50. "A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa."
S. Elsen, G. Efthymiou, P. Peteinatos, G. Diallinas, P. Kyritsis and J.M. Moulis.
BMC Microbiology, vol. 10, pages 271, (2010).  View at publisher's site  Request copy
Abstract:BACKGROUND: Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown. RESULTS: A bioinformatic screening of the sequence features defining very low potential 2[4Fe-4S] ferredoxins has revealed the almost exclusive presence of the corresponding fdx gene in the Proteobacteria phylum, without occurrence in Archaea and Eukaryota. The transcript was found to be monocistronic in Pseudomonas aeruginosa, and not part of an operon in most bacteria. Only fdx genes of bacteria which anaerobically degrade aromatic compounds belong to operons. As this pathway is not present in all bacteria having very low potential 2[4Fe-4S] ferredoxins, these proteins cannot exclusively be reductants of benzoyl CoA reductases. Expression of the ferredoxin gene did not change in response to varying growth conditions, including upon macrophage infection or aerobic growth with 4-hydroxy benzoate as carbon source. However, it increased along the growth curve in Pseudomonas aeruginosa and in Escherichia coli. The sequence immediately 5' upstream of the coding sequence contributed to the promotor activity. Deleting the fdx gene in Pseudomonas aeruginosa abolished growth, unless a plasmid copy of the gene was provided to the deleted strain. CONCLUSIONS: The gene of the very low potential 2[4Fe-4S] ferredoxin displays characteristics of a housekeeping gene, and it belongs to the minority of genes that are essential in Pseudomonas aeruginosa. These data identify a new potential antimicrobial target in this and other pathogenic Proteobacteria.

49. "Dynamic elements at both cytoplasmically and extracellularly facing sides of the UapA transporter selectively control the accessibility of substrates to their translocation pathway."
V. Kosti, I. Papageorgiou and G. Diallinas.
Journal of Molecular Biology, vol. 397, pages 1132-1143, (2010).  View at publisher's site  Request copy
Abstract: In the UapA uric acid-xanthine permease of Aspergillus nidulans, subtle interactions between key residues of the putative substrate binding pocket, located in the TMS8-TMS9 loop (where TMS is transmembrane segment), and a specificity filter, implicating residues in TMS12 and the TMS1-TMS2 loop, are critical for function and specificity. By using a strain lacking all transporters involved in adenine uptake (ΔazgA ΔfcyB ΔuapC) and carrying a mutation that partially inactivates the UapA specificity filter (F528S), we obtained 28 mutants capable of UapA-mediated growth on adenine. Seventy-two percent of mutants concern replacements of a single residue, R481, in the putative cytoplasmic loop TMS10-TMS11. Five missense mutations are located in TMS9, in TMS10 or in loops TMS1-TMS2 and TMS8-TMS9. Mutations in the latter loops concern residues previously shown to enlarge UapA specificity (Q113L) or to be part of a motif involved in substrate binding (F406Y). In all mutants, the ability of UapA to transport its physiological substrates remains intact, whereas the increased capacity for transport of adenine and other purines seems to be due to the elimination of elements that hinder the translocation of non-physiological substrates through UapA, rather than to an increase in relevant binding affinities. The additive effects of most novel mutations with F528S and allele-specific interactions of mutation R481G (TMS10-TMS11 loop) with Q113L (TMS1-TMS2 loop) or T526M (TMS12) establish specific interdomain synergy as a critical determinant for substrate selection. Our results strongly suggest that distinct domains at both sides of UapA act as selective dynamic gates controlling substrate access to their translocation pathway.

48. "Expression and purification of a functional uric acid-xanthine transporter (UapA)."
J. Leung, M. Karachaliou, C. Alves, G. Diallinas and B. Byrne.
Protein Expression and Purification, vol. 72, pages 139-146, (2010).  View at publisher's site  Request copy
Abstract: The Nucleobase-Ascorbate Transporters (NATs) family includes carriers with fundamental functions in uptake of key cellular metabolites, such as uric acid or vitamin C. The best studied example of a NAT transporter is the uric acid-xanthine permease (UapA) from the model ascomycete Aspergillus nidulans. Detailed genetic and biochemical analyses have revealed much about the mechanism of action of this protein; however, the difficulties associated with handling eukaryotic membrane proteins have limited efforts to elucidate the precise structure-function relationships of UapA by structural analysis. In this manuscript, we describe the heterologous overexpression of functional UapA as a fusion with GFP in different strains of Saccharomyces cerevisiae. The UapA-GFP construct expressed to 2.3 mg/L in a pep4Delta deletion strain lacking a key vacuolar endopeptidase and 3.8 mg/L in an npi1-1 mutant strain with defective Rsp5 ubiquitin ligase activity. Epifluorescence microscopy revealed that the UapA-GFP was predominately localized to the plasma membrane in both strains, although a higher intensity of fluorescence was observed for the npi1-1 mutant strain plasma membrane. In agreement with these observations, the npi1-1 mutant strain demonstrated a approximately 5-fold increase in uptake of [(3)H]-xanthine compared to the pep4Delta deletion strain. Despite yielding the best results for functional expression, in-gel fluorescence of the UapA-GFP expressed in the npi1-1 mutant strain revealed that the protein was subject to significant proteolytic degradation. Large scale expression of the protein using the pep4Delta deletion strain followed by purification produced mg quantities of pure, monodispersed protein suitable for further structural and functional studies. In addition, this work has generated a yeast cell based system for performing reverse genetics and other targeted approaches, in order to further understand the mechanism of action of this important model protein.

47. "Transporters, channels, or simple diffusion? Dogmas, atypical roles and complexity in transport systems."
A. Conde, G. Diallinas, F. Chaumont, M. Chaves and H. Gerós.
The International Journal of Biochemistry and Cell Biology, vol. 42, pages 857-868, (2010).  View at publisher's site  Request copy
Abstract: The recent breakthrough discoveries of transport systems assigned with atypical functions provide evidence for complexity in membrane transport biochemistry. Some channels are far from being simple pores creating hydrophilic passages for solutes and can, unexpectedly, act as enzymes, or mediate high-affinity uptake, and some transporters are surprisingly able to function as sensors, channels or even enzymes. Furthermore, numerous transport studies have demonstrated complex multiphasic uptake kinetics for organic and mineral nutrients. The biphasic kinetics of glucose uptake in Saccharomyces cerevisiae, a result of several genetically distinct uptake systems operating simultaneously, is a classical example that is a subject of continuous debate. In contrast, some transporters display biphasic kinetics, being bona fidae dual-affinity transporters, their kinetic properties often modulated by post-translational regulation. Also, aquaporins have recently been reported to exhibit diverse transport properties and can behave as highly adapted, multifunctional channels, transporting solutes such as CO(2), hydrogen peroxide, urea, ammonia, glycerol, polyols, carbamides, purines and pyrimidines, metalloids, glycine, and lactic acid, rather than being simple water pores. The present review provides an overview on some atypical functions displayed by transporter proteins and discusses how this novel knowledge on cellular uptake systems may be related to complex multiphasic uptake kinetics often seen in a wide variety of living organisms and the intriguing diffusive uptake of sugars and other solutes.

46. "Transport-dependent endocytosis and turnover of a uric acid-xanthine permease."
C. Gournas, S. Amillis, A. Vlanti and G. Diallinas.
Molecular Microbiology, vol. 75, pages 246-260, (2010).  View at publisher's site  Request copy
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.

45. "Modelling and mutational evidence identify the substrate binding site and functional elements in APC amino acid transporters."
I. Vangelatos, D. Vlachakis, V. Sophianopoulou and G. Diallinas.
Molecular Membrane Biology, vol. 26, pages 356-370, (2010).  View at publisher's site  Request copy
Abstract: The Amino acid-Polyamine-Organocation (APC) superfamily is the main family of amino acid transporters found in all domains of life and one of the largest families of secondary transporters. Here, using a sensitive homology threading approach and modelling we show that the predicted structure of APC members is extremely similar to the crystal structures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities. All of these proteins, despite having no primary amino acid sequence similarity, share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. Based on this model, we reviewed available data on functional mutations in bacterial, fungal and mammalian APCs and obtained novel mutational data, which provide compelling evidence that the amino acid binding pocket is located in the vicinity of the unwound part of two broken helices, in a nearly identical position to the structures of similar transporters. Our analysis is fully supported by the evolutionary conservation and specific amino acid substitutions in the proposed substrate binding domains. Furthermore, it allows predictions concerning residues that might be crucial in determining the specificity profile of APC members. Finally, we show that two cytoplasmic loops constitute important functional elements in APCs. Our work along with different kinetic and specificity profiles of APC members in easily manipulated bacterial and fungal model systems could form a unique framework for combining genetic, in-silico and structural studies, for understanding the function of one of the most important transporter families.

44. "Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans."
Z. Hamari, S. Amillis, C. Drevet, A. Apostolaki, C. Vágvölgyi, G. Diallinas and C. Scazzocchio.
Molecular Microbiology, vol. 73, pages 43-57, (2009).  View at publisher's site  Request copy
Abstract: IThe 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.

43. "Biochemistry. An almost-complete movie."
G. Diallinas and C. Gournas.
Science, vol. 322, pages 1644-1645, (2008).  View at publisher's site  Request copy
Abstract: Comment on Science. 2008 Dec 12;322(5908):1655-61. No abstract available.

42. "Structure-function relationships in the nucleobase-ascorbate transporter (NAT) family: lessons from model microbial genetic systems."
G. Diallinas and C. Gournas.
Channels (Austin), vol. 2, pages 363-372, (2008).  View at publisher's site  Request copy
Abstract: No abstract available.

41. "Purification and partial characterization of the xanthine-uric acid transporter (UapA) of Aspergillus nidulans".
N. D. Lemuh, G. Diallinas, S. Frillingos, G. Mermelekas, A. D. Karagouni and D. G. Hatzinikolaou.
Protein Expression and Purification, vol. 63, pages 33-39, (2009).  View at publisher's site  Request copy
Abstract: UapA, the uric acid-xanthine permease from the filamentous ascomycete Aspergillus nidulans, is one of the most thoroughly characterized purine/H⁺ transporters in eukaryotes. Detailed studies have addressed its regulation of expression, at both the transcriptional and post-translational levels, in response to physiological and developmental signals. An extensive kinetic profile towards a plethora of purines and mutational analyses have established models on how UapA recognizes the purine ring and revealed specific amino acid residues involved in proper folding, topogenesis, function and specificity. The present work describes for the first time the purification of the UapA transporter of A. nidulans through overexpression via the strong, ethanol-inducible, glucose-repressible, alcA promoter. Purification, almost to homogeneity, was achieved by Ni²⁺ affinity chromatography using a functional His-tagged UapA protein version. It is subsequently shown, by Circular Dichroism (CD) spectroscopy, that the purified protein is structured with a high α-helical content, as expected from the in silico predictions. The result of this work opens the way for further, analytical and biochemical studies on UapA at the protein level.

40. "Specific interdomain synergy in the UapA transporter determines its unique specificity for uric acid among NAT carriers."
I. Papageorgiou, C. Gournas, A. Vlanti, S. Amillis, A. Pantazopoulou and G. Diallinas.
Journal of Molecular Biology, vol. 382, pages 1121-1135, (2008).  View at publisher's site  Request copy
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.

39. "The nucleobase-ascorbate transporter (NAT) family: genomics, evolution, structure-function relationships and physiological role."
C. Gournas, I. Papageorgiou and G. Diallinas.
Molecular Biosystems, vol. 4, pages 404-416, (2008).  View at publisher's site  Request copy
Abstract: This review summarizes knowledge concerning a ubiquitous plasma transmembrane protein family that mediates nucleobase or ascorbate secondary active transport (NAT). We show that prototype bacterial and mostly fungal members have become unique model systems to unravel structure-function relationships and regulation of expression, using classical and reverse genetics, as well as biochemical approaches. We discuss the importance of NAT-mediated ascorbate transport in mammals and how changes in substrate specificity, from different nucleobases to ascorbate, might have evolved at the molecular level. Finally, we also discuss how modelling NAT-purine interactions might constitute a step towards the use of NAT proteins as specific gateways for targeting pathogenic microbes.

38. "The Aspergillus nidulans FcyB cytosine-purine scavenger is highly expressed during germination and in reproductive compartments and is downregulated by endocytosis."
A. Vlanti and G. Diallinas.
Molecular Microbiology, vol. 68, pages 959-977, (2008).  View at publisher's site  Request copy
Abstract: We cloned and characterized an Aspergillus nidulans gene, called fcyB, encoding the closest homologue to the yeast Fcy2p/Fcy21p permeases. Deletion of fcyB (DeltafcyB) does not affect growth, development, reproduction or bulk purine uptake, but eliminates the leaky growth on purines of ΔazgA ΔuapC ΔuapA strains, lacking all known purine transporters, and confers resistance to the antifungal 5-fluorocytosine. Kinetic analyses showed FcyB is a low-capacity, high-affinity, cytosine-purine transporter sharing similar molecular interactions for substrate recognition with the yeast Fcy2p/Fcy21p carriers. fcyB transcription is highly activated during germination but drops at low constitutive levels throughout vegetative development. UaY-mediated purine induction of fcyB transcription is only moderate, while ammonium represses transcription through an AreA-dependent mechanism. A strain expressing FcyB-GFP confirms a low protein expression level in the plasma membrane of vegetative mycelia, but reveals an abundant expression in sexual and asexual compartments. FcyB-GFP was also shown to be downregulated by endocytosis in response to ammonia or the presence of cytosine. The expression profile of FcyB supports that its main physiological role is cytosine-purine scavenging.

37. "Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters."
I. Papageorgiou, H.P. De Koning, K. Soteriadou and G. Diallinas.
International Journal of Parasitology, vol. 38, pages 641-653, (2008).  View at publisher's site  Request copy
Abstract: Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.

36. "Characterization and kinetics of the major purine transporters in Aspergillus fumigatus."
S. Goudela, U. Reichard, S. Amillis and G. Diallinas.
Fungal Genetics and Biology, vol. 45, pages 459-472, (2008).  View at publisher's site  Request copy
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.

35. "Fungal nucleobase transporters."
A. Pantazopoulou and G. Diallinas.
FEMS Microbiol Reviews, vol. 31, pages 657-671, (2007).  View at publisher's site  Request copy
Abstract: Early genetic and physiological work in bacteria and fungi has suggested the presence of highly specific nucleobase transport systems. Similar transport systems are now known to exist in algae, plants, protozoa and metazoa. Within the last 15 years, a small number of microbial genes encoding nucleobase transporters have been cloned and studied in great detail. The sequences of several other putative proteins submitted to databases are homologous to the microbial nucleobase transporters but their physiological functions remain largely undetermined. In this review, genetic, biochemical and molecular data are described concerning mostly the nucleobase transporters of Aspergillus nidulans and Saccharomyces cerevisiae, the two model ascomycetes from which the great majority of data come from. It is also discussed as to what is known on the nucleobase transporters of the two most significant pathogenic fungi: Candida albicans and Aspergillus fumigatus. Apart from highlighting how a basic process such as nucleobase recognition and transport operates, this review intends to highlight features that might be applicable to antifungal pharmacology.

34. "The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H(+) symporter subfamily defines the function of the substrate translocation pathway."
I. Soares-Silva, S. Paiva, G. Diallinas and M. Casal.
Molecular Membrane Biology, vol. 24, pages 464-474, (2007).  View at publisher's site  Request copy
Abstract: In Saccharomyces cerevisiae Jen1p is a lactate/proton symporter belonging to the lactate/pyruvate:H(+) symporter subfamily (TC#2.A.1.12.2) of the Major Facilitator Superfamily. We investigated structure-function relationships of Jen1p using a rational mutational analysis based on the identification of conserved amino acid residues. In particular, we studied the conserved sequence (379)NXX[S/T]HX[S/T]QDXXXT(391). Substitution of amino acid residues N379, H383 or D387, even with very similar amino acids, resulted in a dramatic reduction of lactate and pyruvate uptake, but conserved measurable acetate transport. Acetate transport inhibition assays showed that these mutants conserve the ability to bind, but do not transport, lactate and pyruvate. More interestingly, the double mutation H383D/D387H, while behaving as a total loss-of-function allele for lactate and pyruvate uptake, can fully restore the kinetic parameters of Jen1p for acetate transport. Thus, residues N379, H383 or D387 affect both the transport capacity and the specificity of Jen1p. Substitutions of Q386 and T391 resulted in no or moderate changes in Jen1p transport capacities for lactate, pyruvate and acetate. On the other hand, Q386N reduces the binding affinities for all Jen1p substrates, while Q386A increases the affinity specifically for pyruvate. We also tested Jen1p specificity for a range of monocarboxylates. Several of the mutants studied showed altered inhibition constants for these acids. These results and 3D in silico modelling by homology threading suggest that the conserved motif analyzed is part of the substrate translocation pathway in the lactate/pyruvate:H(+) symporter subfamily.

33. "Regulation of expression and kinetic modeling of substrate interactions of a uracil transporter in Aspergillus nidulans."
S. Amillis, Z. Hamari, K. Roumelioti, C. Scazzocchio and G. Diallinas.
Molecular Membrane Biology, vol. 24, pages 206-214, (2007).  View at publisher's site  Request copy
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.

32. "Differential physiological and developmental expression of the UapA and AzgA purine transporters in Aspergillus nidulans."
A. Pantazopoulou, N.D. Lemuh , D.G. Hatzinikolaou, C. Drevet, G. Cecchetto, C. Scazzocchio and G. Diallinas.
Fungal Genetics and Biology, vol. 44, pages 627-640, (2007).  View at publisher's site  Request copy
Abstract: In this article we study the cellular expression of UapA and AzgA, the two major purine transporters of Aspergillus nidulans, by constructing strains expressing, from their native promoters, fully functional fluorescent (UapA-sGFP, AzgA-sGFP) or immunological (UapA-His) chimeric transporters. Epifluorescence microscopy and immunodetection showed that under different physiological conditions and during Aspergillus development: (i) UapA and AzgA expression in the plasma membrane becomes evident early during germination and remains at a significant basal level in mycelium, (ii) Neither of the two transporters is expressed in the stalk, the vesicle, the phialides and the conidiospores, but surprisingly, UapA is specifically and strongly expressed in the periphery of metulae, (iii) Both transporters are expressed in ascogenous hyphae and in hülle cells but not in cleistothecia or ascospores, (iv) Purine induction leads to approximately 4-fold increase in UapA and AzgA protein content in mycelium, compatible with an analogous increase at the transcriptional level, (v) Ammonium leads to removal of UapA, but not of AzgA, from the plasma membrane by sorting of the protein to the vacuole.

31. "The first transmembrane segment (TMS1) of UapA contains determinants necessary for expression in the plasma membrane and purine transport."
A. Pantazopoulou and G. Diallinas.
Molecular Membrane Biology, vol. 23, pages 337-348, (2006).  View at publisher's site  Request copy
Abstract: UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H+ symporter in Aspergillus nidulans. Determinants critical for substrate binding and transport lie in a highly conserved signature motif downstream from TMS8 and within TMS12. Here we examine the role of TMS1 in UapA biogenesis and function. First, using a mutational analysis, we studied the role of a short motif (Q85H86), conserved in all NATs. Q85 mutants were cryosensitive, decreasing (Q85L, Q85N, Q85E) or abolishing (Q85T) the capacity for purine transport, without affecting physiological substrate binding or expression in the plasma membrane. All H86 mutants showed nearly normal substrate binding affinities but most (H86A, H86K, H86D) were cryosensitive, a phenotype associated with partial ER retention and/or targeting of UapA in small vacuoles. Only mutant H86N showed nearly wild-type function, suggesting that His or Asn residues might act as H donors in interactions affecting UapA topology. Thus, residues Q85 and H86 seem to affect the flexibility of UapA, in a way that affects either transport catalysis per se (Q85), or expression in the plasma membrane (H86). We then examined the role of a transmembrane Leu Repeat (LR) motif present in TMS1 of UapA, but not in other NATs. Mutations replacing Leu with Ala residues altered differentially the binding affinities of xanthine and uric acid, in a temperature-sensitive manner. This result strongly suggested that the presence of L77, L84 and L91 affects the flexibility of UapA substrate binding site, in a way that is necessary for high affinity uric acid transport. A possible role of the LR motif in intramolecular interactions or in UapA dimerization is discussed.

30. "Comparative kinetic analysis of AzgA and Fcy21p, prototypes of the two major fungal hypoxanthine-adenine-guanine transporter families."
S. Goudela, H. Tsilivi and G. Diallinas.
Molecular Membrane Biology, vol. 23, pages 291-303, (2006).  View at publisher's site  Request copy
Abstract: In fungi, uptake of salvageable purines is carried out by members of two evolutionarily distinct protein families, the Purine-Related Transporters (PRT/NCS1) and the AzgA-like Transporters. We carried out a comparative kinetic analysis of two prototypes of these transporter families. The first was Fcy21p, a herein characterized protein of Candida albicans, and the second was AzgA, a transporter of Aspergillus nidulans. Our results showed that: (i) AzgA and Fcy21p are equally efficient high-affinity, high-capacity, purine transporters, (ii) Fcy21p, but not AzgA, is an efficient cytosine and 5-fluorocytosine transporter, interacting with =O2 and C4-NH2 of the pyrimidine ring, (iii) the major interactions of AzgA and Fcy21p with the purine ring are similar, but not identical, involving in all cases positions 6 and 7, and for some substrates, positions 1 and 9 as well, and (iv) in AzgA, bulky groups at position N3 have a detrimental steric effect on substrate binding, while similar substitutions at C2 or N9 are fully or partially tolerated. In contrast, in Fcy21p, C2 and N9 bulky substitutions abolish substrate binding, while similar substitutions in N3 are fully tolerated. These results suggest that all fungal purine transporters might have evolved from a single ancestral protein, and show that fungal transporters use different substrate interactions compared to the analogous protozoan or mammalian proteins. Finally, results are also discussed in respect of the possibility of using fungal purine transporters as specific gateways for the development of targeted antifungal pharmacological therapies.

29. "A novel-type substrate-selectivity filter and ER-exit determinants in the UapA purine transporter."
A. Vlanti, S. Amillis, M. Koukaki and G. Diallinas.
Journal of Molecular Biology, vol. 357, pages 808-819, (2006).  View at publisher's site  Request copy
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.