Functional screening of transmembrane proteins identifies dolichyl pyrophosphate phosphatase CAX-4 as a key regulator of ergosterol biosynthesis and azole resistance in Neurospora crassa

Ergosterol, a key lipid component of fungal membranes, has its homeostasis predominantly maintained through membrane-associated mechanisms, yet the role of the many transmembrane proteins in regulating sterol homeostasis remains poorly understood. Here, we systematically screened 1,042 viable transmembrane protein mutants in Neurospora crassa, and identified 180 that exhibited altered sensitivity to one or more sterol biosynthesis inhibitors. Further analysis pinpointed three genes, cax-4, chol-6 and NCU01826, as required for the upregulation of erg11 upon azole treatment. CAX-4, a conserved dolichyl pyrophosphate phosphatase, was further characterized. Deletion of this gene leads to hypersensitivity to ketoconazole and amorolfine, and impairs the proper expression of membrane and extracellular proteins. Notably, CAX-4 was required for the transcriptional induction of core ergosterol biosynthetic genes (e.g., erg11, erg6, erg24) and sterol metabolism under azole stress. This phenotype was linked to reduced levels of NcSR, a key transcription factor in sterol biosynthesis regulation, at both the mRNA and protein level. Mechanistically, CAX-4 maintains protein homeostasis via N-glycosylation, as Tunicamycin treatment phenocopied its deletion. Above all, by characterizing key transmembrane protein in sterol biosynthesis regulation, our study unveils a CAX-4 integrated crosstalk between dolichol-mediated protein N-glycosylation and sterol biosynthesis regulation, thus offering novel insights into sterol homeostasis and pointing to potential targets for the development of antifungal strategies. Overall design: The samples used for RNA-seq were prepared as described above. After RNA extraction, total RNA was sequenced on a BGISEQ-500 platform. After quality control, the clean reads were mapped to the reference genome of N. crassa (https://fungidb.org/common/downloads/Current_Release/NcrassaOR74A/). Gene expression levels were subsequently quantified as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). To ensure robustness, genes with an FPKM value of less than 1 in all samples were excluded from further analysis. Differentially expressed genes (DEGs) were identified as those exhibiting a fold change greater than 2 or less than 0.5 between two samples.