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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 Innovación

Background: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. Results: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of 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 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., UapA is a uric acid/xanthine H+ symporter from a filamentous fungus. Here, the authors solve the crystal structure of the transporter in complex with xanthine revealing it to be a dimer, and this homodimerisation is proposed to be important for function.