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Lyubomir Dimitrov Stanchev, Juliana Rizzo, Rebecca Peschel, Lilli A. Pazurek, Lasse Bredegaard, Sarina Veit, Sabine Laerbusch, Marcio L. Rodrigues, Rosa Laura López-Marqués, Thomas Günther-Pomorski

• Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen \(\textit {Cryptococcus neoformans}\), the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a \(\beta\)-subunit. Using heterologous expression in \(\textit {S. cerevisiae}\), we demonstrate that Apt1p forms a heterodimeric complex with the \(\textit {C. neoformans}\) Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in \(\textit {C. neoformans}\) is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.