The flip side of ion transport: molecular dissection of a heteromeric lipid pump

Abstract

Flippases catalyze unidirectional phospholipid transport to create transbilayer lipid asymmetry in late secretory and endocytic organelles. Molecular cloning of candidate flippases resulted in the identification of P4-ATPases, a subclass of the superfamily of cation-transporting P-type pumps. Striking structural similarities and a common domain organization imply a transport mechanism that is conserved throughout the P-type ATPase super­family. Understanding how flippases adapted this transport mechanism to translocate phospholipids instead of small cations poses a major challenge. P4-ATPases form heteromeric complexes with Cdc50 proteins. Recent work from our labora­tory suggests that Cdc50 proteins play a vital role in the P4-ATPase transport cycle. While Cdc50 proteins in yeast appear essential for catalytic activity of P4-ATPases, the concept that these accessory subunits are essential components of the P4-ATPase transport machinery is challenged by the fact that Cdc50 proteins in man are greatly outnumbered by P4-ATPases. We find that human Cdc50 subunits are common binding partners of class-1 P4-ATPases and that association with a Cdc50 protein is a prerequisite for P4-ATPase export from the ER. Moreover, we show that phosphorylation of the catalytically important Asp residue in the human class-1 P4-ATPases ATP8B1 and ATP8B2 is critically dependent on their Cdc50 binding partners. These findings indicate that Cdc50 proteins serve a fundamental role in the mechanism by which P4-ATPases translocate phospholipids. To further unravel the inner workings of flippases, we set out to map functional interactions between P4-ATPases and their Cdc50 binding partners. Using domain swapping and side-directed mutagenesis approaches, we find that the Cdc50 ectodomain contains important structural determinants of P4-ATPase binding specificity and function. Moreover, we show that four highly conserved cysteine residues in the Cdc50 ectodomain form two stabilizing disulfide bridges. Intriguingly, loss of these intramolecular disulfide bonds reciprocally affects P4-ATPase binding and flippase activity, consistent with a key function of the Cdc50 ectodomain in P4-ATPase-catalyzed lipid transport. These results provide additional support for the idea that Cdc50 proteins are integral and dynamic compo­nents of the P4-ATPase flippase machinery. We postulate that acquisition of Cdc50 proteins was an essential step in the evolution of flippases from a family of cation pumps. An intriguing question is: which crucial biological role initially necessitated the evolvement of these unidirec­tional lipid pumps? Interestingly, P4-ATPases serve a critical role in the biogenesis of transport vesicles from endocytic and late secretory compartments. Their precise contribution to this fundamental cellular process remains to be established. An attractive hypothesis is that by generating a lipid mass imbalance across bilayers, P4-ATPases assist the cellular coat machinery in promoting membrane curvature during vesicle biogenesis from rigid membranes. To experimentally address this concept, we developed protocols for incorporating P4-ATPase transport machinery into giant unilamellar vesicles (GUVs). We established two complemen­tary methods to generate GUVs from P4-ATPase-containing secretory vesicles (SVs). These SV-derived GUVs provide a novel tool to directly test the membrane-bending capacity of P4-ATPases and to dissect the transport mechanism as well as biological role of these fascinating molecular machines.