Investigating the surface expression of the renal type IIa Na+/P-i-cotransporter in Xenopus laevis oocytes

We have combined a functional assay, surface labeling and immunocytochemica l methods to compare total and surface-exposed renal type IIa Na+/P-i cotra nsporter protein. The wild-type type cotransporter (NaPi-IIa) and its funct ionally comparable cysteine mutant S460C were expressed in Xenopus oocytes. S460C contains a novel cysteine residue that, when modified by preincubati on with methanethiosulfonate reagents, leads to complete suppression of cot ransport function. This allowed surface labeling of the S460C using MTSEA-B iotin and confirmation by electrophysiology on the same cell. Protein was a nalyzed by Western blotting before and after streptavidin precipitation and by immunocytochemistry and immunogold electronmicroscopy. MTSEA-Biotin tre atment resulted in a complete inhibition of S460C-mediated Na+/P-i-cotransp ort activity, which indicated that all transporters at the surface were bio tinylated. After biotinylation, only a small fraction of total S460C protei n 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 t he oocyte membrane. These data indicate that the apparent weak labeling eff iciencies of surface-biotinylation-based assays of membrane proteins hetero logously expressed in oocytes can be related to diminished incorporation of the protein in the oolemma.