[No authors listed]
Co-immunoprecipitation studies using mouse ileal proteins and transfected HEK-293 (human embryonic kidney-293) cells revealed that the two proteins, Ostalpha and Ostbeta, which generate the organic-solute transporter are able to immunoprecipitate each other, indicating a heteromeric complex. Mouse ileal Ostalpha protein appeared on Western blots largely as bands of 40 and 80 kDa, the latter band consistent with an Ostalpha homodimer, and both of these bands were sensitive to digestion by the glycosidase PNGase F (peptide:N-glycosidase F). Ostbeta 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 Ostalpha, and only the 19 kDa form of Ostbeta, were detected among the immunoprecipitated proteins, indicating that the interaction between Ostalpha and Ostbeta is associated with specific post-translational processing. Additional evidence for homodimerization of Ostalpha and for a direct interaction between Ostalpha and Ostbeta was provided by BiFC (bimolecular fluorescence complementation) analysis of HEK-293 cells transfected with Ostalpha and Ostbeta tagged with yellow-fluorescent-protein fragments. BiFC analysis and surface immunolabelling of transfected HEK-293 cells also indicated that the C-termini of both Ostalpha and Ostbeta are facing the intracellular space. The interaction between Ostalpha and Ostbeta 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 Ostalpha-deficient (Ostalpha-/-) mice. In Ostalpha-/- mice, Ostbeta mRNA levels were maintained, yet Ostbeta protein was not detectable, indicating that Ostbeta protein is not stable in the absence of Ostalpha. Overall, these findings identify the membrane topology of Ostalpha and Ostbeta, demonstrate that these proteins are present as heterodimers and/or heteromultimers, and indicate that the interaction between Ostalpha and Ostbeta increases the stability of the proteins and is required for delivery of the heteromeric complex to the plasma membrane.
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