Mutational analysis and modeling reveal functionally critical residues in transmembrane segments 1 and 3 of the UapA transporter

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

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

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

Characterization of AnNce102 and its role in eisosome stability and sphingolipid biosynthesis

The plasma membrane is implicated in a variety of functions, whose coordination necessitates highly dynamic organization of its constituents into domains of distinct protein and lipid composition. Eisosomes, at least partially, mediate this lateral plasma membrane compartmentalization. In this work, we show that the Nce102 homologue of Aspergillus nidulans colocalizes with eisosomes and plays a crucial role in density/number of PilA/SurG foci in the head of germlings. In addition we demonstrate that AnNce102 and PilA negatively regulate sphingolipid biosynthesis, since their deletions partially suppress the thermosensitivity of basA mutant encoding sphingolipid C4-hydroxylase and the growth defects observed upon treatment with inhibitors of sphingolipid biosynthesis, myriocin and Aureobasidin A. Moreover, we show that YpkA repression mimics genetic or pharmacological depletion of sphingolipids, conditions that induce the production of Reactive Oxygen Species (ROS), and can be partially overcome by deletion of pilA and/or annce102 at high temperatures. Consistent with these findings, pilA " and annce102 " also show differential sensitivity to various oxidative agents, while AnNce102 overexpression can bypass sphingolipid depletion regarding the PilA/SurG foci number and organization, also leading to the mislocalization of PilA to septa.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi

Filamentous fungi provide excellent systems for investigating the role of the AP-2 complex in polar growth. Using Aspergillus nidulans we show that AP-2 has a clathrin-independent essential role in polarity maintenance and growth. This is in line with a sequence analysis showing that the AP-2 beta subunit (beta 2) of higher fungi lacks a clathrin-binding domain and experiments showing that AP-2 does not co-localize with clathrin. We provide genetic and cellular evidence that AP-2 interacts with endocytic markers SlaB(End4) and SagA(End3) and the lipid flippases DnfA and DnfB in the sub-apical collar region of hyphae. The role of AP-2 in the maintenance of proper apical membrane lipid and cell wall composition is further supported by its functional interaction with BasA (sphingolipid biosynthesis) and StoA (apical sterol-rich membrane domains) and its essentiality in polar deposition of chitin. Our findings support that the AP-2 complex of dikarya has acquired in the course of evolution a specialized clathrin-independent function necessary for fungal polar growth

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

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

Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins

Informe final del proyecto: Profundización en la identificación de determinantes estructurales y funcionales de UreA

En el hongo filamentoso Aspergillus nidulans el transporte de urea tiene lugar a través de UreA, un simportador urea/H+ con ortólogos en hongos y plantas, perteneciente a la familia de los simportadores de sodio (SSS). Nuestro grupo llevó a cabo un estudio de la relación estructura- función de UreA mediante una estrategia de mutagénesis y modelado tridimensional. El mismo permitió identificar una serie de aminoácidos implicados en la unión, reconocimiento y translocación de la urea por parte del transportador. En este proyecto se propuso ahondar en el conocimiento de los determinantes estructurales y funcionales de UreA, mediante la identificación de nuevos residuos o regiones de UreA implicadas en la unión al sustrato y la selectividad por el mismo. En este sentido pudimos determinar que los aminoácidos W82, W84, N279 y T282 cumplen un rol en la interacción y/o selectividad por el sustrato, proponiendo a su vez, que la región en donde éstos se encuentran comprendería el sitio de unión al sustrato de UreA. Asimismo se realizó un análisis de los “loops” extracelulares 3 e intracelular 7, determinando que éstos poseen un rol estructural y funcional en UreA. Ya que la oligomerización de transportadores es un fenómeno que aparece con frecuencia en la biología de los mismos, pero no se conoce cuán general es ésta ni cuál es su rol para cada clase de proteínas de transporte, nos propusimos aportar a esta temática determinando si UreA es capaz de formar oligómeros. Se obtuvieron resultados preliminares que indicarían que UreA oligomeriza. Actualmente estamos realizando experimentos adicionales para confirmar ésto. Creemos que los resultados obtenidos en este proyecto contribuyen al conocimiento de la relación estructura/función de transportadores de hongos y plantas, así como en la determinación de la especificidad de transportadores en general.Agencia Nacional de Investigación e Innovació

Informe final del proyecto: Profundización en la identificación de determinantes estructurales y funcionales de UreA

En el hongo filamentoso Aspergillus nidulans el transporte de urea tiene lugar a través de UreA, un simportador urea/H+ con ortólogos en hongos y plantas, perteneciente a la familia de los simportadores de sodio (SSS). Nuestro grupo llevó a cabo un estudio de la relación estructura- función de UreA mediante una estrategia de mutagénesis y modelado tridimensional. El mismo permitió identificar una serie de aminoácidos implicados en la unión, reconocimiento y translocación de la urea por parte del transportador. En este proyecto se propuso ahondar en el conocimiento de los determinantes estructurales y funcionales de UreA, mediante la identificación de nuevos residuos o regiones de UreA implicadas en la unión al sustrato y la selectividad por el mismo. En este sentido pudimos determinar que los aminoácidos W82, W84, N279 y T282 cumplen un rol en la interacción y/o selectividad por el sustrato, proponiendo a su vez, que la región en donde éstos se encuentran comprendería el sitio de unión al sustrato de UreA. Asimismo se realizó un análisis de los “loops” extracelulares 3 e intracelular 7, determinando que éstos poseen un rol estructural y funcional en UreA. Ya que la oligomerización de transportadores es un fenómeno que aparece con frecuencia en la biología de los mismos, pero no se conoce cuán general es ésta ni cuál es su rol para cada clase de proteínas de transporte, nos propusimos aportar a esta temática determinando si UreA es capaz de formar oligómeros. Se obtuvieron resultados preliminares que indicarían que UreA oligomeriza. Actualmente estamos realizando experimentos adicionales para confirmar ésto. Creemos que los resultados obtenidos en este proyecto contribuyen al conocimiento de la relación estructura/función de transportadores de hongos y plantas, así como en la determinación de la especificidad de transportadores en general.Agencia Nacional de Investigación e Innovació

Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans

AcpA has been previously characterized as a high-affinity transporter essential for the uptake and use of acetate as sole carbon source in Aspergillus nidulans. Here, we follow the expression profile of AcpA and define its substrate specificity. AcpA-mediated acetate transport is detected from the onset of conidiospore germination, peaks at the time of germ tube emergence, and drops to low basal levels in germlings and young mycelia, where a second acetate transporter is also becoming apparent. AcpA activity also responds to acetate presence in the growth medium, but is not subject to either carbon or nitrogen catabolite repression. Short-chain monocarboxylates (benzoate, formate, butyrate and propionate) inhibit AcpA-mediated acetate transport with apparent inhibition constants (Ki) of 16.89±2.12, 9.25±1.01, 12.06±3.29 and 1.44±0.13mM, respectively. AcpA is also shown not to be directly involved in ammonia export, as proposed for its Saccharomyces cerevisiae homologue Ady2p. In the second part of this work, we search for the unknown acetate transporter expressed in mycelia, and for other transporters that might contribute to acetate uptake. In silico analysis, genetic construction of relevant null mutants, and uptake assays, reveal that the closest AcpA homologue (AN1839), named AcpB, is the 'missing' secondary acetate transporter in mycelia. We also identify two major short-chain carboxylate (lactate, succinate, pyruvate and malate) transporters, named JenA (AN6095) and JenB (AN6703), which however are not involved in acetate uptake. This work establishes a framework for further exploiting acetate and carboxylate transport in filamentous ascomycetes.We are very grateful to Emila Krypotou for her help in the phylogenetic analyses. We are grateful to Dr. Velot for his kind gift of the two strains acpA+ and acpA Delta indispensable for this work. This work was supported by FEDER, through POFC - COMPETE and by Portuguese National Funds from "FCT - Fundacao para a Ciencia e a Tecnologia", in the scope of the projects PEst-C/BIA/UI4050/2011 and PEst-OE/BIA/UI4050/2014. JSP [SFRH/BD/61530/2009] received a fellowship from the Portuguese government from FCT through POPH and FSE.info:eu-repo/semantics/publishedVersio