Investigating the surface expression of the renal type IIa Na+/Pi-cotransporter in Xenopus laevis oocytes.
We have combined a functional assay, surface labeling and immunocytochemical methods to compare total and surface-exposed renal type IIa Na+/Pi 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+/Pi-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.
Traebert
M
M
Institute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Köhler
K
K
Lambert
G
G
Biber
J
J
Forster
I
I
Murer
H
H
D000818:Animals; D001692:Biological Transport; D001710:Biotin / Q000031:analogs & derivatives; D019921:Biotinylation; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004563:Electrochemistry; D007202:Indicators and Reagents; D008698:Mesylates; D009154:Mutation; D009865:Oocytes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D007202:Indicators and Reagents; D008698:Mesylates; C419543:N-biotinylaminoethyl methanethiosulfonate; D011994:Recombinant Proteins; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; C074502:methanethiosulfonate; D001710:Biotin