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P-type ATPases from the P4 subfamily (P4-ATPases) are primary active transporters that maintain lipid asymmetry in eukaryotic cell membranes by flipping lipids from the exoplasmic to the cytosolic leaflet. Mutations in several human P4-ATPase genes are associated with severe diseases. For instance, mutations in the ATP8B1 gene result in progressive familial intrahepatic cholestasis, a rare inherited disorder that usually progresses toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 angstrom resolution. The lipid flippase complex is autoinhibited by the N- and C-termini of ATP8B1, which in concert form extensive interactions with the catalytic sites and flexible domain interfaces of ATP8B1. Consistently, ATP hydrolysis by the ATP8B1-CDC50A complex requires truncation of its C-terminus as well as the presence of phosphoinositides, with a marked preference for phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminus further suggests molecular communication between N- and C-termini in the autoinhibition process and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A conserved (G/A)(Y/F)AFS motif in the C-termini of several P4-ATPase subfamilies suggests that this mechanism is employed widely across P4-ATPase lipid flippases, including both plasma membrane and endomembrane P4-ATPases.

The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast \(\textit {Saccharomyces cerevisiae}\) and are required for sterol uptake. However, the precise mechanism by which these ABC transporters facilitate sterol movement is unknown. In this study, an overexpression and purification procedure was developed with the aim to characterise the Pdr11p transporter. Engineering of Pdr11p variants fused at the C terminus with green fluorescent protein (Pdr11p-GFP) and containing a FLAG tag at the N terminus facilitated expression analysis and one-step purification, respectively. The detergent-solubilised and purified protein displayed a stable ATPase activity with a broad pH optimum near 7.4. Mutagenesis of the conserved lysine to methionine (K788M) in the Walker A motif abolished ATP hydrolysis. Remarkably, and in contrast to Aus1p, ATPase activity of Pdr11p was insensitive to orthovanadate and not specifically stimulated by phosphatidylserine upon reconstitution into liposomes. Our results highlight distinct differences between Pdr11p and Aus1p and create an experimental basis for further biochemical studies of both ABC transporters to elucidate their function.