28. "Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family."
S. Goudela, P. Karatza, M. Koukaki, S. Frillingos and G. Diallinas.
Molecular Membrane Biology, vol. 22, pages 263-275, (2005).  View at publisher's site  Request copy
Abstract: We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.

27. "The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway."
M. Koukaki, A. Vlanti, S. Goudela, A. Pantazopoulou, H. Gioule, S. Tournaviti and G. Diallinas.
Journal of Molecular Biology, vol. 350, pages 499-513, (2005).  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 of Aspergillus nidulans. We have previously presented evidence showing that a highly conserved signature motif ([Q/E/P]408-N-X-G-X-X-X-X-T-[R/K/G])417 is involved in UapA function. Here, we present a systematic mutational analysis of conserved residues in or close to the signature motif of UapA. We show that even the most conservative substitutions of residues Q408, N409 and G411 modify the kinetics and specificity of UapA, without affecting targeting in the plasma membrane. Q408 substitutions show that this residue determines both substrate binding and transport catalysis, possibly via interactions with position N9 of the imidazole ring of purines. Residue N409 is an irreplaceable residue necessary for transport catalysis, but is not involved in substrate binding. Residue G411 determines, indirectly, both the kinetics (K(m), V) and specificity of UapA, probably due to its particular property to confer local flexibility in the binding site of UapA. In silico predictions and a search in structural databases strongly suggest that the first part of the NAT signature motif of UapA (Q(408)NNG(411)) should form a loop, the structure of which is mostly affected by mutations in G411. Finally, substitutions of residues T416 and R417, despite being much better tolerated, can also affect the kinetics or the specificity of UapA. Our results show that the NAT signature motif defines the function of the UapA purine translocation pathway and strongly suggest that this might occur by determining the interactions of UapA with the imidazole part of purines.

26. "Identification of the first pyrimidine nucleobase transporter in Leishmania: similarities with the Trypanosoma brucei U1 transporter and antileishmanial activity of uracil analogues."
I.G. Papageorgiou, L. Yakob, M/I. Al Salabi, G. Diallinas, K.P. Soteriadou and H.P. De Koning.
Parasitology, vol. 130, pages 275-283, (2005).  View at publisher's site  Request copy
Abstract: While purine transport has been widely studied in protozoa, almost nothing is known about their capacity to salvage pyrimidines. Here, we report a Leishmania major transporter with high affinity for uracil (Km=0.32+/-0.07 microM) which we designated LmU1. This transporter displayed a high degree of specificity, as it had virtually no affinity for cytosine, thymine or purine nucleobases, nor did it transport pyrimidine nucleosides. Highest affinity was for 5-fluorouracil. The results show that the permeant binding site of LmU1 interacts strongly with the keto groups of uracil, as shown by a low affinity for 2-thio- and 4-thiouracil. LmU1 appears to further bind uracil through a weak hydrogen bond with N(1)H of the pyrimidine ring in addition to a stronger H-bond with N(3)H. Substrate binding and selectivity were strikingly similar to that of the U1 transporter in the related kinetoplastid Trypanosoma brucei. Uracil analogues likely to be transported by LmU1 were also screened for antileishmanial activity, with 5-fluorouracil displaying strong activity against promastigotes and intracellular amastigotes. Overall, the results show that, like purine nucleobase transport, pyrimidine nucleobase transport function is very similar in L. major and T. brucei insect forms.

25. "Transcription of purine transporter genes is activated during the isotropic growth phase of Aspergillus nidulans conidia."
S. Amillis, G. Cecchetto, V. Sophianopoulou, M. Koukaki, C. Scazzocchio and G. Diallinas.
Molecular Microbiology, vol. 52, pages 205-216, (2004).  View at publisher's site  Request copy
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.

24. "A novel improved method for Aspergillus nidulans transformation."
M. Koukaki, E. Giannoutsou, A. Karagouni and G. Diallinas.
Journal of Microbiological Methods, vol. 55, pages 687-695, (2003).  View at publisher's site  Request copy
Abstract: We systematically investigated the efficiency of Aspergillus nidulans transformation using protoplasts prepared from different stages of conidiospore germination and young mycelium. Using standard integrative plasmids, increased transformation yields were obtained with protoplasts isolated from a specific stage coincident with germ tube emergence. This increase ranged, on the average, from two- to eightfold depending on different plasmids used. Transformation efficiencies with a replicative plasmid were similar to those obtained using previously described methods. Although this observation suggests that elevated transformation efficiencies might be due to increased efficiency of recombination between plasmid and genomic sequences, we cannot exclude other factors associated with the particular developmental stage used. In the course of this study, we also examined the effect of other parameters that might enhance transformation yields. The method described is also significantly easier and faster than other current methods. .

23. "The AzgA purine transporter of Aspergillus nidulans. Characterization of a protein belonging to a new phylogenetic cluster."
G. Cecchetto, S. Amillis, G. Diallinas, C. Scazzocchio and C. Drevet.
Journal of Biological Chemistry, vol. 279, pages 3132–3141, (2004).  View at publisher's site  Request copy
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.

22. "Mutational analysis of the major proline transporter (PrnB) of Aspergillus nidulans."
S.N. Tavoularis, U.H. Tazebay, G. Diallinas, M. Sideridou, A. Rosa A, C. Scazzocchio and V. Sophianopoulou.
Molecular Membrane Biology, vol. 20, pages 285-297, (2003).  View at publisher's site  Request copy
Abstract: PrnB, the l-proline transporter of Aspergillus nidulans, belongs to the Amino acid Polyamine Organocation (APC) transporter family conserved in prokaryotes and eukaryotes. In silico analysis and limited biochemical evidence suggest that APC transporters comprise 12 transmembrane segments (TMS) connected with relatively short hydrophilic loops (L). However, very little is known on the structure-function relationships in APC transporters. This work makes use of the A. nidulans PrnB transporter to address structure-function relationships by selecting, constructing and analysing several prnB mutations. In the sample, most isolated missense mutations affecting PrnB function map in the borders of cytoplasmic loops with transmembrane domains. These are I119N and G120W in L2-TMS3, F278V in L6-TMS7, NRT378NRTNRT and PY382PYPY in L8-TMS9 and T456N in L10-TMS11. A single mutation (G403E) causing, however, a very weak phenotype, maps in the borders of an extracellular loop (L9-TMS10). An important role of helix TMS6 for proline binding and transport is supported by mutations K245L and, especially, F248L that clearly affect PrnB uptake kinetics. The critical role of these residues in proline binding and transport is further shown by constructing and analysing isogenic strains expressing selected prnB alleles fused to the gene encoding the Green Fluorescent Protein (GFP). It is shown that, while some prnB mutations affect proper translocation of PrnB in the membrane, at least two mutants, K245E and F248L, exhibit physiological cellular expression of PrnB and, thus, the corresponding mutations can be classified as mutations directly affecting proline binding and/or transport. Finally, comparison of these results with analogous studies strengthens conclusions concerning amino acid residues critical for function in APC transporters.

21. "Substitution F569S converts UapA, a specific uric acid-xanthine transporter, into a broad specificity transporter for purine-related solutes."
S. Amillis, M. Koukaki and G. Diallinas.
Journal of Molecular Biology, vol. 313, pages 765-774, (2001).  View at publisher's site  Request copy
Abstract: UapA, a highly specific uric acid-xanthine transporter in Aspergillus nidulans, is a member of a large family of nucleobase-ascorbate transporters conserved in all domains of life. We have investigated structure-function relationships in UapA, by studying chimeric transporters and missense mutations, and showed that specific polar or charged amino acid residues (E412, E414, Q449, N450, T457) on either side of an amphipathic alpha-helical transmembrane segment (TMS10) are critical for purine binding and transport. Here, the mutant Q449E, having no uric acid-xanthine transport activity at 25 degrees C, was used to isolate second-site revertants that restore function. Seven of them were found to have acquired the capacity to transport novel substrates (hypoxanthine and adenine) in addition to uric acid and xanthine. All seven revertants were found to carry the mutation F569S within the last transmembrane segment (TMS14) of UapA. Further kinetic analysis of a selected suppressor showed that UapA-Q449E/F569S transports with high affinity (K(M) values of 4-10 microM) xanthine, hypoxanthine and uracil. Uptake competition experiments suggested that UapA-Q449E/F569S also binds guanine, 6-thioguanine, adenosine or ascorbic acid. A strain carrying mutation F569S by itself conserves high-capacity, high-affinity (K(M) values of 1.5-15 microM), transport activity for purine-uracil transport. Compared to UapA-Q449E/F569S, UapA-F569S has a distinct capacity to bind several nucleobase-related compounds and different kinetic parameters of transport. These results show that molecular determinants external to the central functional domain (L9-TMS10-L10) are critical for the uptake specificity and transport kinetics of UapA.

20. "A novel putative reductase (Cpd1p) and the multidrug exporter Snq2p are involved in resistance to cercosporin and other singlet oxygen-generating photosensitizers in Saccharomyces cerevisiae."
P. Ververidis, F. Davrazou, G. Diallinas, D. Georgakopoulos, A.K. Kanellis and N. Panopoulos.
Current Genetics, vol. 39, pages 127-136, (2001).  View at publisher's site  Request copy
Abstract: Phytopathogenic Cercospora species produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers which absorb light energy and produce extremely cytotoxic, reactive oxygen species. In this work, we used Saccharomyces cerevisiae as a model system for the identification and cloning of genes whose products mediate cercosporin detoxification. Two genesexpressed in high-copy number vectors conferred cercosporin resistance to an otherwise sensitive strain. One gene codes for Snq2p, a well-characterized multidrug, ABC-type, efflux protein. The other, designated CPD1 (Cercosporin Photosensitizer Detoxification), encodes a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases. We showed that over-expression of either of these proteins can also mediate resistance to other singlet oxygen-generating compounds. The involvement of Snq2p and Cpd1p in photosensitizer detoxification reinforces previous observations which suggested that singlet oxygen acts on membrane lipids and that cellular resistance to cercosporin is mediated by a mechanism involving toxin efflux and/or toxin reduction.

19. "Functional characterization of a maize purine transporter by expression in Aspergillus nidulans."
E. Argyrou, V. Sophianopoulou, N. Schultes and G. Diallinas.
Plant Cell, vol. 13, pages 953-964, (2001).  View at publisher's site  Request copy
Abstract: We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport-deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (K(m) = 30 +/- 2.5 microM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.