P4-ATPases, or flippases, translocate phospholipids between the two leaflets of eukaryotic biological membranes. They are essential to the physiologically crucial phospholipid asymmetry and involved in severe diseases, but their molecular structure and mechanism are still unresolved. Here, we show that in an extensive mutational alanine screening of the mammalian flippase ATP8A2 catalytic subunit, five mutations stand out by leading to reduced glycosylation of the accessory subunit CDC50A. These mutations may disturb the interaction between the subunits.