Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
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 SM
,
Kittanakom S
,
Fuster D
,
Wong V
,
Bertic M
,
Radanovic T
,
Hall RA
,
Murer H
,
Biber J
,
Markovich D
,
Moe OW
,
Stagljar I
.
???displayArticle.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.
Beck,
Molecular aspects of renal tubular handling and regulation of inorganic sulfate.
2001, Pubmed
Beck,
Molecular aspects of renal tubular handling and regulation of inorganic sulfate.
2001,
Pubmed
Beck,
The mouse Na(+)-sulfate cotransporter gene Nas1. Cloning, tissue distribution, gene structure, chromosomal assignment, and transcriptional regulation by vitamin D.
2000,
Pubmed
,
Xenbase
Bezprozvanny,
Classification of PDZ domains.
2001,
Pubmed
Blasco,
Rat kidney MAP17 induces cotransport of Na-mannose and Na-glucose in Xenopus laevis oocytes.
2003,
Pubmed
,
Xenbase
Breeden,
Regulation of the yeast HO gene.
1985,
Pubmed
Burckhardt,
The Na+/H+ exchanger gene family.
2002,
Pubmed
Cheng,
Modulation of mature cystic fibrosis transmembrane regulator protein by the PDZ domain protein CAL.
2004,
Pubmed
Cheng,
A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.
2002,
Pubmed
Christopherson,
PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain.
1999,
Pubmed
Daram,
Functional analysis and cell-specific expression of a phosphate transporter from tomato.
1998,
Pubmed
Dawson,
Hyposulfatemia, growth retardation, reduced fertility, and seizures in mice lacking a functional NaSi-1 gene.
2003,
Pubmed
de Jong,
The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer.
2003,
Pubmed
,
Xenbase
Fam,
P2Y1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2.
2005,
Pubmed
Fetchko,
Application of the split-ubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions.
2004,
Pubmed
Fields,
A novel genetic system to detect protein-protein interactions.
1989,
Pubmed
Gietz,
Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.
2002,
Pubmed
Gisler,
Interaction of the type IIa Na/Pi cotransporter with PDZ proteins.
2001,
Pubmed
Gisler,
PDZK1: I. a major scaffolder in brush borders of proximal tubular cells.
2003,
Pubmed
Gotta,
The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae.
1996,
Pubmed
Guarente,
Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast.
1983,
Pubmed
Harris,
Mechanism and role of PDZ domains in signaling complex assembly.
2001,
Pubmed
Hernando,
PDZ proteins and proximal ion transport.
2004,
Pubmed
Hershko,
The ubiquitin system for protein degradation.
1992,
Pubmed
Hillier,
Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex.
1999,
Pubmed
Hung,
PDZ domains: structural modules for protein complex assembly.
2002,
Pubmed
Iyer,
Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins.
2005,
Pubmed
Johnsson,
Split ubiquitin as a sensor of protein interactions in vivo.
1994,
Pubmed
Kocher,
Targeted disruption of the PDZK1 gene by homologous recombination.
2003,
Pubmed
Kocher,
Identification and partial characterization of a novel membrane-associated protein (MAP17) up-regulated in human carcinomas and modulating cell replication and tumor growth.
1996,
Pubmed
Kocher,
Identification and partial characterization of PDZK1: a novel protein containing PDZ interaction domains.
1998,
Pubmed
Kocher,
PDZK1, a novel PDZ domain-containing protein up-regulated in carcinomas and mapped to chromosome 1q21, interacts with cMOAT (MRP2), the multidrug resistance-associated protein.
1999,
Pubmed
Lamprecht,
The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3.
1998,
Pubmed
Markovich,
Physiological roles and regulation of mammalian sulfate transporters.
2001,
Pubmed
Martinez,
Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae.
1998,
Pubmed
Murer,
Proximal tubular phosphate reabsorption: molecular mechanisms.
2000,
Pubmed
Obrdlik,
K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions.
2004,
Pubmed
Paumi,
Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.
2007,
Pubmed
Persson,
Regulation of phosphate acquisition in Saccharomyces cerevisiae.
2003,
Pubmed
Plant,
Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast.
1999,
Pubmed
Pribanic,
Interactions of MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells.
2003,
Pubmed
Reinders,
Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays.
2002,
Pubmed
Runyon,
Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3.
2007,
Pubmed
Scheper,
Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein.
2003,
Pubmed
Silver,
Identification of small PDZK1-associated protein, DD96/MAP17, as a regulator of PDZK1 and plasma high density lipoprotein levels.
2003,
Pubmed
Stagljar,
Analysis of membrane protein interactions using yeast-based technologies.
2002,
Pubmed
Stagljar,
A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo.
1998,
Pubmed
Thaminy,
Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system.
2003,
Pubmed
Thomson,
Role of PDZK1 in membrane expression of renal brush border ion exchangers.
2005,
Pubmed
Weinman,
Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by PKA.
2000,
Pubmed
Weinman,
NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes.
2003,
Pubmed
Weinman,
A C-terminal PDZ motif in NHE3 binds NHERF-1 and enhances cAMP inhibition of sodium-hydrogen exchange.
2003,
Pubmed
Wong,
Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled.
2003,
Pubmed
,
Xenbase
Yun,
NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin.
1998,
Pubmed
Yun,
cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein.
1997,
Pubmed
