Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae

The type IV P-type ATPases (P4-ATPases) thus far characterized are lipid flippases that transport specific substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the exofacial leaflet to the cytofacial leaflet of membranes. This transport activity generates compositional asymmetry between the two leaflets important for signal transduction, cytokinesis, vesicular transport, and host-pathogen interactions. Most P4-ATPases function as a heterodimer with a β-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a β-subunit requirement and it is unclear how these proteins transport substrate. Here we tested if residues linked to lipid substrate recognition in other P4-ATPases also contribute to Neo1 function in budding yeast. Point mutations altering entry gate residues in the first (Q209A) and fourth (S457Q) transmembrane segments of Neo1, where phospholipid substrate would initially be selected, disrupt PS and PE membrane asymmetry, but do not perturb growth of cells. Mutation of both entry gate residues inactivates Neo1, and cells expressing this variant are inviable. We also identified a gain-of-function mutation in the second transmembrane segment of Neo1 (Neo1[Y222S]), predicted to help form the entry gate, that substantially enhances Neo1's ability to replace the function of a well characterized phospholipid flippase, Drs2, in establishing PS and PE asymmetry. These results suggest a common mechanism for substrate recognition in widely divergent P4-ATPases. •The P4-ATPase Neo1 transports phospholipid to control membrane asymmetry.•Positionally conserved residues in Neo1 are critical for substrate recognition.•A new entry gate residue (Y222) identified in Neo1 second transmembrane segment.•Neo1 mutations enhancing phosphatidylserine recognition suppress drs2∆.