A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

Shuhua Xu,Albert Mennone,James L Boyer,An-Qiang Sun,Frederick J Suchy,Donald S Backos,Carol J Soroka,Matthew Seaman

doi:10.1371/journal.pone.0158269

Shuhua Xu, Albert Mennone + Show 6 more

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https://doi.org/10.1371/journal.pone.0158269

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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.

Highlights

Organic Solute Transporter beta (OSTβ, SLC51b) is associated with Organic Solute Transporter alpha (OSTα, SLC51a) subunit to form a novel heteromeric transporter (OSTα-OSTβ) that was discovered in 2003 [1]
The expression level of the LL/AA OSTβ mutant was similar to the WT OSTβ, little OSTα was detected in the total cell lysate, suggesting that the dileucine motif at the N-terminus of OSTβ might play an important role in stability of the OSTα subunit
The organic solute transporter alpha-beta (OSTα-OSTβ, SLC51) is a unique heteromeric, facilitated organic solute transporter that is expressed on the basolateral membrane of epithelium where sterol transport is essential [20]

Summary

Introduction

Organic Solute Transporter beta (OSTβ, SLC51b) is associated with Organic Solute Transporter alpha (OSTα, SLC51a) subunit to form a novel heteromeric transporter (OSTα-OSTβ) that was discovered in 2003 [1] These two proteins are co-expressed at the basolateral membranes in the ileum, kidney, and liver, as well as other epithelia, and function together to transport bile acids and other important sterol-derived molecules via a sodium-independent mechanism characterized by facilitated diffusion [2,3,4]. The mechanisms involved in the polarized membrane trafficking of OST proteins are not fully understood It remains to be determined which domain of OSTβ is required for the formation of a functional heterodimer with OSTα and which amino acids affect this interaction [7]

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