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PMID:22535958

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Citation

Christian, WV, Li, N, Hinkle, PM and Ballatori, N (2012) β-Subunit of the Ostα-Ostβ organic solute transporter is required not only for heterodimerization and trafficking but also for function. J. Biol. Chem. 287:21233-43

Abstract

The organic solute transporter, Ost/Slc51, is composed of two distinct proteins that must heterodimerize to generate transport activity, but the role of the individual subunits in mediating transport activity is unknown. The present study identified regions in Ostβ required for heterodimerization with Ostα, trafficking of the Ostα-Ostβ complex to the plasma membrane, and bile acid transport activity in HEK293 cells. Bimolecular fluorescence complementation analysis revealed that a 25-amino acid peptide containing the Ostβ transmembrane (TM) domain heterodimerized with Ostα, although the resulting complex failed to reach the plasma membrane and generate cellular [(3)H]taurocholate transport activity. Deletion of the single TM domain of Ostβ abolished interaction with Ostα, demonstrating that the TM segment is necessary and sufficient for formation of a heteromeric complex with Ostα. Mutation of the highly conserved tryptophan-asparagine sequence within the TM domain of Ostβ to alanines did not prevent cell surface trafficking, but abolished transport activity. Removal of the N-terminal 27 amino acids of Ostβ resulted in a transporter complex that reached the plasma membrane and exhibited transport activity at 30 °C. Complete deletion of the C terminus of Ostβ abolished [(3)H]taurocholate transport activity, but reinsertion of two native arginines immediately C-terminal to the TM domain rescued this defect. These positively charged residues establish the correct N(exo)/C(cyt) topology of the peptide, in accordance with the positive inside rule. Together, the results demonstrate that Ostβ is required for both proper trafficking of Ostα and formation of the functional transport unit, and identify specific residues of Ostβ critical for these processes.

Links

PubMed PMC3375545 Online version:10.1074/jbc.M112.352245

Keywords

Bile Acids and Salts/genetics; Bile Acids and Salts/metabolism; Cell Membrane/genetics; Cell Membrane/metabolism; HEK293 Cells; Humans; Ion Transport/physiology; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Mutation; Protein Multimerization/physiology; Protein Structure, Tertiary; Protein Transport/physiology

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

MOUSE:OSTB

involved_in

GO:0060050: positive regulation of protein glycosylation

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:OSTB

part_of

GO:0032991: protein-containing complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:OSTB

involved_in

GO:0015721: bile acid and bile salt transport

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:OSTB

involved_in

GO:0090314: positive regulation of protein targeting to membrane

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:OSTB

involved_in

GO:0031647: regulation of protein stability

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:OSTB

involved_in

GO:0070863: positive regulation of protein exit from endoplasmic reticulum

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:OSTB

part_of

GO:0016021: integral component of membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:OSTB

part_of

GO:0005886: plasma membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:OSTA

part_of

GO:0032991: protein-containing complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:OSTA

involved_in

GO:0015721: bile acid and bile salt transport

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:OSTA

part_of

GO:0005789: endoplasmic reticulum membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:OSTA

part_of

GO:0005886: plasma membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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