title: A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.
abstract: The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.
authors.affiliation: Department of Internal Medicine and Yale Liver Center, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Shuhua
authors.initials: S
authors.lastname: Xu
authors.affiliation: Department of Internal Medicine and Yale Liver Center, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Soroka
authors.affiliation: Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, United States of America.
authors.forename: An-Qiang
authors.initials: AQ
authors.lastname: Sun
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, United States of America.
authors.forename: Donald S
authors.initials: DS
authors.lastname: Backos
authors.affiliation: Department of Internal Medicine and Yale Liver Center, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Albert
authors.initials: A
authors.lastname: Mennone
authors.affiliation: Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, United States of America.
authors.forename: Frederick J
authors.initials: FJ
authors.lastname: Suchy
authors.affiliation: Department of Internal Medicine and Yale Liver Center, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: James L
authors.initials: JL
authors.lastname: Boyer
mesh_terms: D020816:Amino Acid Motifs; D002462:Cell Membrane / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D000072417:Protein Domains; D021381:Protein Transport
chemical_list: D026901:Membrane Transport Proteins; C476577:SLC51B protein, human