Journal
BIOCHEMICAL JOURNAL
Volume 407, Issue -, Pages 363-372Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20070716
Keywords
bimolecular fluorescence complementation (BiFC); co-immunoprecipitation (co-IP); heterodimer; membrane topology; organic-solute transporter (Ost); Ost alpha(-/-) mouse
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Funding
- NIDDK NIH HHS [R01 DK048823, R01 DK067214, DK48823, DK067214] Funding Source: Medline
- NIEHS NIH HHS [ES07026, R01 ES007026, T32 ES007026, P30 ES003828, P30 ES001247, ES01247, ES03828] Funding Source: Medline
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Co-immunoprecipitation studies using mouse ileal proteins and transfected HEK-293 (human embryonic kidney-293) cells revealed that the two proteins, Ost alpha and Ost ss, which generate the organic-solute transporter are able to immunoprecipitate each other, indicating a heteromeric complex. Mouse ileal Ost alpha protein appeared on Western blots largely as bands of 40 and 80 kDa, the latter band consistent with an Osta alpha homodimer, and both of these bands were sensitive to digestion by the glycosidase PNGase F (peptide:N-glycosidase F). Ost ss appeared as bands of 17 and 19 kDa, and these bands were not sensitive to PNGase F. Both the 40 and 80 kDa forms of Ost alpha, and only the 19 kDa form of Ost ss were detected among the immunoprecipitated proteins, indicating that the interaction between Ost alpha and Ost ss is associated with specific post-translational processing. Additional evidence for homodimerization of Ost alpha and for a direct interaction between Ost alpha and Ost ss was provided by BiFC (bimolecular fluorescence complementation) analysis of HEK-293 cells transfected with Ost alpha and Ost ss tagged with yellow-fluorescent-protein fragments. BiFC analysis and surface immunolabelling of transfected HEK293 cells also indicated that the C-termini of both Ost alpha and Ost ss are facing the intracellular space. The interaction between Ost alpha and Ost ss was required not only for delivery of the proteins to the plasma membrane, but it increased their stability, as noted in transfected HEK-293 cells and in tissues from Ost alpha-deficient (Osta(-/-)) mice. In Osta(-/-) mice, Ost ss mRNA levels were maintained, yet Ost ss protein was not detectable, indicating that Ost ss protein is not stable in the absence of Ost alpha. Overall, these findings identify the membrane topology of Ost alpha and Ost ss, demonstrate that these proteins are present as heterodimers and/or heteromultimers, and indicate that the interaction between Ost alpha and Ost ss increases the stability of the proteins and is required for delivery of the heteromeric complex to the plasma membrane.
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