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 employing 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 C-terminus of NaS1. Moreover, NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally, we used the MYTH 2.0 to demonstrate that the NaPi-IIa transporter homo-dimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomic studies of membrane protein complexes.