Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system.
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.
Gisler
Serge M
SM
Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Kittanakom
Saranya
S
Fuster
Daniel
D
Wong
Victoria
V
Bertic
Mia
M
Radanovic
Tamara
T
Hall
Randy A
RA
Murer
Heini
H
Biber
Jürg
J
Markovich
Daniel
D
Moe
Orson W
OW
Stagljar
Igor
I
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D008322:Mammals / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009865:Oocytes / Q000737:chemistry; D054731:PDZ Domains; D010957:Plasmids / Q000138:chemical synthesis; D011485:Protein Binding; D051381:Rats; D012441:Saccharomyces cerevisiae; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D014981:Xenopus
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D025801:Ubiquitin