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The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane.

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.

SUBMITTER: Segawa K

PROVIDER: S-EPMC5808776 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane.

Segawa Katsumori K Kurata Sachiko S Nagata Shigekazu S

The Journal of biological chemistry 20171224 6

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 func ...[more]

PMID: 29276178

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Project description:P4-ATPases comprise a family of P-type ATPases that actively transport or flip phospholipids across cell membranes. This generates and maintains membrane lipid asymmetry, a property essential for a wide variety of cellular processes such as vesicle budding and trafficking, cell signaling, blood coagulation, apoptosis, bile and cholesterol homeostasis, and neuronal cell survival. Some P4-ATPases transport phosphatidylserine and phosphatidylethanolamine across the plasma membrane or intracellular membranes whereas other P4-ATPases are specific for phosphatidylcholine. The importance of P4-ATPases is highlighted by the finding that genetic defects in two P4-ATPases ATP8A2 and ATP8B1 are associated with severe human disorders. Recent studies have provided insight into how P4-ATPases translocate phospholipids across membranes. P4-ATPases form a phosphorylated intermediate at the aspartate of the P-type ATPase signature sequence, and dephosphorylation is activated by the lipid substrate being flipped from the exoplasmic to the cytoplasmic leaflet similar to the activation of dephosphorylation of Na(+)/K(+)-ATPase by exoplasmic K(+). How the phospholipid is translocated can be understood in terms of a peripheral hydrophobic gate pathway between transmembrane helices M1, M3, M4, and M6. This pathway, which partially overlaps with the suggested pathway for migration of Ca(2+) in the opposite direction in the Ca(2+)-ATPase, is wider than the latter, thereby accommodating the phospholipid head group. The head group is propelled along against its concentration gradient with the hydrocarbon chains projecting out into the lipid phase by movement of an isoleucine located at the position corresponding to an ion binding glutamate in the Ca(2+)- and Na(+)/K(+)-ATPases. Hence, the P4-ATPase mechanism is quite similar to the mechanism of these ion pumps, where the glutamate translocates the ions by moving like a pump rod. The accessory subunit CDC50 may be located in close association with the exoplasmic entrance of the suggested pathway, and possibly promotes the binding of the lipid substrate. This review focuses on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms.

Dataset Information

The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane.

Publications

The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane.

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