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ABSTRACT: The recent elucidation of crystal structures of a bacterial member of the NCS1 family, the Mhp1 benzyl-hydantoin permease from Microbacterium liquefaciens, allowed us to construct and validate a 3D model of the Aspergillus nidulans purine-cytosine/H+ FcyB symporter. The model consists of 12 transmembrane α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct core of 10 TMSs is made of two intertwined inverted repeats (TMS1-5 and TMS6-10), which are followed by two additional TMSs. TMS1, TMS3, TMS6 and TMS8 form an open cavity, which is predicted to host the substrate binding site. Based on primary sequence alignment, 3D topology and substrate docking, we identified five residues as potentially essential for substrate binding in FcyB; S85 (TMS1), W159, N163 (TMS3), W259 (TMS6) and N354 (TMS8). To validate the role of these and other putatively critical residues, we performed a systematic functional analysis of relevant mutants. We show that the proposed substrate binding residues, plus N350, N351 and P353 are irreplaceable for FcyB function. Among these residues, S85, N163, N350, N351 and N354 are critical for determining the substrate binding affinity and/or the specificity of FcyB. Our results suggest that S85, N163 and N354 directly interact with substrates, W159 and W259 stabilise binding through pi-pi stacking interactions, P353 affects the local architecture of substrate binding site, whereas N350 and N351 probably affect substrate binding indirectly. Our work is the first systematic approach to address structure-function-specificity relationships in a eukaryotic member of NCS1 family, by combining genetic and computational approaches.
Journal of Biological Chemistry 09/2012; · 4.77 Impact Factor
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ABSTRACT: Type I casein kinases are highly conserved among Eukaryotes. Of the two Aspergillus nidulans casein kinases I, CkiA is related to the δ/ε mammalian kinases and to Saccharomyces cerevisiae Hrr25p. CkiA is essential. Three recessive ckiA mutations leading to single residue substitutions, and downregulation using a repressible promoter, result in partial loss-of-function, which leads to a pleiotropic defect in amino acid utilization and resistance to toxic amino acid analogues. These phenotypes correlate with miss-routing of the YAT plasma membrane transporters AgtA (glutamate) and PrnB (proline) to the vacuole under conditions that, in the wild type, result in their delivery to the plasma membrane. Miss-routing to the vacuole and subsequent transporter degradation results in a major deficiency in the uptake of the corresponding amino acids that underlies the inability of the mutant strains to catabolize them. Our findings may have important implications for understanding how CkiA, Hrr25p and other fungal orthologues regulate the directionality of transport at the ER-Golgi interface.
Molecular Microbiology 04/2012; 84(3):530-49. · 5.01 Impact Factor
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ABSTRACT: Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA.
Journal of Molecular Biology 06/2011; 411(3):567-80. · 4.00 Impact Factor
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ABSTRACT: We have previously identified by classical genetics and biochemistry, all the genes of Aspergillus nidulans predicted to be involved in purine utilisation, together with cognate regulatory genes and one gene encoding a novel xanthine hydroxylation activity. In this article we complete the description of the purine utilisation pathway with the identification of the two genes (uaX and uaW) encoding the enzymes catalysing the conversion of the product of urate oxidation by urate oxidase, 5-hydroxyisourate, to optically active allantoin. The identification of these additional genes confirms the complete absence of clustering of the genes involved in purine utilisation in A. nidulans.
Fungal Genetics and Biology 03/2011; 48(8):840-8. · 3.74 Impact Factor
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ABSTRACT: We report here the characterization of UreA, a high-affinity urea/H+ symporter of Aspergillus nidulans. The deletion of the encoding gene abolishes urea transport at low substrate concentrations, suggesting that in these conditions UreA is the sole transport system specific for urea in A. nidulans. The ureA gene is not inducible by urea or its precursors, but responds to nitrogen metabolite repression, necessitating for its expression the AreA GATA factor. In contrast to what was observed for other transporters in A. nidulans, repression by ammonium is also operative during the isotropic growth phase. The activity of UreA is down-regulated post-translationally by ammonium-promoted endocytosis. A number of homologues of UreA have been identified in A. nidulans and other Aspergilli, which cluster in four groups, two of which contain the urea transporters characterized so far in fungi and plants. This phylogeny may have arisen by gene duplication events, giving place to putative transport proteins that could have acquired novel, still unidentified functions.
Fungal Genetics and Biology 12/2010; 47(12):1023-33. · 3.74 Impact Factor
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ABSTRACT: In this work we unmask a novel downregulation mechanism of the uric acid/xanthine transporter UapA, the prototype member of the ubiquitous Nucleobase-Ascorbate Transporter family, directly related to its function. In the presence of substrates, UapA is endocytosed, sorted into the multivesicular body pathway and degraded in vacuoles. Substrate-induced endocytosis, unlike ammonium-induced turnover, is absolutely dependent on UapA activity and several lines of evidence showed that the signal for increased endocytosis is the actual translocation of substrates through the UapA protein. The use of several UapA functional mutants with altered kinetics and specificity has further shown that transport-dependent UapA endocytosis occurs through a mechanism, which senses subtle conformational changes associated with the transport cycle. We also show that distinct mechanisms of UapA endocytosis necessitate ubiquitination of a single Lys residue (K572) by HulA(Rsp5). Finally, we demonstrate that in the presence of substrates, non-functional UapA versions can be endocytosed in trans if expressed in the simultaneous presence of active UapA versions, even if the latter cannot be endocytosed themselves.
Molecular Microbiology 12/2009; 75(1):246-60. · 5.01 Impact Factor
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ABSTRACT: Early genetic evidence suggested that A. nidulans possesses at least one uracil transporter. A gene, named furD, was recently identified by reverse genetics and in silico approaches and we confirm here that it encodes a high-affinity, high-capacity, uracil transporter. In this work, we study the regulation of expression of FurD and develop a kinetic model describing transporter-substrate interactions. The furD gene is not expressed in resting conidiospores, is transcriptionally activated and reaches a peak during the isotropic growth phase of conidiospore germination, and stays at a basic low level in mycelium. Transcriptional expression is correlated to uracil transport activity. Expression in a strain blocked in uracil biosynthesis (pyrG-) is moderately increased and extended to later stages of germination. The presence of excess uracil in the medium leads to down-regulation of furD expression and FurD activity. A detailed kinetic analysis using a number of pyrimidine and purine analogues showed that FurD is able to recognize with high-affinity uracil (Km 0.45 microM), thymine (Ki 3.3 microM) and several 5-substituted analogues of uracil, and with moderate affinity uric acid and xanthine (Ki 94-99 microM). Kinetic evidence supports a model in which the positions N1-H, =O2, N3-H, =O4, as well as planarity play a central role for the substrate binding. This model, which rationalizes the unique specificity of FurD for uracil, is compared to and found to be very similar to analogous models for protozoan uracil transporters.
Molecular Membrane Biology 07/2009; 24(3):206-14. · 2.86 Impact Factor
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ABSTRACT: The function of seven paralogues phylogenetically related to the Saccharomyces cerevisiae Fur4p together with a number of functionally related transporters present in Aspergillus nidulans has been investigated. After deletion of the cognate genes we checked the incorporation of radiolabelled substrates, utilization of nitrogen sources, resistance to toxic analogues and supplementation of auxotrophies. FurA and FurD encode allantoin and uracil transporters respectively. No function was found for FurB, FurC, FurE, FurF and FurG. As we failed to identify Fur-related transporters for uridine, pyridoxine or thiamine, we deleted other possible candidates for these functions. A FCY2-like gene carrying in its 5' UTR a putative thiamine pyrophosphate riboswitch, and which encodes a protein similar to the pyridoxine transporter of yeast (Tpn1p), does not encode either a major thiamine or a pyridoxine transporter. CntA, a member of the concentrative nucleoside transporter family, is a general nucleoside permease, while no function was found for PnpA, a member of the equilibrative transporter family. Phylogenetic analysis shows that within the ascomycetes, the same transport activity could be catalysed by totally unrelated proteins and that within the Fur subfamily convergent evolution towards uracil and allantoin transport activity has occurred at least three and two independent times respectively.
Molecular Microbiology 06/2009; 73(1):43-57. · 5.01 Impact Factor
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ABSTRACT: UapA, a uric acid-xanthine permease of Aspergillus nidulans, has been used as a prototype to study structure-function relationships in the ubiquitous nucleobase-ascorbate transporter (NAT) family. Using novel genetic screens, rational mutational design, chimeric NAT molecules, and extensive transport kinetic analyses, we show that dynamic synergy between three distinct domains, transmembrane segment (TMS)1, the TMS8-9 loop, and TMS12, defines the function and specificity of UapA. The TMS8-9 loop includes four residues absolutely essential for substrate binding and transport (Glu356, Asp388, Gln408, and Asn409), whereas TMS1 and TMS12 seem to control, through steric hindrance or electrostatic repulsion, the differential access of purines to the TMS8-9 domain. Thus, UapA specificity is determined directly by the specific interactions of a given substrate with the TMS8-9 loop and indirectly by interactions of this loop with TMS1 and TMS12. We finally show that intramolecular synergy among UapA domains is highly specific and propose that it forms the basis for the evolution of the unique specificity of UapA for uric acid, a property not present in other NAT members.
Journal of Molecular Biology 09/2008; 382(5):1121-35. · 4.00 Impact Factor
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ABSTRACT: Three genes encoding putative purine transporters have been identified in silico in the genome of Aspergillus fumigatus by their very close similarity of their translation products to well-studied homologues in A. nidulans. Two of these transporters, called AfUapC and AfAzgA, were found responsible for bulk uptake of purines and studied in detail herein. Genetic knock-out analysis, regulation of transcription, direct purine uptake assays and heterologous expression in A. nidulans have unequivocally shown that AfUapC and AfAzgA are high-affinity, high-capacity, purine/H(+) symporters, the first being specific for xanthine, uric acid and oxypurinol, whereas the second for adenine, hypoxanthine, guanine and purine. The expression of these transporters is primarily controlled at the level of transcription. Transcription of both genes is purine-inducible, albeit with different efficiencies, whereas AfuapC is also ammonium-repressible. We characterised in detail the kinetics of the AfUapC and AfAzgA transporters, both in A. fumigatus and in A. nidulans, using a plethora of possible purine substrates. This analysis led us to propose kinetic models describing the molecular interactions of AfUapC and AfAzgA with purines. These models are discussed comparatively with analogous models from other purine transporters from fungi, bacteria and humans, and within the frame of a systematic development of novel purine-related antifungals.
Fungal Genetics and Biology 05/2008; 45(4):459-72. · 3.74 Impact Factor
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ABSTRACT: We present a functional analysis of the last alpha-helical transmembrane segment (TMS12) of UapA, a uric acid-xanthine/H+ symporter in Aspergillus nidulans and member of the nucleobase-ascorbate transporter (NAT) family. First, we performed a systematic mutational analysis of residue F528, located in the middle of TMS12, which was known to be critical for UapA specificity. Substitution of F528 with non-aromatic amino acid residues (Ala, Thr, Ser, Gln, Asn) did not affect significantly the kinetics of UapA for its physiological substrates, but allowed high-capacity transport of several novel purines and pyrimidines. Allele-specific combinations of F528 substitutions with mutations in Q408, a residue involved in purine binding, led to an array of UapA molecules with different kinetic and specificity profiles. We propose that F528 plays the role of a novel-type selectivity filter, which, in conjunction with a distinct purine-binding site, control UapA-mediated substrate translocation. We further studied the role of TMS12 by analysing the effect of its precise deletion and chimeric molecules in which TMS12 was substituted with analogous domains from other NATs. The presence of any of the TMS12 tested was necessary for ER-exit while their specific amino acid composition affected the kinetics of chimeras.
Journal of Molecular Biology 04/2006; 357(3):808-19. · 4.00 Impact Factor
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ABSTRACT: Aspergillus nidulans possesses three well-characterized purine transporters encoded by the genes uapA, uapC and azgA. Expression of these genes in mycelium is induced by purines and repressed by ammonium or glutamine through the action of the pathway-specific UaY regulator and the general GATA factor AreA respectively. Here, we describe the regulation of expression of purine transporters during conidiospore germination and the onset of mycelium development. In resting conidiospores, mRNA steady-state levels of purine transporter genes and purine uptake activities are undetectable or very low. Both mRNA steady-state levels and purine transport activities increase substantially during the isotropic growth phase of conidial germination. Both processes occur in the absence of purine induction and independently of the nitrogen source present in the medium. The transcriptional activator UaY is dispensable for the germination-induced expression of the three transporter genes. AreA, on the other hand, is essential for the expression of uapA, but not for that of azgA or uapC, during germination. Transcriptional activation of uapA, uapC and azgA during germination is also independent of the presence of a carbon source in the medium. This work establishes the presence of a novel system triggering purine transporter transcription during germination. Similar results have been found in studies on the expression of other transporters in A. nidulans, suggesting that global expression of transporters might operate as a general system for sensing solute availability.
Molecular Microbiology 05/2004; 52(1):205-16. · 5.01 Impact Factor
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ABSTRACT: The azgA gene of Aspergillus nidulans encodes a hypoxanthine-adenine-guanine transporter. It has been cloned by a novel transposon methodology. The null phenotype of azgA was defined by a number of mutations, including a large deletion. In mycelia, the azgA gene is, like other genes of purine catabolism, induced by uric acid and repressed by ammonium. Its transcription depends on the pathway-specific UaY zinc binuclear cluster protein and the broad domain AreA GATA factor. AzgA is not closely related to any other characterized membrane protein, but many close homologues of unknown function are present in fungi, plants, and prokaryotes but not metazoa. Two of three data bases and the phylogeny presented in this article places proteins of this family in a cluster clearly separated (but perhaps phylogenetically related) from the NAT family that includes other eukaryotic and prokaryotic nucleobase transporters. Thus AzgA is the first characterized member of this family or subfamily of membrane proteins.
Journal of Biological Chemistry 02/2004; 279(5):3132-41. · 4.77 Impact Factor
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ABSTRACT: The azgA gene of Aspergillus nidulans encodes a hypoxanthine-adenine-guanine transporter. It has been cloned by a novel transposon methodology. The null phenotype
of azgA was defined by a number of mutations, including a large deletion. In mycelia, the azgA gene is, like other genes of purine catabolism, induced by uric acid and repressed by ammonium. Its transcription depends
on the pathway-specific UaY zinc binuclear cluster protein and the broad domain AreA GATA factor. AzgA is not closely related
to any other characterized membrane protein, but many close homologues of unknown function are present in fungi, plants, and
prokaryotes but not metazoa. Two of three data bases and the phylogeny presented in this article places proteins of this family
in a cluster clearly separated (but perhaps phylogenetically related) from the NAT family that includes other eukaryotic and
prokaryotic nucleobase transporters. Thus AzgA is the first characterized member of this family or subfamily of membrane proteins.
Journal of Biological Chemistry 01/2004; 279(5):3132-3141. · 4.77 Impact Factor
