ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

Abstract

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1. Previously, we have shown in mice that Atp8b1 deficiency leads to enhanced biliary excretion of phosphatidylserine, and we hypothesized that ATP8B1 is a flippase for phosphatidylserine. However, direct evidence for this function is still lacking. In Saccharomyces cerevisiae, members of the Cdc50p/Lem3p family are essential for proper function of the ATP8B1 homologs. We have studied the role of two human members of this family, CDC50A and CDC50B, in the routing and activity of ATP8B1. When only ATP8B1 was expressed in Chinese hamster ovary cells, the protein localized to the endoplasmic reticulum. Coexpression with CDC50 proteins resulted in relocalization of ATP8B1 from the endoplasmic reticulum to the plasma membrane. Only when ATP8B1 was coexpressed with CDC50 proteins was a 250%-500% increase in the translocation of fluorescently labeled phosphatidylserine observed. Importantly, natural phosphatidylserine exposure in the outer leaflet of the plasma membrane was reduced by 17%-25% in cells coexpressing ATP8B1 and CDC50 proteins in comparison with cells expressing ATP8B1 alone. The coexpression of ATP8B1 and CDC50A in WIF-B9 cells resulted in colocalization of both proteins in the canalicular membrane. Conclusion: Our data indicate that CDC50 proteins are pivotal factors in the trafficking of ATP8B1 to the plasma membrane and thus may be essential determinants of ATP8B1-related disease. In the plasma membrane, ATP8B1 functions as a flippase for phosphatidylserine. Finally, CDC50A may be the potential ␤-subunit or chaperone for ATP8B1 in hepatocytes. (HEPATOLOGY 2008;47: 268-278.)

CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells

“…Confocal microscopy studies revealed that CDC50A-V5 was retained in the intracellular membrane compartments in HeLa cells when over-expressed alone, whereas when it was co-expressed with Atp8b1 in the same cells (Fig. 9), both proteins co-localized at the plasma membrane, in a similar manner to that reported for the pair ATP8B1 + 17 CDC50A [29]. Co-localization of these two proteins was also observed when coexpressed in HEK-293T cells (data not shown).…”

“…However, we found that the uptake of perifosine and NBD-aminophospholipids (but not NBD-PC) increased remarkably when both HeLa and HEK-293T cells were co-transfected with Atp8b1-myc and CDC50A-V5. Moreover, CDC50A-V5 remained intracellular when overexpressed alone, but when co-expressed with Atp8b1 both proteins exported from the ER and co-localized at the plasma membrane, as is the case with human ATP8B1 + CDC50A [29]. CDC50A may be coupled with a P4 ATPase to drive perifosine and aminophospholipids uptake and could contribute to the transport specificity of the complex.…”

“…In contrast, CDC50A exports from the ER only to the TGN in KB PER-R clone10 cells and does not reach the cell surface, thereby suggesting that these perifosine-resistant cells harbor a defect that results in the incorrect trafficking of CDC50A at this point of the exocytic pathway. Since CDC50A must couple with a P4-ATPase for correct trafficking of the full APLT complex to the plasma membrane [29], the observed traffic defect of CDC50A in KB PER-R cells could explain the inability of this cell line to translocate perifosine. In contrast to KB 19 cells, over-expression of CDC50A in the human cell lines HeLa and HEK-293T did not modulate perifosine uptake, thus suggesting that, in this case, the alpha subunit rather than CDC50A may be the limiting component of the putative APLT.…”

“…Cdc50-Lem3p proteins play a role as both molecular chaperones needed for the correct folding and export of the alpha subunit P4-ATPase from the endoplasmic reticulum (ER) [28,29], and also as catalytically relevant, integral components of the phospholipid translocation machinery [30]. Since we have found previously that human CDC50A appears to play a role in perifosine uptake in KB cells, we decided to establish the cellular localization of this protein.…”

Clinical utility gene card for: Progressive familial intrahepatic cholestasis type 1

“…5,7 A chaperone protein, CDC50A, may be required for normal trafficking and function of the product of ATP8B1. 15 No data are available concerning molecular study of the gene encoding CDC50A in patient with a PFIC1 phenotype. So whether mutation of CDC50A may be implicated in patients with a PFIC1 phenotype remains an open question.…”