ABSTRACT: 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.
