Genetic dissection of the secretory route followed by a fungal extracellular glycosyl hydrolase

Hyphal tip cells of Aspergillus nidulans are > 100 µm‐long, which challenges intracellular traffic. In spite of the basic and applied interest of the secretory pathway of filamentous fungi, only recently has it been investigated in detail. We used InuA, an inducible and highly glycosylated inulinase, and mutations affecting different intracellular membranous compartments, to investigate the route by which the enzyme traffics to the extracellular medium. InuA is core‐N‐glycosylated in the ER and hyperglycosylated during transit across the Golgi. Hyperglycosylation was prevented by ts mutations in sarA^SAR1 impeding ER exit, and in sedV^SED5 and rabO^RAB1 dissipating the early Golgi, but not by mutations in the TGN regulators hypA^TRS120 and hypB^SEC7, implicating the early Golgi in cargo glycosylation. podB1^ts (cog2^ts ) affecting the COG complex also prevents glycosylation, without disassembling early Golgi cisternae. That InuA exocytosis is prevented by inactivation of any of the above genes shows that it follows a conventional secretory pathway. However, ablation of RabB^RAB5 regulating early endosomes (EEs), but not of RabS^RAB7, its equivalent in late endosomes, also prevents InuA accumulation in the medium, indicating that EEs are specifically required for InuA exocytosis. This work provides a framework to understand the secretion of enzyme cargoes by industrial filamentous fungi.