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.
Highlights
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
Except for NaS1, all required target proteins were not expressed from the pCLB-1 vector in which the expression of the transcription factor (TF)-Cub-Bait chimeric protein is under control of a weak CYC1 promoter
The membrane yeast two-hybrid (MYTH) technology has led to the dissection of quaternary structures from protein complexes such as sucrose transporters [49]; a yeast protein translocation machinery [25, 50]; a receptor tyrosine kinase, ErbB3 [24]; physical interactions between plant Kϩ channels [31]; and a yeast ATP-binding cassette transporter Ycf1p [26]
Summary
Cloning of the MYTH 2.0 Bait Vectors pTLB-1 and pCLB-1—The MYTH bait vector pTMBV4 (Dualsystems Biotech AG, Schlieren, Switzerland) allows the fusion of a bait to the N terminus of a Cub(LexAϩVP16) (Cub-TF) reporter module (conventional MYTH system) whose LexA is mutated at R156G to abate the affinity toward the exogenous reporter gene promoters (Dualsystems Biotech AG). Construction of a NubG-X Human Kidney cDNA Library and MYTH 2.0 Screen Using the TF-Cub-NaS1 as a Bait—An oligo(dT)-primed, size-selected (0.6 – 8 kb; average insert size of 1.8 kb) human kidney cDNA library with 2.6 ϫ 106 independent clones was custom constructed in the prey vector pPR3-N by Dualsystems Biotech AG and was a kind gift of Dr Reinhart Reithmeier This cDNA library was transformed into the yeast reporter strain THY AP4 expressing the human TF-Cub-NaS1 bait. Yeast PM971 (⌬pho89::TRP1 ⌬pho84::HIS3 ade leu his trp ura3), a knock-out strain of both high affinity Pi transporters [38], was transformed with TF-Cub-NaPi-IIa in pTLB-1 and grown overnight in 30 ml of SDϪLeu. Cells from 10.5 A600 units were collected (700 ϫ g for 5 min) and washed in SDϪLeu/ϪPi (20 mM Mes at pH 7.4, SDϪLeu devoid of KH2PO4 (ForMedium, CYN0801)). Primary antibodies were as follows: mouse anti-HA (clone HA-7, 1:10,000; Sigma), rabbit anti-MYC (2 g/ml; Upstate, Waltham, MA), or rabbit anti-NaPi-IIa (1:6000)
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