P4-ATPases are members of the P-type ATPase superfamily that drive the inward translocation (flipping) of lipids within the membrane. These lipid flippase largely function as binary complexes with an auxiliary protein from the CDC50 family. The bulk of our knowledge has derived genetic and biochemical studies of yeast and mammalian P4-ATPases, in particular the phosphatidylserine (PS) transporting Drs2p/Cdc50p (Saccharomyces cerevisiae) and bATP8A2/CDC50A (Bos taurus). However, questions surrounding the mechanism of lipid translocation remain.

To address this deficit in knowledge and to provide a basis for the analysis of reported mutagenesis data, we aim to solve the first molecular structures of the PS transporting P4-ATPases using electron microscopy. To date, negative stain EM analysis, on detergent, amphipol and saposin-lipoprotein nanoparticle (Salipro) reconstituted of both Drs2p/CDC50p and bATP8A2/CDC50A, has yielded comparable low-resolution envelopes of these two transporters, highlighting the bulk architecture of the complex. Current efforts and progress on the functional characterization and cryo-EM analysis of both lipid transporters reconstituted in Salipro are described.

References
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Figure 1. Negative stain analysis of Drs2p/Cdc50p and bATP8A2/CDC50A in Salipro. a-b) Representative 2D class averages of both proteins c) low resolution negative stain envelope of Drs2p/Cdc50p in salipro. The Na-K ATPase is used to highlight the architecture of the lipid flippase in the lipo-protein nanoparticle. d) Comparison of the low-resolution envelopes for Drs2p/Cdc50p and bATP8A2/CDC50A in Salipro.