Abstract
Flippases are enzymes that translocate phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) from the outer to the inner leaflet in the lipid bilayer of the plasma membrane, leading to the asymmetric distribution of aminophospholipids in the membrane. One mammalian phospholipid flippase at the plasma membrane is ATP11C, a type IV P-type ATPase (P4-ATPase) that forms a heterocomplex with the transmembrane protein CDC50A. However, the structural features in CDC50A that support the function of ATP11C and other P4-ATPases have not been characterized. Here, using error-prone PCR-mediated mutagenesis of human CDC50A cDNA followed by functional screening and deep sequencing, we identified 14 amino acid residues that affect ATP11C's flippase activity. These residues were all located in CDC50A's extracellular domain and were evolutionarily well-conserved. Most of the mutations decreased CDC50A's ability to chaperone ATP11C and other P4-ATPases to their destinations. The CDC50A mutants failed to form a stable complex with ATP11C and could not induce ATP11C's PtdSer-dependent ATPase activity. Notably, one mutant variant could form a stable complex with ATP11C and transfer ATP11C to the plasma membrane, yet the ATP11C complexed with this CDC50A variant had very weak or little PtdSer- or PtdEtn-dependent ATPase activity. These results indicated that the extracellular domain of CDC50A has important roles both in CDC50A's ability to chaperone ATP11C to the plasma membrane and in inducing ATP11C's ATP hydrolysis-coupled flippase activity.
Highlights
Flippases are enzymes that translocate phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) from the outer to the inner leaflet in the lipid bilayer of the plasma membrane, leading to the asymmetric distribution of aminophospholipids in the membrane
To investigate CDC50A’s functions, mutations were randomly introduced into the human CDC50A (hCDC50A) cDNA sequence (1083 bp) by error-prone PCR using low-fidelity DNA polymerase
We examined the PtdSer flippase activity of the transformants expressing hATP11C with or without wildtype or mutant hCDC50A and found that the 18 CDC50A mutants could be categorized into three groups (Fig. 3B)
Summary
One mutant variant could form a stable complex with ATP11C and transfer ATP11C to the plasma membrane, yet the ATP11C complexed with this CDC50A variant had very weak or little PtdSer- or PtdEtn-dependent ATPase activity These results indicated that the extracellular domain of CDC50A has important roles both in CDC50A’s ability to chaperone ATP11C to the plasma membrane and in inducing ATP11C’s ATP hydrolysis-coupled flippase activity. The flippase activity at plasma membranes was assayed by the incorporation of a fluorescently labeled PtdSer analogue, and the cells exhibiting low flippase activity were enriched by repetitive cell sorting The cells in this population, designated as lost-flippase (LF) cells, were subjected to deep sequencing analysis, which identified 14 amino acid residues that were important for CDC50A’s ability to support ATP11C’s flippase activity. Its phospholipid-dependent ATPase activity was dramatically decreased, indicating that, in addition to its chaperone function, CDC50A plays a direct role in ATP11C’s flippase activity
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