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. 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. 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.

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.

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 ascorbatetransport 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 NATproteins as specific gateways for targeting pathogenic microbes.

Substrate-transport–elicited endocytosis is a common control mechanism of membrane transporters avoiding excess uptake of external compounds, though poorly understood at the molecular level. In yeast, endocytosis of transporters is triggered by their ubiquitylation mediated by the Rsp5 ubiquitin-ligase, recruited by α-arrestin–family adaptors. We here report that transport-elicited ubiquitylation of the arginine transporter Can1 is promoted by transition to an inward-facing state. This conformational change unveils a region of the N-terminal cytosolic tail targeted by the Art1 α-arrestin, which is activated via the TORC1 kinase complex upon arginine uptake. Can1 mutants altered in the arginine-binding site or a cytosolic tripeptide sequence permanently expose the α-arrestin–targeted region so that Art1 activation via TORC1 is sufficient to trigger their endocytosis. We also provide evidence that substrate-transport elicited endocytosis of other amino acid permeases similarly involves unmasking of a cytosolic Art1-target region coupled to activation of Art1 via TORC1. Our results unravel a mechanism likely involved in regulation of many other transporters by their own substrates. They also support the emerging view that transporter ubiquitylation relies on combinatorial interaction rules such that α-arrestins, stimulated via signaling cascades or in their basal state, recognize transporter regions permanently facing the cytosol or unveiled during transport.

The plasma membrane (PM) performs a plethora of physiological processes, the coordination of which requires spatial and temporal organization into specialized domains of different sizes, stability, protein/lipid composition and overall architecture. Compartmentalization of the PM has been particularly well studied in the yeast Saccharomyces cerevisiae, where five non-overlapping domains have been described: The Membrane Compartments containing the arginine permease Can1 (MCC), the H+-ATPase Pma1 (MCP), the TORC2 kinase (MCT), the sterol transporters Ltc3/4 (MCL), and the cell wall stress mechanosensor Wsc1 (MCW). Additional cortical foci at the fungal PM are the sites where clathrin-dependent endocytosis occurs, the sites where the external pH sensing complex PAL/Rim localizes, and sterol-rich domains found in apically grown regions of fungal membranes. In this review, we summarize knowledge from several fungal species regarding the organization of the lateral PM segregation. We discuss the mechanisms of formation of these domains, and the mechanisms of partitioning of proteins there. Finally, we discuss the physiological roles of the best-known membrane compartments, including the regulation of membrane and cell wall homeostasis, apical growth of fungal cells and the newly emerging role of MCCs as starvation-protective membrane domains.