Previous investigation revealed that hepatic uptake or organic anions such as bilirubin and sulfobromophthalein (BSP) has carrier-mediated kinetics. We have shown that uptake of these ligands by cultured rat hepatocytes and isolated perfused rat liver requires Cl in the medium. We have also identified and purified an organic anion binding protein (OABP) from a sinusoidal enriched rat liver cell plasma membrane subfraction. The presence of OABP on the cell surface correlates with its ability to transport bilirubin and BSP. Using monospecific antibodies, we cloned OABP from a rat liver lambda gt11 expression library and found marked homology with the B-subunit of F1-ATPase. Our long-term goal is to understand the mechanism of organic anion transport and its relationship to specific proteins in normal liver and in various acquired and inheritable disorders. The specific aims of the proposed studies are: (1) To investigate the role of inorganic anions in liver cell organic anion uptake. Uptake kinetics of BSP and bilirubin will be defined under conditions of unidirectional inorganic anion gradients (e.g., Cl , OH ) in cultured hepatocytes and plasma membrane vesicles. The influence of Cl on interaction of BSP with specific plasma membrane proteins will be studied by photoaffinity labelling and ligand binding. (2) To define the time course and intracellular events associated with sorting of OABP to the cell surface. These studies will include metabolic labelling and determination of the oligomeric state of OABP in the plasma membrane. (3) To define by techniques of molecular biology the relationship of surface OABP to the B-subunit of mitochondrial F1-ATPase. Distribution of mRNA between free and membrane-bound polysomes will be determined and cell-free synthesis and translocation into microsomes and mitochondria will be studied. Whether specific mRNAs coding for mitochondrial and cell surface proteins exist will be studied by primer extension methodology. if specific surface OABP mRNA is isolated, cells lacking the antigen on the surface will be transfected and studies will be performed to determine whether transport of BSP is restored.