title: A two-hybrid assay to study protein interactions within the secretory pathway.
abstract: Interactions of transcriptional activators are difficult to study using transcription-based two-hybrid assays due to potent activation resulting in false positives. Here we report the development of the Golgi two-hybrid (G2H), a method that interrogates protein interactions within the Golgi, where transcriptional activators can be assayed with negligible background. The G2H relies on cell surface glycosylation to report extracellularly on protein-protein interactions occurring within the secretory pathway. In the G2H, protein pairs are fused to modular domains of the reporter glycosyltransferase, Och1p, and proper cell wall formation due to Och1p activity is observed only when a pair of proteins interacts. Cells containing interacting protein pairs are identified by selectable phenotypes associated with Och1p activity and proper cell wall formation: cells that have interacting proteins grow under selective conditions and display weak wheat germ agglutinin (WGA) binding by flow cytometry, whereas cells that lack interacting proteins display stunted growth and strong WGA binding. Using this assay, we detected the interaction between transcription factor MyoD and its binding partner Id2. Interfering mutations along the MyoD:Id2 interaction interface ablated signal in the G2H assay. Furthermore, we used the G2H to detect interactions of the activation domain of Gal4p with a variety of binding partners. Finally, selective conditions were used to enrich for cells encoding interacting partners. The G2H detects protein-protein interactions that cannot be identified via traditional two-hybrid methods and should be broadly useful for probing previously inaccessible subsets of the interactome, including transcriptional activators and proteins that traffic through the secretory pathway.
authors.affiliation: Department of Chemistry and Biochemistry, Bowdoin College, Brunswick, Maine, United States of America.
authors.forename: Danielle H
authors.initials: DH
authors.lastname: Dube
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Ethan J
authors.initials: EJ
authors.lastname: Greenblatt
authors.forename: Sadeieh
authors.initials: S
authors.lastname: Nimer
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Raymond
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Kohler
mesh_terms: D005189:False Positive Reactions; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2 / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017570:MyoD Protein / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D014909:Wheat Germ Agglutinins / Q000737:chemistry
chemical_list: C497450:ID2 protein, human; D051797:Inhibitor of Differentiation Protein 2; D008562:Membrane Glycoproteins; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; C075636:OCH1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014909:Wheat Germ Agglutinins; D008364:Mannosyltransferases