AbstractsBiology & Animal Science

The heteromeric organic solute and steroid transporter (Ostα-Ostβ) is a major basolateral transporter of bile acids and endogenous and exogenous conjugated steroids in a variety of tissues, including the small intestine, kidney, adrenal gland, and liver. The functional Ostα-Ostβ transporter requires co-expression of two distinct gene products, a predicted 340 amino acid, seven-transmembrane (TM) domain protein, Ostα, and a 128 amino acid, single TM domain polypeptide, Ostβ. The purpose of this study was to identify the mechanism by which these two gene products interact to generate transport activity. Direct evidence for heterodimerization between Ostα and Ostβ, as well as for homodimerization of Ostα, was provided by studies that utilized co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) analysis. BiFC analysis and surface immunolabeling of transfected HEK293 cells also indicated that the carboxyl termini of both Ostα and Ostβ are facing towards the intracellular space. The interaction between Ostα and Ostβ was found to be required not only for delivery of the proteins to the plasma membrane, but for maintaining protein stability, as assessed in transfected HEK293 cells and in tissues from Ostα-deficient mice. By characterizing multiple Ostβ mutant constructs, the TM domain of Ostβ was identified as a major site of interaction with Ostα. Of significance, the complex formed between Ostα and a 25-amino acid peptide consisting largely of the TM domain of Ostβ was trapped in the endoplasmic reticulum, suggesting that this peptide or related peptides may be used as inhibitors of the transporter. Additional studies revealed that the cysteine-rich region in Ostα appeared to be required for transport activity, but not for heterodimerization or trafficking. In contrast, mutation of the putative endoplasmic reticulum retrieval sequences (Arg-Arg-Lys and Arg- Asn-Arg) had only small effects on function. Overall, these findings provide important insights into the structure and function of the heterodimeric Ostα-Ostβ transporter, and suggest an approach for inhibiting this transporter. Because Ostα- Ostβ is critical for intestinal bile acid and lipid absorption, these findings have important implications for the regulation of bile acid and lipid levels in humans.