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Identification of P4-ATPase Phospholipid Flippases in Red Blood Cells and Functional Analysis of Disease-associated Mutants

ABSTRACT: ATP-dependent phospholipid flippase activity crucial for generating lipid asymmetry in membranes was first detected in red blood cells (RBCs), but the P4-ATPases responsible have not been directly determined. Using affinity-based mass spectrometry, we show that ATP11C is the only abundant P4-ATPase in human RBCs, while ATP11C and ATP8A1 are the major P4-ATPases in mouse RBCs. ATP11A and ATP11B are detectable at low levels. Mutations in ATP11C are responsible for blood and liver disorders, but the mechanisms are not known. Using heterologous expression, we show that the T418N mutation localized in the conserved phosphorylation motif of ATP11C and responsible for human congenial hemolytic anemia expresses at low levels, is retained in the endoplasmic reticulum, and has 33% of wild-type ATP11C activity. The I355K mutant in the transmembrane domain associated with cholestasis and anemia in mice expresses at wild-type levels and traffics to the plasma membrane, but is devoid of activity. We conclude that the T418N mutation of ATP11C causes significant protein misfolding, whereas the I355K mutant folds normally, but lacks key contacts required for functional activity.

INSTRUMENT(S): Bruker Daltonics instrument model

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Blood Cell, Blood

SUBMITTER: Jenny Moon

LAB HEAD: Robert S. Molday

PROVIDER: PXD011841 | Pride | 2019-03-08

REPOSITORIES: Pride

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