Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system

Gisler, Serge M; Kittanakom, Saranya; Fuster, Daniel Guido; Wong, Victoria; Bertic, Mia; Radanovic, Tamara; Hall, Randy A; Murer, Heini; Biber, Jürg; Markovich, Daniel; Moe, Orson W; Stagljar, Igor (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(7), pp. 1362-77. Bethesda, Md.: American Society for Biochemistry and Molecular Biology 10.1074/mcp.M800079-MCP200

[img] Text
SplitpaperfinalPDF.pdf - Published Version
Restricted to registered users only
Available under License Publisher holds Copyright.

Download (733kB) | Request a copy

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 (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Dermatology, Urology, Rheumatology, Nephrology, Osteoporosis (DURN) > Clinic of Nephrology and Hypertension

UniBE Contributor:

Fuster, Daniel Guido

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1535-9476

ISBN:

18407958

Publisher:

American Society for Biochemistry and Molecular Biology

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 15:05

Last Modified:

19 Jan 2016 15:27

Publisher DOI:

10.1074/mcp.M800079-MCP200

PubMed ID:

18407958

Web of Science ID:

000257463000013

BORIS DOI:

10.7892/boris.28102

URI:

https://boris.unibe.ch/id/eprint/28102 (FactScience: 116694)

Actions (login required)

Edit item Edit item
Provide Feedback