P4-ATPase BbCrpa2 detoxifies cyclosporine a via AP2-clathrin-dependent endocytic pathway in Beauveria bassiana.

Cyclosporine A (CsA) exerts potent intracellular toxicity in fungi, yet the mechanisms underlying fungal CsA tolerance remain poorly understood. Here, we identify BbCrpa2, a P4-ATPase in the entomopathogenic fungus Beauveria bassiana, as a key determinant of CsA resistance. Unlike the trans-Golgi network-localized BbCrpa, which sequesters cytosolic CsA, BbCrpa2 functions as a plasma membrane gatekeeper. It captures CsA at the cell surface and mediates its internalization into vesicles through AP2-clathrin-dependent endocytosis. Genetic and functional analyses reveal that BbCrpa2 and BbCrpa act independently and additively to protect against CsA toxicity. BbCrpa2 constitutes the primary defense by limiting the free entry of CsA into the cytoplasm, whereas BbCrpa serves as a secondary safeguard by sequestering the residual cytosolic CsA. Together, these findings demonstrate that spatially distinct P4-ATPases cooperate to coordinate vesicular CsA transport, revealing a previously unrecognized, multi-tiered defense mechanism that enables fungal tolerance to toxic secondary metabolites.