Mechanism of P-type ATPase-catalyzed phospholipid transport : tackling the giant substrate problem

Abstract

Mechanism of P-type ATPase-catalyzed phospholipid transport: tackling the giant substrate problem After the initial discovery of an ATP-driven transporter of aminophospholipids as a principal generator of phospholipid asymmetry in the erythrocyte membrane in 1984, it took another 25 years before flippase activity was demonstrated directly by functional reconstitution of the purified enzyme. Surprisingly, the enzymes that catalyze flippase activity belong to the P-type ATPase superfamily of cation pumps. These so-called P4-ATPases display extensive sequence similarity with the other family members. The high similarity between the quaternary structures of three distinct P-type ATPases that have been crystallized thus far has led to a single concept of the transport mechanism thought to be applicable to all P-type ATPases. Hence, a challenging problem is to understand how this mechanism is adapted in P4-ATPases to translocate phospholipids instead of simple ions. Their kinship to cation pumps raised the possibility that P4-ATPases require additional machinery to accomplish this task. Here, we used a proteomics-based strategy to identify binding partners of the P4-ATPase Drs2p, a protein previously implicated in vesicle budding at the Golgi complex in yeast. By using in vivo chemical cross-linking, we set out to capture also transient or weak protein interactions that occur in live cells prior to cell lysis. This led to the identification of one known and eight novel Drs2p-binding partners. Using a genetic interaction assay, we confirmed the majority of the identified proteins as true interactors of Drs2p. The PI(4)P phosphastase Sac1p is of particular interest since a physical interaction between Drs2p and Sac1p may interconnect flippase activity with coat recruitment during biogenesis of secretory vesicles at the Golgi. Among the previously established interaction partners of P4-ATPases are Cdc50 proteins. These proteins have been suggested to function as chaperones that promote the proper maturation and intracellular targeting of P4-ATPases. We now provide the first experimental evidence that Cdc50 proteins play a more direct and intimate role in P4-ATPase-catalyzed phospholipid transport. We show that specific interactions between P4-ATPases and Cdc50 subunits are required to render the enzyme competent for phosphorylation at the catalytically important aspartate residue. Moreover, we find that the affinity of the P4-ATPase for its Cdc50 binding partner fluctuates during the reaction cycle, with the strongest interaction occurring at a point where the enzyme is loaded with phospholipid ligand. Together, these findings suggest that acquiring Cdc50 subunits was a key step in the evolution of flippases from a family of cation pumps. In view of these findings, we next explored the structural basis of the dynamic association between P4-ATPases and Cdc50 proteins. We show that the ectodomain of Cdc50 proteins is a critical mediator of P4-ATPase binding and function. Moreover, we identified a disulfide bridge between two conserved cysteines in the Cdc50 ectodomain as a critical determinant of productive transporter-subunit interactions. These findings are consistent with a role of the subunit in loading the transporter with phospholipid ligand and/or in creating a sizeable pathway for its translocation across the bilayer.