Mapping Functional Interactions in a Heterodimeric Phospholipid Pump

Type 4 P-type ATPases (P4-ATPases) catalyze phospholipid transport to generate phospholipid asymmetry across membranes of late secretory and endocytic compartments, but their kinship to cation-transporting P-type transporters raised doubts about whether P4-ATPases alone are sufficient to mediate flippase activity. P4-ATPases form heteromeric complexes with Cdc50 proteins. Studies of the enzymatic properties of purified P4-ATPase·Cdc50 complexes showed that catalytic activity depends on direct and specific interactions between Cdc50 subunit and transporter, whereas in vivo interaction assays suggested that the binding affinity for each other fluctuates during the transport reaction cycle. The structural determinants that govern this dynamic association remain to be established. Using domain swapping, site-directed, and random mutagenesis approaches, we here show that residues throughout the subunit contribute to forming the heterodimer. Moreover, we find that a precise conformation of the large ectodomain of Cdc50 proteins is crucial for the specificity and functionality to transporter/subunit interactions. We also identified two highly conserved disulfide bridges in the Cdc50 ectodomain. Functional analysis of cysteine mutants that disrupt these disulfide bridges revealed an inverse relationship between subunit binding and P4-ATPase-catalyzed phospholipid transport. Collectively, our data indicate that a dynamic association between subunit and transporter is crucial for the transport reaction cycle of the heterodimer. Background: P4-ATPases form heterodimeric complexes with Cdc50 proteins and catalyze phospholipid transport to generate membrane asymmetry. Results: Disruption of disulfide bridges in the ectodomain of Cdc50 proteins has reciprocal effects on P4-ATPase binding and phospholipid transport. Conclusion: Cdc50 proteins are critical components of the P4-ATPase flippase machinery. Significance: Our data begin to define the function of different regions of the Cdc50 polypeptide in supporting P4-ATPase-catalyzed phospholipid transport.