Sotiris Amillis

Specific Interdomain Synergy in the UapA Transporter Determines Its Unique Specificity for Uric Acid among NAT Carriers

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.

Transport‐dependent endocytosis and turnover of a uric acid‐xanthine permease

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 HulARsp5. 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.

Transcription of purine transporter genes is activated during the isotropic growth phase of Aspergillus nidulans conidia

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.

The arrestin-like protein ArtA is essential for ubiquitination and endocytosis of the UapA transporter in response to both broad-range and specific signals.

We investigated the role of all arrestin‐like proteins of Aspergillus nidulans in respect to growth, morphology, sensitivity to drugs and specifically for the endocytosis and turnover of the uric acid‐xanthine transporter UapA. A single arrestin‐like protein, ArtA, is essential for HulARsp5‐dependent ubiquitination and endocytosis of UapA in response to ammonium or substrates. Mutational analysis showed that residues 545–563 of the UapA C‐terminal region are required for efficient UapA endocytosis, whereas the N‐terminal region (residues 2–123) and both PPxY motives are essential for ArtA function. We further show that ArtA undergoes HulA‐dependent ubiquitination at residue Lys‐343 and that this modification is critical for UapA ubiquitination and endocytosis. Lastly, we show that ArtA is essential for vacuolar turnover of transporters specific for purines (AzgA) or l‐proline (PrnB), but not for an aspartate/glutamate transporter (AgtA). Our results are discussed within the frame of recently proposed mechanisms on how arrestin‐like proteins are activated and recruited for ubiquitination of transporters in response to broad range signals, but also put the basis for understanding how arrestin‐like proteins, such as ArtA, regulate the turnover of a specific transporter in the presence of its substrates.

Convergent evolution and orphan genes in the Fur4p-like family and characterization of a general nucleoside transporter in Aspergillus nidulans

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.

Regulation of expression and kinetic modeling of substrate interactions of a uracil transporter in Aspergillus nidulans

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 µM), thymine (Ki 3.3 µM) and several 5-substituted analogues of uracil, and with moderate affinity uric acid and xanthine (Ki 94–99 µM). 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.

Structure of eukaryotic purine/H(+) symporter UapA suggests a role for homodimerization in transport activity.

The uric acid/xanthine H+ symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1–11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

Modeling, Substrate Docking, and Mutational Analysis Identify Residues Essential for the Function and Specificity of a Eukaryotic Purine-Cytosine NCS1 Transporter

Background: The purine-cytosine FcyB transporter is a prototype member of the NCS1 family. Results: Using homology modeling, substrate docking, and rational mutational analysis, we identify residues critical for function and specificity. Conclusion: Important aspects concerning the molecular mechanism and evolution of transporter specificity are revealed. Significance: The first systematic approach on structure-function-specificity relationships in a eukaryotic NCS1 member is shown. 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 three-dimensional 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) that are followed by two additional TMSs. TMS1, TMS3, TMS6, and TMS8 form an open cavity that is predicted to host the substrate binding site. Based on primary sequence alignment, three-dimensional topology, and substrate docking, we identified five residues as potentially essential for substrate binding in FcyB; Ser-85 (TMS1), Trp-159, Asn-163 (TMS3), Trp-259 (TMS6), and Asn-354 (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 Asn-350, Asn-351, and Pro-353 are irreplaceable for FcyB function. Among these residues, Ser-85, Asn-163, Asn-350, Asn-351, and Asn-354 are critical for determining the substrate binding affinity and/or the specificity of FcyB. Our results suggest that Ser-85, Asn-163, and Asn-354 directly interact with substrates, Trp-159 and Trp-259 stabilize binding through π-π stacking interactions, and Pro-353 affects the local architecture of substrate binding site, whereas Asn-350 and Asn-351 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.

Completing the purine utilisation pathway of Aspergillus nidulans

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.

UreA, the major urea/H+ symporter in Aspergillus nidulans

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.