Members of the nucleobase ascorbate transporter (NAT) family (SLC23) are elevator-type transporters that are responsible for the uptake of nucleobases and ascorbate. In fungi, NAT members are also responsible for the specific uptake of antifungal nucleobase analogues, such as oxypurinol, allopurinol, or 8-azaguanine. Here, we report nearly full-length cryo-EM structures of UapA, a high-affinity purine transporter from the model fungus , in inward-facing apo- and substrate-loaded conformations at 2.06 to 3.5 Å in detergent and lipid nanodiscs. The high-resolution structures reveal the role of water molecules and lipids in substrate binding, specificity, transporter dimerization, and activity. Notably, the N-tail of UapA is found to be structured, interacting with both the core and scaffold domains, which in combination with functional data suggests a dual role in trafficking and transport dynamics. Overall, our study provides unprecedented structural and functional insights into an elevator-type fungal transporter, which may well contribute to the exploitation of NAT transporters as specific gateways for targeted pharmacological antifungal approaches.

Growth of filamentous fungi is highly polarized requiring the coordinated apical delivery of cell wall components and plasma membrane (PM) material, primarily lipids and proteins, to hyphal tips via conventional vesicular secretion. Fungal growth also requires the tight coordination of exocytosis (secretion) with endocytosis and recycling of proteins and lipids, which occurs in a defined region behind the growing tip known as the endocytic collar. Here, we genetically characterized proteins tentatively implicated in the formation of endoplasmic reticulum-plasma membrane (ER-PM) contact sites, including Scs2/VAP, tricalbins and Ist2 homologues, in . We showed that among these proteins, only the single Scs2/VapA homologue is essential for normal fungal growth, and this requirement is due to the critical role of VapA in maintaining the polarized localization of apical cargoes, such as the lipid flippases DnfA and DnfB or the SNARE protein SynA. In mutants, these cargoes lose their polarized localization, a phenotype that correlates with the mislocalization of the AP-2 cargo adaptor complex, which is essential for the endocytosis and recycling of apical membrane components. Further analysis provides evidence linking the defect in apical cargo endocytosis observed in mutants to altered membrane lipid partitioning, suggesting that VapA contributes to lipid domain organization critical for cargo recycling. Strikingly, deletion of VapA does not impair the localization or endocytosis of non-polarized (subapical) plasma membrane transporters, indicating that the trafficking and biogenesis of polarized (apical) versus non-polarized (subapical) cargoes are differentially dependent on membrane lipid composition and domain-specific organization.