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.

In-Frame cDNA Library Combined with Protein Complementation Assay Identifies ARL11-Binding Partners

“…A more recently developed approach to reduce false-positive reactions is based on the concept of permutated fusion proteins, which contain a mixture of N- and C-terminal bait fusion proteins [34]. Finally, because the cells used in the detection systems can activate the transcription of reporter genes by their internal proteins, the method of interrogating protein-protein interactions within the Golgi center, where internal transcriptional activation is reduced to a negligible background level, has been utilized [35]. All these approaches were designed to improve the specificity of transcriptional activation as well as the specificity of bait-and-prey interactions.…”

Saccharomyces cerevisiae as a Tool to Investigate Plant Potassium and Sodium Transporters

“…The absence of this mannose structure renders yeast cells sensitive to high temperatures and to cell wall damaging agents, such as the benzidine-type dye Congo red. Upon interaction of both proteins, the function of the Och1 protein is reconstituted and the och1 mutant will be able to grow at the non-permissive temperature (37 °C) or in the presence of Congo red [144].…”

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

“…The recently developed Golgi complex two-hybrid system (Fig. 6C) is based on the complementation of the Golgi complex-resident mannosyltransferase Och1 (146). Like many Golgi complexbased enzymes, Och1 consists of two modular domains: the N-terminal LOC domain for membrane attachment and the C-terminal CAT domain, which performs the mannose transfer reaction within the Golgi complex lumen, an essential reaction for the production of large-chain cell wall mannans.…”

“…Like many Golgi complexbased enzymes, Och1 consists of two modular domains: the N-terminal LOC domain for membrane attachment and the C-terminal CAT domain, which performs the mannose transfer reaction within the Golgi complex lumen, an essential reaction for the production of large-chain cell wall mannans. Deletion of OCH1 results in increased cell binding of chitin-binding reagents, such as wheat germ agglutinin, and in strongly reduced growth at a nonpermissive temperature (37°C) or in the presence of the benzidine-type dye Congo red (146). Fusion of the two modular fragments of Och1 to the human transcription factor MyoD and the inhibitor of differentiation protein 2 (Id2) reverses all of the och1 phenotypes through the reassembly of Och1 upon MyoD-Id2 interaction.…”

“…Hac1 activity can be sensed by detection of inositol synthesis and by activation of reporters regulated by Hac1-dependent promoters. (C) Golgi two-hybrid system (146). The catalytic (CAT) and membrane-attaching (LOC) domains of the mannosyltransferase Och1 are fused to the bait protein X and prey protein Y, respectively.…”