Membrane Yeast Two-hybrid Research Articles

Twinstar is a chitin synthase interacting protein with an essential role in insect cuticle biosynthesis

Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton. The membrane-integral β-glycosyltransferase, chitin synthase, has been identified as the central component in chitin biosynthesis. However, the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear. In this study, we employed split-ubiquitin membrane yeast two-hybrid (MYTH) and pull-down assays to demonstrate the physical interaction between Twinstar (Tsr), a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family, and chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster in vitro. The RNA interference (RNAi)-mediated global knockdown of Tsr in D. melanogaster resulted in larval lethality. Furthermore, targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality, while knocking down Tsr in the wing tissues led to wrinkled wings. Additionally, silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure. To validate the functional conservation of Tsr in other insect orders, the two agricultural pests Ostrinia furnacalis and Tribolium castaneum, representing lepidoptera and coleoptera insects, respectively, were investigated. Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O. furnacalis and TcTsr in T. castaneum produced abnormal larvae during molting or pupation in O. furnacalis and lethality in T. castaneum. Our findings not only improve our knowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.

Identification and characterization of protein interactions with the major Niemann-Pick type C disease protein in yeast reveals pathways of therapeutic potential.

Niemann-Pick type C (NP-C) disease is a rare lysosomal storage disease caused by mutations in NPC1 (95% cases) or NPC2 (5% cases). These proteins function together in cholesterol egress from the lysosome, whereby upon mutation, cholesterol and other lipids accumulate causing major pathologies. However, it is not fully understood how cholesterol is transported from NPC1 residing at the lysosomal membrane to the endoplasmic reticulum (ER) and plasma membrane. The yeast ortholog of NPC1, Niemann-Pick type C-related protein-1 (Ncr1), functions similarly to NPC1; when transfected into a mammalian cell lacking NPC1, Ncr1 rescues the diagnostic hallmarks of cholesterol and sphingolipid accumulation. Here, we aimed to identify and characterize protein-protein interactions (PPIs) with the yeast Ncr1 protein. A genome-wide split-ubiquitin membrane yeast two-hybrid (MYTH) protein interaction screen identified 11 ER membrane-localized, full-length proteins interacting with Ncr1 at the lysosomal/vacuolar membrane. These highlight the importance of ER-vacuole membrane interface and include PPIs with the Cyb5/Cbr1 electron transfer system, the ceramide synthase complex, and the Sec61/Sbh1 protein translocation complex. These PPIs were not detected in a sterol auxotrophy condition and thus depend on normal sterol metabolism. To provide biological context for the Ncr1-Cyb5 PPI, a yeast strain lacking this PPI (via gene deletions) exhibited altered levels of sterols and sphingolipids including increased levels of glucosylceramide that mimic NP-C disease. Overall, the results herein provide new physical and genetic interaction models to further use the yeast model of NP-C disease to better understand human NP-C disease.

Identification and Functional Analyses of Host Proteins Interacting with the P3a Protein of Brassica Yellows Virus

Viruses are obligate parasites that only undergo genomic replication in their host organisms. ORF3a, a newly identified non-AUG-initiated ORF encoded by members of the genus Polerovirus, is required for long-distance movement in plants. However, its interactions with host proteins still remain unclear. Here, we used Brassica yellows virus (BrYV)-P3a as bait to screen a plant split-ubiquitin-based membrane yeast two-hybrid (MYTH) cDNA library to explain the functional role of P3a in viral infections. In total, 138 genes with annotations were obtained. Bioinformatics analyses revealed that the genes from carbon fixation in photosynthetic, photosynthesis pathways, and MAPK signaling were affected. Furthermore, Arabidopsis thaliana purine permease 14 (AtPUP14), glucosinolate transporter 1 (AtGTR1), and nitrate transporter 1.7 (AtNRT1.7) were verified to interact with P3a in vivo. P3a and these three interacting proteins mainly co-localized in the cytoplasm. Expression levels of AtPUP14, AtGTR1, and AtNRT1.7 were significantly reduced in response to BrYV during the late stages of viral infection. In addition, we characterized the roles of AtPUP14, AtGTR1, and AtNRT1.7 in BrYV infection in A. thaliana using T-DNA insertion mutants, and the pup14, gtr1, and nrt1.7 mutants influenced BrYV infection to different degrees.

IFITM3 Interacts with the HBV/HDV Receptor NTCP and Modulates Virus Entry and Infection.

The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1–3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein–protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.

Lysine crotonylation of DgTIL1 at K72 modulates cold tolerance by enhancing DgnsLTP stability in chrysanthemum.

SummaryLysine crotonylation of proteins is a recently identified post‐translational modification (PTM) in plants. However, the function of lysine‐crotonylated proteins in response to abiotic stress in plants has not been reported. In this study, we identified a temperature‐induced lipocalin‐1‐like gene (DgTIL1) from chrysanthemum and showed that it was notably induced in response to cold stress. Overexpression of DgTIL1 enhanced cold tolerance in transgenic chrysanthemum. Ubiquitin membrane yeast two‐hybrid (MYTH) system and bimolecular fluorescence complementation (BIFC) assays showed that DgTIL1 interacts with a nonspecific lipid transfer protein (DgnsLTP), which can promote peroxidase (POD) gene expression and POD activity to reduce the accumulation of reactive oxygen species (ROS) and improve resistance to cold stress in DgnsLTP transgenic chrysanthemum. In addition, we found that DgTIL1 was lysine crotonylated at K72 in response to low temperature in chrysanthemum. Moreover, lysine crotonylation of DgTIL1 prevented DgnsLTP protein degradation in tobacco and chrysanthemum. Inhibition of DgnsLTP degradation by lysine crotonylation of DgTIL1 further enhanced POD expression and POD activity, reduced the accumulation of ROS under cold stress in DgTIL1 transgenic chrysanthemum, thus promoting the cold resistance of chrysanthemum.

High-Throughput Identification of Nuclear Envelope Protein Interactions in Schizosaccharomyces pombe Using an Arrayed Membrane Yeast-Two Hybrid Library.

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins from the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two -hybrid system. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nuclear membrane: Cut11/Ndc1, Lem2 and Ima1/Samp1/Net5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11-ts mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

Characterization of Deletion Mutants Encoding Protein Interactors of the Stress Sensor Mtl1p of Saccharomyces cerevisiae

We identified nine full‐length proteins that interact physically with the cytosolic domain of the Cell Wall Integrity (CWI) Pathway sensor Mtl1p in Saccharomyces cerevisiae using membrane yeast two hybrid (MYTH) assays. We tested eight viable null mutants for functionality when exposed to 0.5mM H2O2 or to a low‐ glucose environment.ObjectivesThe objectives of our study are to identify protein interactors of Mtl1p that exhibit growth phenotypes in the null mutant and may therefore be useful for designing novel antifungal pharmacotherapies.MethodsTo assess the roles of these protein interactors in stress resistance, cells bearing a deletion of these genes were challenged either by exposure to 0.5mM H2O2 or when grown on 0.05% glucose medium and compared for growth defects with a wild‐type strain. Likewise, double deletion combinations of sensor and interactors were tested in growth assays. The phosphorylation status of the CWI MAPK, Slt2p, was assessed by Western blot (WB) under each condition.ResultsIn low glucose concentration (0.05% w/v) all strains grew slower over a period of 8 hours compared to growth in medium with 2% glucose. We observed a marked decrease in the phosphorylation levels of Slt2p for all strains, including the mtl1Δ strain when cells were put in low glucose for two hours, suggesting a generalized effect which is not dependent on the expression of MTL1. When exposed to 0.5mM H2O2, the single deletion strains egd2Δ and ras2Δ experience enhanced sensitivity over the wild type and their untreated strains.ConclusionsWe successfully identified interactors of the cytosolic domain of Mtl1p whose deletions confer particular susceptibility phenotypes upon exposure to H2O2 and for growth on low glucose suggesting functionality under these conditions. By using sensor‐interactor double mutants we also identified a genetic interaction with the sensor protein Mtl1p.Support or Funding InformationThis project is supported by NIH Grants G12MD007600, U54MD007587, R25GM061838, and by Grant NIGMS INBRE P20GM103475 to the University of Puerto Rico, Medical Sciences Campus.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and Functional Testing of Novel Interacting Protein Partners for the Stress Sensors Wsc1p and Mid2p of Saccharomyces cerevisiae.

Wsc1p and Mid2p are transmembrane signaling proteins of cell wall stress in the budding yeast Saccharomyces cerevisiae. When an environmental stress compromises cell wall integrity, they activate a cell response through the Cell Wall Integrity (CWI) pathway. Studies have shown that the cytoplasmic domain of Wsc1p initiates the CWI signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor. Binding of Rom2p to the cytoplasmic tail of Wsc1p requires dephosphorylation of specific serine residues but the mechanism by which the sensor is dephosphorylated and how it subsequently interacts with Rom2p remains unclear. We hypothesize that Wsc1p and Mid2p must be physically associated with interacting proteins other than Rom2p that facilitate its interaction and regulate the activation of CWI pathway. To address this, a cDNA plasmid library of yeast proteins was expressed in bait strains bearing membrane yeast two-hybrid (MYTH) reporter modules of Wsc1p and Mid2p, and their interacting preys were recovered and sequenced. 14 previously unreported interactors were confirmed for Wsc1p and 29 for Mid2p. The interactors’ functionality were assessed by cell growth assays and CWI pathway activation by western blot analysis of Slt2p/Mpk1p phosphorylation in null mutants of each interactor under defined stress conditions. The susceptibility of these strains to different stresses were tested against antifungal agents and chemicals. This study reports important novel protein interactions of Wsc1p and Mid2p that are associated with the cellular response to oxidative stress induced by Hydrogen Peroxide and cell wall stress induced by Caspofungin.

Identification and Functional Testing of Six Interactors of the Transmembrane Sensor Mtl1p in the Budding Yeast Saccharomyces cerevisiae

Protein–protein interactions (PPIs) are often central to their cellular functions. These functions are often influenced by PPIs at all levels, including structural, energy metabolism, and signaling processes among others. The latter are crucial, as signaling functions often promote cell viability. We hypothesize that cytosolic protein interacting partners of the transmembrane sensor Mtl1p in Saccharomyces cerevisiae have a role in the stress response, promoting cell viability by modulation of the signaling process through the PKC1–dependent Cell Wall Integrity (CWI) Pathway.ObjectiveThe objective of our study is to identify cytosolic interactors of Mtl1p and to assess their relative importance in promoting cell viability upon exposure to diverse environmental stresses.MethodologyUsing membrane yeast two hybrid (MYTH) assays we have identified nine full–length proteins, and six other partial clones with C–terminals of various lengths that interact physically with the cytosolic domain of Mtl1p. In addition, using Western blots and growth curves we have made functional tests of deletion mutants corresponding to five of the identified full–length interactors, FPR1, ZEO1, RPL40a, EGD2, and CPR1, and one of the truncated interactors, RAS2, to assess their relative importance in promoting cell viability under oxidative the stress produced by exposure to H2O2, and cell wall stress produced by the antifungal Caspofungin.ResultsIn CSM broth cultures at 27°C, growth of the ras2Δ and zeo1Δ strains was severely impaired by 8 hours treatment with 0.5 mM H2O2, with a 50% reduction of cell concentration yields compared to the untreated wild type strain (WT) which is our reference standard. As determined by WB analyses, the ras2Δ strain treated with 1 mM H2O2 for two hours showed reduced levels of PKC1–Slt2p accumulation, indicative of CWI Pathway signaling, when compared to both the untreated ras2Δ and WT strains. Compared to the untreated WT, all three strains exhibit severely impaired growth when treated with 10 ng/ml Caspofungin at 27°C, especially the zeo1Δ strain. Moreover, all three strains show increased levels of PKC1–Slt2p signaling when treated with 75 ng/ml Caspofungin for two hours.ConclusionsOur results show that selective deletion of Mtl1p interactors like Ras2p or Zeo1p in S. cerevisiae are required to confer stress–specific susceptibility phenotypes. We propose that Ras2p modulates PKC1‐dependent Slt2p signaling on exposure to H2O2, perhaps directly by interacting with Mtl1p or indirectly by exerting an influence on other signaling pathways through a cross‐talk mechanism.Support or Funding InformationThis work is supported in part by NIH grants from NIGMS‐INBRE P20GM103475, NIGMSRISE R25GM061838, and NIMHD‐RCMI G12 MD007600.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

Analysis of the Bile Salt Export Pump (ABCB11) Interactome Employing Complementary Approaches

The bile salt export pump (BSEP, ABCB11) plays an essential role in the formation of bile. In hepatocytes, BSEP is localized within the apical (canalicular) membrane and a deficiency of canalicular BSEP function is associated with severe forms of cholestasis. Regulation of correct trafficking to the canalicular membrane and of activity is essential to ensure BSEP functionality and thus normal bile flow. However, little is known about the identity of interaction partners regulating function and localization of BSEP. In our study, interaction partners of BSEP were identified in a complementary approach: Firstly, BSEP interaction partners were co-immunoprecipitated from human liver samples and identified by mass spectrometry (MS). Secondly, a membrane yeast two-hybrid (MYTH) assay was used to determine protein interaction partners using a human liver cDNA library. A selection of interaction partners identified both by MYTH and MS were verified by in vitro interaction studies using purified proteins. By these complementary approaches, a set of ten novel BSEP interaction partners was identified. With the exception of radixin, all other interaction partners were integral or membrane-associated proteins including proteins of the early secretory pathway and the bile acyl-CoA synthetase, the second to last, ER-associated enzyme of bile salt synthesis.

Membrane Yeast Two-Hybrid (MYTH) Mapping of Full-Length Membrane Protein Interactions.

Mapping of protein interaction networks is a major strategy for obtaining a global understanding of protein function in cells and represents one of the primary goals of proteomics research. Membrane proteins, which play key roles in human disease and as drug targets, are of considerable interest; however, because of their hydrophobic nature, mapping their interactions presents significant technical challenges and requires the use of special methodological approaches. One powerful approach is the membrane yeast two-hybrid (MYTH) assay, a split-ubiquitin-based system specifically suited to the study of full-length membrane protein interactions in vivo using the yeast Saccharomyces cerevisiae as a host. The system can be used in both low- and high-throughput formats to study proteins from a wide range of different organisms. There are two primary variants of MYTH: integrated (iMYTH), which involves endogenous expression and tagging of baits and is suitable for studying native yeast membrane proteins, and traditional (tMYTH), which involves ectopic plasmid-based expression of tagged baits and is suitable for studying membrane proteins from other organisms. Here we provide an introduction to the MYTH assay, including both the iMYTH and tMYTH variants. MYTH can be set up in almost any laboratory environment, with results typically obtainable within 4 to 6 wk.

MYTH Screening: iMYTH and tMYTH Variants

Once a bait has been generated and validated for the membrane yeast two-hybrid (MYTH) assay, it can be used for either high-throughput screening to generate a detailed interaction map (interactome) or in low-throughput experiments to examine interactions with specific targets. Here we describe how to carry out high-throughput MYTH library screening of a validated bait generated using integrated or traditional MYTH. The principles herein can be easily adapted for use in a smaller-scale format if required. A typical MYTH library screen can be completed in ∼3-4 wk.

Generation and Validation of MYTH Baits: iMYTH and tMYTH Variants.

Generation of baits for membrane yeast two-hybrid (MYTH) screening differs depending on the nature of the protein(s) being studied. When using native yeast proteins with cytoplasmic carboxyl termini, the integrated form of MYTH (iMYTH) is the method of choice. iMYTH involves endogenous carboxy-terminal tagging of the gene of interest within the yeast chromosome, leaving the gene under the control of its natural promoter. When studying proteins not native to yeast, or native yeast proteins with only cytoplasmic amino termini, traditional MYTH (tMYTH) must be used. In the tMYTH approach, amino- or carboxy-terminally tagged proteins are expressed ectopically from a plasmid. In this protocol, we describe the generation and validation of iMYTH and tMYTH baits. MYTH bait generation can typically be completed in ∼1-2 wk.

Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.

We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae

Sho1p, an integral membrane protein, plays a vital role in the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in the yeast Saccharomyces cerevisiae. Activated under conditions of high osmotic stress, it interacts with other HOG pathway proteins to mediate cell signaling events, ensuring that yeast cells can adapt and remain viable. In an attempt to further understand how the function of Sho1p is regulated through its protein–protein interactions (PPIs), we identified 49 unique Sho1p PPIs through the use of membrane yeast two-hybrid (MYTH), an assay specifically suited to identify PPIs of full-length integral membrane proteins in their native membrane environment. Secondary validation by literature search, or two complementary PPI assays, confirmed 80% of these interactions, resulting in a high-quality Sho1p interactome. This set of putative PPIs included both previously characterized interactors, along with a large subset of interactors that have not been previously identified as binding to Sho1p. The SH3 domain of Sho1p was found to be important for binding to many of these interactors. One particular novel interactor of interest is the glycerol transporter Fps1p, which was shown to require the SH3 domain of Sho1p for binding via its N-terminal soluble regulatory domain. Furthermore, we found that Fps1p is involved in the positive regulation of Sho1p function and plays a role in the phosphorylation of the downstream kinase Hog1p. This study represents the largest membrane interactome analysis of Sho1p to date and complements past studies on the HOG pathway by increasing our understanding of Sho1p regulation.

Mapping the dynamic Interactomes of “druggable” membrane proteins: roles in human health and disease (1095.20)

Despite extensive research in the past decade, there is a lack of in‐depth understanding of protein networks associated with integral membrane proteins because of their unique biochemical features, enormous complexity and multiplicity. This is a major obstacle to understanding the biology of deregulation of these integral membrane proteins which leads to numerous human diseases, and consequently hinders our development of improved and more targeted therapies to help treat these diseases. To address this challenge, we previously developed an in vivo genetic system, called the Membrane Yeast Two‐Hybrid (MYTH) assay, to identify and characterize protein interactors of all yeast ABC transporters and human receptor tyrosine kinases (RTKs), as well as selected cancer stem cell receptors (CSCRs)5 and G‐protein coupled receptors (GPCRs). However, despite MYTH being a robust technology suitable for mapping the PPIs of a wide‐range of membrane proteins, we have found that a significant percentage of mammalian integral membrane proteins cannot be properly analyzed using this system.To address this, we have recently developed a new variant of MYTH suitable for use in mammalian cells, which we have called the Mammalian Membrane Two‐Hybrid (MaMTH) system. During my talk, I will discuss exciting new findings indicating that MaMTH can detect stimuli (hormone/agonist)‐ and phosphorylation‐dependent PPIs. In addition, I will show that MaMTH allows for monitoring of the phosphorylation states of ErbB‐receptor mutants and drug‐induced activity changes of oncogenic variants of the Epidermal Growth Factor Receptor (EGFR). In conclusion, our study illustrates that MaMTH is a powerful tool for investigating dynamic interactomes of human integral membrane proteins and promises significant contributions to therapeutic research.

CHIP-MYTH: A novel interactive proteomics method for the assessment of agonist-dependent interactions of the human β2-adrenergic receptor

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human β2-adrenergic receptor (β2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting β2AR-agonist. Our results suggest that β2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαβγ, into Gα and Gβγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel β2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.

G protein modulation of K2P potassium channel TASK-2

TASK-2 (K2P5.1) is a background K+ channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gβγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-β-S instead. Purified Gβγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-β-S was present. The effects of GTP-γ-S and Gβγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257–K258 or K296–K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gβ1 and Gβ2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257–K258 (but not K296–K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-β-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gβγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions.

Cpr1 cyclophilin and Ess1 parvulin prolyl isomerases interact with the tombusvirus replication protein and inhibit viral replication in yeast model host

To identify host proteins interacting with the membrane-bound replication proteins of tombusviruses, we performed membrane yeast two-hybrid (MYTH) screens based on yeast cDNA libraries. The screens led to the identification of 57 yeast proteins interacting with replication proteins of two tombusviruses. Results from a split ubiquitin assay with 12 full-length yeast proteins and the viral replication proteins suggested that the replication proteins of two tombusviruses interact with a similar set of host proteins. Follow-up experiments with the yeast Cpr1p cyclophilin, which has prolyl isomerase activity that catalyzes cis–trans isomerization of peptidyl–prolyl bonds, confirmed that Cpr1p interacted with the viral p33 replication protein in yeast and in vitro. Replication of Tomato bushy stunt virus replicon RNA increased in cpr1Δ yeast, while over-expression of Cpr1p decreased viral replication. We also show that the Ess1p parvulin prolyl isomerase partly complements Cpr1p function as an inhibitor of tombusvirus replication.