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. 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. The mammalian renal proximal tubule sustains reabsorption of fluid and solutes of the highest variety and magnitude. Vectorial transport in this epithelium is mediated by the polarized distribution of polytopic transport proteins. Strikingly many membrane proteins located at the apical but not at the basolateral side harbor a C-terminal PDZ-binding motif. The acronym PDZ denotes a conserved modular domain of 90 amino acids that recognizes an octapeptide PDZ-binding motif primarily residing at the C termini of target proteins (1Hung A.Y. Sheng M. PDZ domains: structural modules for protein complex assembly.J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text and functional of the PDZ of and of PDZ domain by of PDZ are to their binding modules PDZ proteins that bind the where is and is a of PDZ is an of PDZ-binding membrane proteins in the apical membrane of proximal PDZ proteins and proximal a in of proximal Full Text Full Text The used C C-terminal of for protein of membrane yeast MYTH to the of the MYTH 2.0 to the N terminus of the of a for the of MYTH sulfate of domain in and PDZ protein from transcription yeast amino to and of and The 2002; The NaPi-IIa and NaS1 uptake of phosphate and sulfate and of mammalian sulfate The protein of has is this of apical PDZ-binding proteins as and of a novel protein in and and M. of and in Xenopus Conventional yeast two-hybrid and such as identified proximal tubule PDZ proteins as binding partners to the apical proteins and PDZ proteins and proximal The factor NHERF-1 and NHERF-2 as that of NHERF-1 the that in renal apical The of and in the of Biol. Chem. Full Text Full Text two PDZ modules in and to to with complex of renal transport and of by from the C-terminal an PDZ-binding in has been protein the and the protein Biol. Chem. Full Text Full Text their NHERF-1 and NHERF-2 with the C-terminal motif of NaPi-IIa M. of the with PDZ Biol. Chem. Full Text Full Text PDZ proteins PDZK1 and PDZ modules of the PDZ domain with the NaPi-IIa C-terminal as the of PDZ a in of proximal Full Text Full Text M. of the with PDZ Biol. Chem. Full Text Full Text In and of PDZK1 but not NHERF-1 NHERF-2 bind to a in of proximal Full Text Full Text of with the protein complex in renal proximal a novel PDZ domain-containing protein in and to with the a between PDZ proteins and the sulfate transporter NaS1 has not been in have the of the of these PDZ proteins. the of and in proximal PDZ proteins and proximal was to between the and the membrane of protein by the PDZ domain protein Biol. Chem. Full Text Full Text M. M. M. PDZ domain protein membrane Biol. Chem. 2002; 277: Full Text Full Text In these apical of NaPi-IIa on NHERF-1 PDZ proteins and proximal and of NHERF-1 the that in renal apical The of and in the of Biol. Chem. Full Text Full Text M. M. of the an PDZK1 was to an apical for the of renal transporters a in of proximal Full Text Full Text M. of PDZK1 in membrane of renal the protein is at the apical membrane of is that as an apical of PDZK1 of with the protein complex in renal proximal in was to the of the in of protein by the PDZ domain protein Biol. Chem. 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Chem. 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NaPi-IIa with NaPi-IIa full-length and proteins in yeast as in In PDZ proteins their target membrane proteins. The of that not of the PDZ-binding motifs was for PDZ proteins with of yeast yeast and not as as with the In with a in of proximal Full Text Full Text of with the protein complex in renal proximal with PDZK1 with NHERF-1 NHERF-2 when based on of and in and in a functional the apical NaS1 membrane transporter to a in proximal tubule sulfate reabsorption in its the of NaS1 interactions has not been by of the available interactive proteomics this we a MYTH 2.0 using a to NaS1 protein that from of in to the the in the where for was by the yeast the MYTH 2.0 of yeast novel NHERF-2, and PDZK1 this study on the PDZ proteins, the of the identified NaS1 in the MYTH 2.0 was from NHERF-1 and with PDZK1 NHERF-2 interactions specific for the NaS1 protein of using an NaS1 in the MYTH from a MYTH 2.0 on a of identified protein domain-containing protein protein protein protein protein factor protein protein domain-containing protein in a of NaS1 are for binding to and NHERF-2, full-length and and for their to with the identified PDZ proteins in MYTH The and of by and by using an not to our full-length NaS1 MYTH with and NHERF-2 of the C-terminal amino acids of NaS1 not the binding of PDZ domain proteins. However, of an amino acids of NaS1 the binding of PDZK1 the to for PDZK1 The of NHERF-1 and NHERF-2 was with this NaS1 but not with the an interacting for in the between the and on NaS1 not with of the and thus the of the and from the MYTH 2.0 is an to binding motifs on proteins as by the NaS1 sulfate to their with NHERF-1 and NHERF-2 NaS1 this we a transport assay in Xenopus oocytes and NaS1 with NHERF-1 NHERF-2, sulfate uptake was a functional of NaS1 by protein-protein interactions between mammalian transporters and their PDZ the MYTH 2.0 system is to the of membrane proteins. in and NaPi-IIa proteins in a on the on the transporters the at for NaPi-IIa a in as a with the of NaPi-IIa with its C-terminal and in was specific of the in the of renal that the of NaPi-IIa not the C-terminal of NaPi-IIa was in oocytes of and The and of the NaPi-IIa protein found in the membrane from oocytes from of oocytes PDZ is not for in of NaPi-IIa was specific as from a with was The that of the used for protein-protein interactions soluble from proteins a to our of proteins. the of tails from integral membrane proteins. soluble tails of proteins when from the of their the previously MYTH in the this its the MYTH technology has to the of from protein such as transporters and interactions in transporters at the membrane by the split-ubiquitin system and functional 2002; Full Text Full Text a yeast protein system based on split-ubiquitin for the of interactions between membrane proteins in of and protein the membrane by Biol. Chem. Full Text Full Text a of novel using the split-ubiquitin membrane yeast two-hybrid interactions between M. interactions by a system for studies of membrane protein and a yeast transporter protein-protein interactions for the yeast transporter by split-ubiquitin membrane yeast two-hybrid Full Text Full Text PDZ proteins a amino acids at the of soluble C-terminal tails from proteins (1Hung A.Y. Sheng M. PDZ domains: structural modules for protein complex assembly.J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text studies showed that PDZ domain-containing proteins and bind motifs and functional of the PDZ of and and of PDZ in complex assembly.J. M. binding of the PDZ domain of to a conserved in the C-terminal of Full Text Full Text However, the of a full-length membrane-inserted protein on a and the of binding the C-terminal domain have thus not been with interactive proteomics this we modified a MYTH to a where the C-terminal of a polytopic membrane protein is to for with PDZ proteins five proteins to the location and in we interactions of PDZ proteins from proximal of identified in but we applied full-length membrane proteins of their termini as baits. The developed MYTH 2.0 system the PDZ-binding with we interacting such as for NaPi-IIa and NHERF-2 for NaS1 by a by In this is the and of NaS1 to is the of the modified MYTH 2.0 we used the MYTH 2.0 assay to and the on NaS1 for binding to the PDZ proteins. Finally the modified system of NaPi-IIa that was by its the MYTH 2.0 a tool to binding of membrane proteins and establishes a novel for interactive proteomics studies of membrane protein complexes. the proteins and NaS1 in the MYTH 2.0 with their C termini from and in yeast that the of to the transporter proteins in a of of transporters in a distribution of and as in a to their native distribution in mammalian and with We not to in this of a of transporters that are in yeast as previously of in Biol. Chem. Full Text Full Text The PDZ proteins NHERF-2, and by using soluble C termini from and as a in of proximal Full Text Full Text M. of the with PDZ Biol. Chem. Full Text Full Text of with the protein complex in renal proximal M. M. of the an interactions have been to C-terminal PDZ motifs of the transporters a in of proximal Full Text Full Text M. of the with PDZ Biol. Chem. Full Text Full Text of with the protein complex in renal proximal interacting PDZ proteins have been for and is for NaPi-IIa C terminus was on an of to different PDZ proteins in the detection of and In the MYTH 2.0 of was for of NHERF-1 and NHERF-2 with of the C-terminal PDZ motif of the C terminus with binding within the C-terminal domain of for NHERF-1 but not for NHERF-2 target binding on is C-terminal from the C-terminal PDZ was to by in protein the and the protein Biol. Chem. Full Text Full Text NaPi-IIa a binding PDZ proteins, and to at the in the MYTH 2.0 The that of NaPi-IIa with NHERF-1 and have binding to that of when by from MYTH have to and The of binding not to NaPi-IIa the C-terminal amino acids for PDZ interactions in the MYTH 2.0 and in the PDZ not with protein used in this study in specific of NaPi-IIa in a to that for the of of protein by the PDZ domain protein Biol. Chem. Full Text Full Text Furthermore NaPi-IIa was found to was by from an system and was by membrane-inserted of termini a PDZ the MYTH system in the of PDZK1 with NHERF-1 and NHERF-2 that a specific for the protein PDZK1 from proximal that the of proteins such as NaPi-IIa and of a novel protein in and and M. of and in Xenopus of as a of PDZK1 and Biol. Chem. Full Text Full Text Moreover MYTH a to the of NaS1 to with and NHERF-2 a that was not by interactive The of and was by a functional assay that NHERF-1 and to a NHERF-2 sulfate uptake in Xenopus oocytes when with NaS1. is that NHERF-1 NHERF-2 the protein of NaS1 on the the to are our from the MYTH 2.0 using of NaS1 as baits. The of PDZ-binding on this sulfate transporter the C-terminal upstream of the C terminus, was for PDZK1 and of the of such PDZ-binding on integral membrane proteins have been previously of PDZ domain by of a complex with the and a PDZ Biol. Chem. Full Text Full Text demonstrate that and NaPi-IIa N and C termini the whereas at the N termini of and NaS1 of the with in yeast in a MYTH and with was the In the MYTH 2.0 applied to the of a integral membrane protein. In we have developed an membrane system that to study polytopic membrane proteins, are in to the lipid as for the detection of protein interactions this of proteins. system interactions that have been identified previously and binding partners that not by protein-protein we that our the to of membrane proteins and their interacting partners. We the from the for the yeast for and and for of and for for the and for

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