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Traebert, M; Köhler, K; Lambert, G; Biber, J; Forster, I C; Murer, H (2001). Investigating the surface expression of the renal type IIa Na+/Pi-cotransporter in Xenopus laevis oocytes. Journal of Membrane Biology, 180(1):83-90.

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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+/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.

Item Type:Journal Article, refereed
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
DDC:570 Life sciences; biology
Language:English
Date:01 March 2001
Deposited On:11 Feb 2008 13:23
Last Modified:27 Nov 2013 17:50
Publisher:Springer
ISSN:0022-2631
Publisher DOI:10.1007/s002320010059
PubMed ID:11284206
Citations:Web of Science®. Times Cited: 6
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