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.)

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one ofthe following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp9l-phox and p22-phox, constituting the membrane-bound cytochrome b55s. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp --Gly substitution at residue 500 of gp9l-phox, associated with normal amounts of nonfunctional cytochrome b559 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp9l-phox inhibited NADPH oxidase activity in the cell-free assay (IC5o about 10 ,uM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp9l-phox resides in a region critical for stable binding of p47-phox and p67-phox. (J. Clin. Invest. 1994.95:2120-2126

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This study shows that, although PS exposure remains low, prolonged storage does compromise the RBC membrane by affecting flippase activity. When the metabolic changes induced by storage are corrected, flippase activity can be restored.

SummarySrc homology 3 (SH3) domains have been suggested to play an important role in the assembly of the superoxide-forming nicotinamide adenine dinudeotide phosphate (NADPH) oxidase upon activation of phagocytes, which involves the association of membrane-bound and cytosolic components. We studied the translocation of the cytosolic proteins to the plasma membrane in neutrophils of a patient with a point mutation in the gene encoding the light chain ofcytochrome bsss. This mutation leads to a substitution at residue 156 of a proline into a glutamine in a putative SH3 binding domain of p22-phox (Dinauer, M., E. A. Pierce, K. W. Erickson, T. Muhlebach, H. Messner, K. A. Seger, S. H. Orkin, and J. T. Curnutte. 1991. Proc. Natl. Acad. Sci. 88:11231). In PMA-stimulated neutrophils and in a eell-free translocation assay with neutrophfl membranes and cytosol, association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient's neutrophils was virtually absent. In contrast, when solubilized membranes of the patient's neutrophils were activated with phospholipids in the absence ofcytosol (Koshkin, V., and E. Pick. 1993. FEBS[Fed. Eur. Biochem. Soa]Lett. 327:57), the rate of NADPHdependent oxygen uptake was observed at a rate similar to that of control membranes. We suggest that the binding of an SH3 domain of p47-phox to p22-phox, and thus activation of the oxidase, does not occur in the neutrophils of this patient, although under artificial conditions, electron flow from NADPH to oxygen in cytochrome bsss is possible.