Generation of a yeast two-hybrid strain suitable for competitive protein binding analysis
2005; Future Science Ltd; Volume: 39; Issue: 2 Linguagem: Inglês
10.2144/05392bm01
ISSN1940-9818
AutoresMarkus Ralser, Heike Goehler, Erich E. Wanker, Hans Lehrach, Sylvia Krobitsch,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoBioTechniquesVol. 39, No. 2 BenchmarksOpen AccessGeneration of a yeast two-hybrid strain suitable for competitive protein binding analysisMarkus Ralser, Heike Goehler, Erich E. Wanker, Hans Lehrach & Sylvia KrobitschMarkus RalserMax Planck Institute for Molecular Genetics, Heike GoehlerMax Delbrueck Center for Molecular Medicine, Berlin, Germany, Erich E. WankerMax Delbrueck Center for Molecular Medicine, Berlin, Germany, Hans LehrachMax Planck Institute for Molecular Genetics & Sylvia Krobitsch*Address correspondence to: Sylvia Krobitsch, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. e-mail: E-mail Address: krobitsc@molgen.mpg.deMax Planck Institute for Molecular GeneticsPublished Online:30 May 2018https://doi.org/10.2144/05392BM01AboutSectionsView ArticleSupplemental MaterialPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail View article The yeast two-hybrid (Y2H) system is one of the most common applied genetic systems utilized for the identification of protein-protein interactions in vivo (1). Over the years this system has been systematically exploited for analyzing the physiological function of proteins via the generation of huge protein interaction networks (2). It is based on the principle that different transcription factors can be split into a DNA binding and a polymerase II activation domain. When these independently functional domains are brought into close proximity, the activity of the transcription factor is restored. If an interaction of a protein fused to the DNA binding domain (bait protein) and another protein fused to the activation domain (prey protein) occurs, a functionally transcription factor is reconstructed, and transcription of certain reporter genes involved in amino acid biosynthesis is initiated. The colorimetric reporter LacZ is often exploited as well, because it allows the relative quantification of its activity and, consequently, the relative strength of protein-protein interactions in vivo (3–5).Since proteins are multifunctional and interact with many factors, it would be obviously advantageous and in particular less time-consuming to characterize the interplay between proteins in vivo by the Y2H system in comparison to existing in vitro approaches (6,7). Therefore, the Y2H technology has been broadened and applied for further characterization of protein-protein interactions. Reverse approaches have been developed allowing the identification of particular protein mutations, peptides, or agents that inhibit the protein-protein interaction of interest (8,9). Furthermore, Y2H systems have been generated in which the interaction between proteins is enhanced or depends upon expression of a third protein or ligand (5,10,11). However, the simultaneous expression of a third protein or factor to analyze competitive protein binding in the Y2H background can be difficult, because often the auxotrophic marker genes available have been exhausted in the course of generating the strains for a particular Y2H system. Moreover, many Y2H systems are incompatible to each other because of the different promoters and the auxotrophic markers that have to be used. Thus, switching between the available Y2H systems and strategies is often impossible or very time-consuming due to required cloning efforts for the creation of different bait and prey plasmids needed.Hence, to facilitate competitive protein binding approaches in a quite frequent Y2H system, we aimed to create a strain still exploitable for Y2H screens, but additionally allowing the simultaneous expression of a third protein (Figure 1A). Given that most yeast expression vectors are based on auxothropic markers, we decided to replace the URA3 gene in the standard strain L40c [MATa his3Δ200 trp1–901 leu2–3,112 ade2 lys2–801am can1 URA3::(lexAop)8-GAL1TATA-lacZ LYS2::(lexAop)4-HIS3TATA-HIS3] (12) with the Kan4MX marker gene. For this purpose, the respective DNA fragment was amplified from plasmid pUG6 (13) with primers Kan4MX::ura3 sense and Kan4MX:: ura3 antisense (see Supplementary Table S1). Since the yield of the resulting 1.5-kb DNA fragment was very low, we performed a subsequent PCR using this fragment as template and primers URA3 sense and URA3 antisense. After transformation (14), recombinants were selected on yeast extract/peptone/dextrose (YPD) media supplemented with 200 µg/mL G418 sulfate (Invitrogen, Carlsbad, CA, USA). Yeast clones were tested for re-growth on YPD/G418 medium and on media lacking uracil to verify the replacement of the URA3 gene. Clones that were deficient for uracil biosynthesis were transformed with bait and prey plasmids encoding interacting proteins and tested for activity of the LacZ reporter by a β-galactosidase assay (data not shown). One of the yeast clones that exhibited activity of the LacZ reporter was selected and named L40KMX. To investigate competitive effects between proteins, we aimed to quantify the relative activity of the colorimetric reporter LacZ rather than to determine the activation of the auxotrophic markers, because such a growth assay would be biased due to positive selection of yeast that exhibits better growth under the chosen conditions.Figure 1. Applying the yeast two-hybrid (Y2H) system for competitive protein binding analysis.(A) Illustration of the basic principle of the Y2H system. An interaction of bait and prey protein leads to the activation of reporter genes (left panel). If a third factor competes for binding the bait (middle panel) or prey protein (right panel), the expression level of reporter genes is reduced. (B) Competitive protein binding effects on LacZ activity. Strain L40KMX was transformed with the corresponding plasmids as indicated, and the relative activity of the LacZ reporter was measured as described. Error bars indicate standard deviations. (C) Expression levels of bait and prey proteins. Yeast were grown to an A600 of 0.6–1.0, and ethanol lysates were prepared. Cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). After blocking, the membranes were incubated with α-lexA serum (1:5000) or α-Gal4AD serum (1:10,000). The polyclonal antibodies against lexA or Gal4AD were generated by injecting HIS6-tagged lexA (amino acids 1–202) or glutathione S-transferase (GST)-tagged Gal4AD (amino acid 768–881), respectively, into a rabbit. Afterwards, the membranes were incubated with alkaline phosphatase (AP)-conjugated (lexA) or peroxidase-conjugated (Gal4AD) anti-rabbit immunoglobulin G (IgG; Sigma, St. Louis, MO, USA). Proteins were visualized using nitroblue tetrazolium (NBT)/X-phosphate or Western Lightning™ chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA, USA).For our study, we focused on the interactome of huntingtin (htt) (15), a protein encoded by the IT15 gene in which an expansion of the trinucleotide repeat CAG is causative for the neurodegenerative disorder Huntington's disease (16). From this network, we selected three proteins that directly interact with huntingtin: (i) the transcription factor CA150 (17); (ii) the actin cytoskeleton organization regulating protein PFN2 (15); and (iii) the formin binding protein HYPA (15). Furthermore, we generated plasmids for expression of huntingtin exon 1 protein (HD1) and endophilin-A3, another interaction partner of huntingtin (12), to analyze their potential competitive effects. Since the bait and prey proteins are expressed under the control of the ADH1 promoter, the proteins tested for competition were placed under the control of the GPD promoter to minimize the risk of promoter effects. The generation of expression plasmids is outlined in supplementary Table S1, while plasmids encoding the prey proteins have been described earlier (15). After transformation of strain L40KMX with the relevant bait and prey plasmids, as well as with expression plasmids encoding either HD1 (p426-htt) or endophilin-A3 (p426-EndA3), transformants were selected on media lacking amino acids tryptophan, leucine, and uracil. As controls, we used plasmids p426GPD (empty vector) or p426-GFP encoding the green fluorescent protein (GFP).For measurements of the LacZ reporter activity, four yeast colonies of each transformation were grown overnight. Then, the same number of cells was transferred into fresh media and grown to an A600 of 0.6–1.0. Yeast cells were lysed by freezing/thawing cycles and resuspended in buffer containing 100 mM HEPES/KOH, pH 7.0, 150 mM NaCl, 2 mM MgCl2, 1% bovine serum albumin (BSA) supplied with 55 µL chloroform and 55 µL 0.1% sodium dodecyl sulfate (SDS). After intensive mixing, 500 ng ortho-nitrophenyl-β-D-galactopyranoside (ONPG; Applichem GmbH, Darmstadt, Germany) were added to each lysate, and samples were incubated at 37°C. After addition of 400 µL 1 M Na2CO3, samples were centrifuged, and A420 was measured for each sample. The relative β-galactosidase activity in Miller Units was calculated by the formula [UR=(1000×A420)/(vol(mL)×t(min)×A600)] (18). The standard deviation was calculated from four measurements, and the Nalimov method was used with an offset of 1.982 to eliminate outliers with statistical probability larger than 99.9% (19). In addition, each competitive binding experiment was repeated twice.As Figure 1B shows, the relative activity of the LacZ reporter was greatly decreased in yeast expressing lexA-HD1 and AD-PFN2 in the presence of HD1 or endophilin-A3, respectively, indicating a strong competitive effect of HD1 and endophilin-A3, respectively. A weaker competitive effect was observed in yeast expressing lexA-HD1 and AD-HYPA or lexA-HD1 and AD-CA150, respectively. Here, the expression of endophilin-A3 and HD1, respectively, did not result in a strong reduction of LacZ reporter gene activity, suggesting that the competitive effect for both proteins is weaker as detected in the case of the interacting pair lex A-HD1 and AD-PFN2. However, the competitive effect of endophilin-A3 on the bait/prey pair lexA-HD1 and AD-CA150 cannot be regarded as significant due to the calculated standard deviation. Since the measurement of LacZ reporter gene activity has often been exploited to determine the relative strength of protein-protein interactions in yeast, we noticed that the interference of the third protein correlated with the relative strength of the protein-protein interaction. However, the LacZ reporter gene activity cannot be correlated with the absolute binding affinity between interacting proteins (4). Moreover, we observed that expression of the unrelated GFP protein did not reduce LacZ activity in comparison to the control (empty vector), demonstrating that GFP expression did not affect interactions between the respective bait/prey pairs. To further exclude that the reduction in the relative activity of LacZ is not a general artifact, but is specifically due to competition of HD1 or endophilin-A3, respectively, we have also selected the protein pair BRCA1-associated RING domain protein 1 (BARD) and BARD interacting protein 1 (BAIP1) from the huntingtin network. Both proteins do not directly interact with huntingtin or endophilin-A3 (15). No competitive effect of HD1 or endophilin-A3 was observed in yeast expressing lexA-BARD1 and AD-BAIP1. Furthermore, to verify that the differences of LacZ reporter activity did not result from a reduced expression of the respective bait and prey proteins, we analyzed their intracellular level by immunoblotting (Figure 1C). In comparison to controls, no alterations in expression levels of bait and prey proteins were detected in the presence of additional expressed HD1 or endophilin-A3. Additionally, we tested the stability of the different plasmids by spotting dilution series of the various yeast transformants onto plates lacking the amino acids tryptophan, leucine, or uracil, respectively, to exclude that loss of expression plasmids had occurred. No discrepancy in colony number was observed between the different transformants (data not shown).Taken together, our data show that the differences of LacZ reporter activity of the respective yeast clones resulted from competitive binding of HD1 or endophilin-A3 and, consequently, clearly demonstrated that this modified Y2H strain can be exploited for analyzing competitive protein binding in vivo. 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Ehrenhofer-Murray (Max Planck Institute for Molecular Genetics, Germany) and M. Breitenbach (University of Salzburg, Austria) for providing yeast strains and plasmids. We are grateful to the Max Planck Society for funding part of the research.Competing Interests StatementThe authors declare no competing interests.Supplementary dataTo view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/suppl/10.2144/05392BM01PDF download
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