Investigating the Surface Expression of the Renal Type IIa Na+/P i -Cotransporter in Xenopus laevis Oocytes

Investigating the Surface Expression of the Renal Type IIa Na+/P i -Cotransporter in Xenopus... We have combined a functional assay, surface labeling and immunocytochemical methods to compare total and surface-exposed renal type IIa Na+/P i cotransporter protein. The wild-type type cotransporter (NaPi-IIa) and its functionally comparable cysteine mutant S460C were expressed in Xenopus oocytes. S460C contains a novel cysteine residue that, when modified by preincubation with methanethiosulfonate reagents, leads to complete suppression of cotransport function. This allowed surface labeling of the S460C using MTSEA-Biotin and confirmation by electrophysiology on the same cell. Protein was analyzed by Western blotting before and after streptavidin precipitation and by immunocytochemistry and immunogold electronmicroscopy. MTSEA-Biotin treatment resulted in a complete inhibition of S460C-mediated Na+/P i -cotransport activity, which indicated that all transporters at the surface were biotinylated. After biotinylation, only a small fraction of total S460C protein was precipitated by streptavidin compared with the total amount of S460C protein detected in the lysate. Light- and electron-microscopy analysis of oocytes showed a large amount of WT and S460C transporter protein beneath the oocyte membrane. These data indicate that the apparent weak labeling efficiencies of surface-biotinylation-based assays of membrane proteins heterologously expressed in oocytes can be related to diminished incorporation of the protein in the oolemma. The Journal of Membrane Biology Springer Journals

Investigating the Surface Expression of the Renal Type IIa Na+/P i -Cotransporter in Xenopus laevis Oocytes

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Copyright © Inc. by 2001 Springer-Verlag New York
Life Sciences; Biochemistry, general; Human Physiology
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