Structural and functional properties of the N- and C-terminal segments of the P4-ATPase phospholipid flippase ATP8A2

ATP8A2 is a P4-ATPase that actively flips phosphatidylserine and to a lesser extent phosphatidylethanolamine across cell membranes to generate and maintain transmembrane phospholipid asymmetry. The importance of this flippase is evident in the finding that loss-of-function mutations in ATP8A2 are known to cause the neurodevelopmental disease known as cerebellar ataxia, intellectual disability, and dysequilibrium syndrome 4 (CAMRQ4) in humans and related neurodegenerative disorders in mice. Although significant progress has been made in understanding mechanisms underlying phospholipid binding and transport across the membrane domain, little is known about the structural and functional properties of the cytosolic N- and C-terminal segments of this flippase. In addition, there has been uncertainty regarding the methionine start site of ATP8A2 and accordingly the size of the N-terminal segment. Here, we have used mass spectrometry to show that bovine ATP8A2 like its human counterpart has an extended N-terminal segment not apparent in the mouse ortholog. This segment greatly enhances the expression of ATP8A2 without affecting its cellular localization or phosphatidylserine-activated ATPase activity. Using a cleavable C-terminal protein and site-directed mutagenesis, we further show that the conserved GYAFS motif in the C-terminal segment plays a role in autoinhibition as well as efficient folding of ATP8A2 into a functional protein.