Investigating the Surface Expression of the Renal Type IIa Na+/P
i
-Cotransporter in Xenopus laevis Oocytes |
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Authors: | M Traebert K Köhler G Lambert J Biber I Forster H Murer |
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Institution: | (1) Institute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland, CH |
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Abstract: | 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.
Received: 18 August 2000/Revised: 1 December 2000 |
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Keywords: | :Xenopus laevis oocytes — Surface labeling — NaPi-IIa — Electronmicroscopy — Immunocytochemistry — Biotinylation |
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