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.

A GFP-tagged version of the pseudorabies virus protein UL56 localizes to the Golgi and trans-Golgi network through a predicted C-terminal leucine-rich helix in transfected cells

“…4a). Among them, the dileucine motif is of high interest because its importance in targeting transmembrane proteins to the endosome/lysosome system and TGN [33–38]. Although several di-leucine motifs are present in PRV pUL56, our data showed that none had the ability to target pUL56 to the TGN.…”

The calmodulin‐binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3

“…In addition, the dileucine determinant at the C terminus of the luteinizing hormone b subunit prevents its increased trafficking and exocytosis (48). In contrast, the substitution of a dileucine motif impairs the membrane localization of the Organic Soluble Transporter (44). Our study now introduces CaM into this debate because Kv1.3, KCNE4, and CaM are in a tight relationship.…”

“…We identified a tetraleucine motif within the C-terminal domain of KCNE4 that mediates the association with Kv1.3. Interestingly, dileucine motifs promote endocytosis, protein-protein interactions, and basolateral trafficking (43)(44)(45)(46). Altering the KCNE4 tetraleucine motif, as well as the suppression of the ERRM, improved the peptide targeting to the plasma membrane.…”

Organic solute transporter OSTα/β is overexpressed in nonalcoholic steatohepatitis and modulated by drugs associated with liver injury

“…Two other studies reported a modest one-to-fivefold increase in OST␣ mRNA in extrahepatic cholestasis and PBC (7,51). The constant OST␣ mRNA, but increased OST␣ protein, can be explained by the fact that OST␣ protein is unstable without OST␤ protein (14,40,60). In liver tissue from NASH patients and patients with PBC, OST␤ mRNA and protein are remarkably induced, which enables OST␣-OST␤ complex formation.…”