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Functional aminophospholipid translocases are composed of at least two proteins: an alpha subunit from the P4 subfamily of P-type ATPases and a beta subunit from the CDC50-Lem3p family. Over-expression and knockdown of the human beta subunit CDC50A in KB cells enhanced and decreased, respectively, the uptake of both fluorescent aminophospholipid analogues and the anticancer alkyl-phospholipid perifosine. Confocal microscopy showed that CDC50A-V5 was localized at the endoplasmic reticulum and the Golgi complex of both KB (perifosine-sensitive) and KB PER-R (perifosine-resistant, alkyl-phospholipid uptake deficient) cells, but was only widely distributed in the early and late endosomes in KB cells. Biotinylation of cell surface proteins allowed CDC50A-V5 to be detected in the plasma membrane of KB cells but not in KB PER-R cells, thereby suggesting a defect in CDC50A trafficking that could explain the inability of KB PER-R to uptake perifosine. Over-expression of CDC50A in HeLa and HEK293T cells did not increase uptake, since the protein was retained at the endoplasmic reticulum and Golgi. However, when CDC50A was co-expressed with the P4-ATPase Atp8b1, the two proteins co-localized at the plasma membrane and the uptake of aminophospholipids and perifosine increased strikingly in both cell lines. These findings suggest that CDC50A plays a key role in perifosine uptake in human cells, presumably by forming a functional plasma membrane translocator in combination with a P4-ATPase.