Biochemical Characterization of P4-ATPase Mutations Identified in Patients with Progressive Familial Intrahepatic Cholestasis

• Published in: The Journal of biological chemistry 2012-11, Vol.287 (49), p.41139-41151
• Main Authors: Stone, Alex, Chau, Christopher, Eaton, Christian, Foran, Emily, Kapur, Mridu, Prevatt, Edward, Belkin, Nathan, Kerr, David, Kohlin, Torvald, Williamson, Patrick
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Amino Acid Sequence; ATP-Binding Cassette Transporters - genetics; ATPases; Cdc50; Cholestasis; Cholestasis, Intrahepatic - genetics; Humans; Kinetics; Lipid Asymmetry; Membrane Enzymes; Membrane proteins; Membrane Transport; Molecular Bases of Disease; Molecular Sequence Data; Mutation; P4-ATPase; Phenotype; Phospholipid; Phospholipid Transport; Phospholipids - chemistry; Regression Analysis; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sequence Homology, Amino Acid; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.413039

Mutations in the P4-ATPase ATP8B1 cause the inherited liver disease progressive familial intrahepatic cholestasis. Several of these mutations are located in conserved regions of the transmembrane domain associated with substrate binding and transport. Assays for P4-ATPase-mediated transport in living yeast cells were developed and used to characterize the specificity and kinetic parameters of this transport. Progressive familial intrahepatic cholestasis mutations were introduced into the yeast plasma membrane P4-ATPase Dnf2p, and the effect of these mutations on its catalysis of phospholipid transport were determined. The results of these measurements have implications for the basis of the disease and for the mechanism of phospholipid transit through the enzyme during the reaction cycle. Background: Mutations in the P4-ATPase ATP8B1 cause progressive familial intrahepatic cholestasis (PFIC). Results: Homologous mutations in yeast P4-ATPase Dnf2p alter enzyme activity and subunit interaction phenotypes. Conclusion: This approach provides a method for characterizing the pathological basis of PFIC mutations. Significance: This approach identifies residues involved in substrate binding and a potential path for phospholipid movement.