CDC50 proteins are critical components of the human class-1 P 4-ATPase transport machinery

Abstract

Members of the P4 subfamily of P-type ATPases catalyze phospholipid transport and create membrane lipid asymmetry in late secretory and endocytic compartments. P-type ATPases usually pump small cations and the transport mechanism involved appears conserved throughout the family. How this mechanism is adapted to flip phospholipids remains to be established. P 4-ATPases form heteromeric complexes with CDC50 proteins. Dissociation of the yeast P4-ATPase Drs2p from its binding partner Cdc50p disrupts catalytic activity (Lenoir, G., Williamson, P., Puts, C. F., and Holthuis, J. C. (2009) J. Biol. Chem. 284, 17956-17967), suggesting that CDC50 subunits play an intimate role in the mechanism of transport by P 4-ATPases. The human genome encodes 14 P4-ATPases while only three human CDC50 homologues have been identified. This implies that each human CDC50 protein interacts with multiple P4-ATPases or, alternatively, that some human P4-ATPases function without a CDC50 binding partner. Here we show that human CDC50 proteins each bind multiple class-1 P4-ATPases, and that in all cases examined, association with a CDC50 subunit is required for P4-ATPase export from the ER. Moreover, we find that phosphorylation of the catalytically important Asp residue in human P 4-ATPases ATP8B1 and ATP8B2 is critically dependent on their CDC50 subunit. These results indicate that CDC50 proteins are integral part of the P4-ATPase flippase machinery. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc