UZH-Logo

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


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.

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.

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.

Citations

6 citations in Web of Science®
5 citations in Scopus®
Google Scholar™

Altmetrics

Additional indexing

Item Type:Journal Article, refereed
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:1 March 2001
Deposited On:11 Feb 2008 12:23
Last Modified:05 Apr 2016 12:18
Publisher:Springer
ISSN:0022-2631
Publisher DOI:10.1007/s002320010059
PubMed ID:11284206

Download

Full text not available from this repository.View at publisher

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations