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

Serge M. Gisler, Saranya Kittanakom, Daniel Fuster, Victoria Wong, Mia Bertic, Tamara Radanovic, Randy A. Hali, Heini Murer, Jürg Biber, Daniel Markovich, Orson W. Moe, Igor Stagljar

Research output: Contribution to journalArticle

58 Citations (Scopus)

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 (NaPilla) 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-lla 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.

Original languageEnglish (US)
Pages (from-to)1362-1377
Number of pages16
JournalMolecular and Cellular Proteomics
Volume7
Issue number7
DOIs
StatePublished - Jul 2008

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Two-Hybrid System Techniques
Membrane Transport Proteins
Ubiquitin
Hybrid systems
Yeast
PDZ Domains
Membranes
Monitoring
Membrane Proteins
Proteins
Yeasts
Sulfates
Transcription Factors
Lipid Bilayers
Lipid bilayers
Xenopus
Protein Binding
Proteomics
Oocytes
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

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; Wong, Victoria; Bertic, Mia; Radanovic, Tamara; Hali, Randy A.; Murer, Heini; Biber, Jürg; Markovich, Daniel; Moe, Orson W.; Stagljar, Igor.

In: Molecular and Cellular Proteomics, Vol. 7, No. 7, 07.2008, p. 1362-1377.

Research output: Contribution to journalArticle

Gisler, Serge M. ; Kittanakom, Saranya ; Fuster, Daniel ; Wong, Victoria ; Bertic, Mia ; Radanovic, Tamara ; Hali, Randy A. ; Murer, Heini ; Biber, Jürg ; Markovich, Daniel ; Moe, Orson W. ; Stagljar, Igor. / Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system. In: Molecular and Cellular Proteomics. 2008 ; Vol. 7, No. 7. pp. 1362-1377.
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AU - Fuster, Daniel

AU - Wong, Victoria

AU - Bertic, Mia

AU - Radanovic, Tamara

AU - Hali, Randy A.

AU - Murer, Heini

AU - Biber, Jürg

AU - Markovich, Daniel

AU - Moe, Orson W.

AU - Stagljar, Igor

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