Abstract / Summary
ABSTRACT Azole antifungals are widely used to control fungal infection, yet resistance mechanisms beyond drug efflux and target modification remain insufficiently understood. Here, we identified the Guided Entry of Tail-anchored proteins (GET) pathway as a conserved regulator of azole susceptibility in Neurospora crassa and Aspergillus fumigatus . Deletion or dysfunction of the core GET components Get-3 or Get-4 confers resistance to multiple azoles without impairing hyphal growth or sporulation. Mechanistically, GET deficiency neither reduced intracellular accumulation nor altered drug efflux and target expression or total ergosterol levels. Instead, GET-deficient strains displayed significantly reduced accumulation of the toxic sterol intermediate 14α-methyl-3,6-diol under azole stress, a change that may limit azole-induced sterol toxicity and contribute to reduced azole susceptibility. Proximity labeling and functional validation indicated that GET deficiency perturbs the trafficking of multiple azole-resistance-associated proteins, including the key tail-anchored protein transporter Emp-47 and mitochondrial Cox subunits Cox-4 and Cox-15, which deletion enhanced azole resistance. Transcriptomic and functional analyses further revealed that GET disruption altered the expression of some azole resistance-associated genes, which involve in membrane transport, metabolism, and cell wall organization, independent of canonical stress pathways. In this study, 19 previously uncharacterized genes modulating azole susceptibility are uncovered. Collectively, our findings identify the GET pathway as a non-classical regulator of azole susceptibility associated with protein-trafficking defects and metabolic adaptation in filamentous fungi. IMPORTANCE Understanding non-canonical mechanisms of azole resistance is crucial given the rising failure of antifungal therapies. Here, we identify the GET pathway as an evolutionarily conserved regulator of azole susceptibility in both a model fungus and the major pathogen Aspergillus fumigatus . We uncover 19 previously unknown resistance-modulating genes and reveal a conceptually distinct mechanism whereby GET disruption confers resistance independently of classical efflux pumps or target alterations. Instead, GET dysfunction impairs trafficking of Emp-47 and Cox subunits, reduces toxic sterol intermediate accumulation, and alters resistance-associated gene expression. These findings establish protein trafficking as a new layer of stress adaptation and provide a mechanistic framework for further investigation of fungal azole susceptibility.