Quick Search:

uzh logo
Browse by:
bullet
bullet
bullet
bullet

Zurich Open Repository and Archive 

Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-3094

Gisler, S M; Kittanakom, S; Fuster, D; Wong, V; Bertic, M; Randanovic, T; Hall, R A; Murer, H; Biber, J; Markovich, D; Moe, O W; Stagljar, I (2008). Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system. Molecular & Cellular Proteomics, 7:1362-1377.

[img]
Preview
Accepted Version
PDF
3MB

Abstract

PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Center for Integrative Human Physiology
04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
DDC:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2008
Deposited On:09 Sep 2008 09:02
Last Modified:28 Nov 2013 00:41
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:1535-9476
Additional Information:This research was originally published in Gisler, S M; Kittanakom, S; Fuster, D; Wong, V; Bertic, M; Randanovic, T; Hall, R A; Murer, H; Biber, J; Markovich, D; Moe, O W; Stagljar, I (2008). Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system. Molecular & Cellular Proteomics, 7:1362-1377. © the American Society for Biochemistry and Molecular Biology.
Publisher DOI:10.1074/mcp.M800079-MCP200
PubMed ID:18407958
Citations:Web of Science®. Times Cited: 38
Google Scholar™
Scopus®. Citation Count: 42

Users (please log in): suggest update or correction for this item

Repository Staff Only: item control page