of the chemical defense. Several studies have shown that glucosinolates play a role in interactions between Arabidopsis and the green peach aphid. In this work, we used a recently identified Arabidopsis glucosinolate transporter mutant (gtr1gtr2 dKO), with altered glucosinolate content in the vasculature, to investigate the role of defense compound transport in aphid infestation. By monitoring aphid performance on caged leaves and analyzing glucosinolates in leaf tissue and phloem sap, as well as inside aphids, we examined if a change in spatial distribution of glucosinolates within a leaf influences aphid performance. Based on reduced glucosinolate content in the phloem sap of the transporter mutant, we hypothesized that aphids would perform better on gtr1gtr2 dKO leaves compared to WT. Unexpectedly, aphids performed poorly on gtr1gtr2 dKO leaves. Our data suggest that higher glucosinolate content in tissues surrounding the phloem of the double transporter mutant may play a role in reducing aphid performance on this genotype.Electronic supplementary materialThe online version of this article (doi:10.1007/s10886-015-0641-3) contains supplementary material, which is available to authorized users.

Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.

An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria.

A Mutation in Transmembrane Domain 7 (TM7) of Excitatory Amino Acid Transporters Disrupts the Substrate-dependent Gating of the Intrinsic Anion Conductance and Drives the Channel into a Constitutively Open State

In the mammalian central nervous system, excitatory amino acid transporters (EAATs) are responsible for the clearance of glutamate after synaptic release. This energetically demanding activity is crucial for precise neuronal communication and for maintaining extracellular glutamate concentrations below neurotoxic levels. In addition to their ability to recapture glutamate from the extracellular space, EAATs exhibit a sodium- and glutamate-gated anion conductance. Here we show that substitution of a conserved positively charged residue (Arg-388, hEAAT1) in transmembrane domain 7 with a negatively charged amino acid eliminates the ability of glutamate to further activate the anion conductance. When expressed in oocytes, R388D or R388E mutants show large anion currents that display no further increase in amplitude after application of saturating concentrations of Na(+) and glutamate. They also show a substantially reduced transport activity. The mutant transporters appear to exist preferentially in a sodium- and glutamate-independent constitutive open channel state that rarely transitions to complete the transport cycle. In addition, the accessibility of cytoplasmic residues to membrane-permeant modifying reagents supports the idea that this substrate-independent open state correlates with an intermediate outward facing conformation of the transporter. Our data provide additional insights into the mechanism by which substrates gate the anion conductance in EAATs and suggest that in EAAT1, Arg-388 is a critical element for the structural coupling between the substrate translocation and the gating mechanisms of the EAAT-associated anion channel.

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

Transgenic tools for proteomic analysis of ciliary transport

Vision begins as photons are captured by photoreceptor cilia and light is converted into electrical signals that are then sent to the brain. As the photoreceptor cilium is not able to make its own proteins, all polypeptides needed for converting photons into electrical signals are synthetized in the cell body. How these molecules move from the cell body to cilia is still unclear. Opsin is one of the best-characterized transmembrane proteins. Our goal is to understand the mechanism of opsin transport into photoreceptor cilia. In this project, we use a combination of genetic and proteomic approaches in the zebrafish model. As the first step, we are constructing a transgenic line that expresses EGFP-opsin fusion from an inducible promoter specifically in photoreceptors. For this purpose, we will mate two transgenic lines: 1. A line that specifically expresses Cre recombinase in photoreceptors, and 2. A line that conditionally expresses EGFP-opsinCT44 (EGFP fused with the 44 C-terminal residues of opsin, which are sufficient to mediate ciliary transport) from the heat-shock promoter. A lox-mCherry-STOP cassette is inserted upstream of EGFP-opsinCT44. In the long run, we plan to purify photoreceptors via FACS sorting, pull down EGFP-opsinCT44 using the EGFP tag, and analyze opsin C-terminal binding proteins by mass spectrometry. In parallel, we will perform analysis of opsin transport in cilia mutants. We hope this will allow us to formulate a general model of how transmembrane proteins, such as GPCRs and TRP channels, are transported into cilia.

Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B)

The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4–5 fold more weakly than TetA(B), with Kds of 28 μM and 36μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination.

Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3.

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.