Mating Based Split-ubiquitin Assay for Detection of Protein Interactions

Abstract

The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitindegradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are cytosolic or membrane-bound. Here we describe the mbSUS assay protocol which has been used for detecting the interaction between K + channel and

Identification and Characterization of Shaker K+ Channel Gene Family in Foxtail Millet (Setaria italica) and Their Role in Stress Response

“…The mbSUS assays were performed as described before ( Grefen et al, 2009 ; Horaruang and Zhang, 2017 ). Bait destination clones were transformed into yeast strain THY.AP4 and prey clones were transformed into THY.AP5.…”

K+ Channel-SEC11 Binding Exchange Regulates SNARE Assembly for Secretory Traffic

“…We used the mating-based split-ubiquitin system (mbSUS) assay, previously employed successfully to identify K 1 channel binding motifs (Honsbein et al, 2009;Grefen et al, 2010;Karnik et al, 2015;Zhang et al, 2015Zhang et al, , 2017Horaruang and Zhang, 2017), to assess channel binding with the cognate SNAREs and identify the relevant binding motifs. The mbSUS assay takes advantage of protein fusions with the N-and C-terminal halves (Nub-X and Y-Cub) of ubiquitin.…”

“…mbSUS assays were performed as described before (Karnik et al, 2013b;Grefen et al, 2015;Horaruang and Zhang, 2017). Bait constructs were transformed into yeast strain THY.AP4 and prey constructs were transformed into THY.AP5.…”

A GPI Signal Peptide-Anchored Split-Ubiquitin (GPS) System for Detecting Soluble Bait Protein Interactions at the Membrane

“…Techniques based on heterologous expression of bait and prey proteins in yeast have gained much popularity because they are quick, easy to perform, and inexpensive. Compared with the conventional yeast two-hybrid system, commonly used for screening interactors from a prey library, the yeast mating-based split-ubiquitin system (mbSUS;Grefen et al, 2007Grefen et al, , 2009Horaruang and Zhang, 2017) has proven effective, especially for studying interactions between membrane proteins. The technology incorporates several advantages over conventional yeast two-hybrid approaches, including mating-based recombination, which greatly enhances transformation efficiencies, and bait suppression by Met, which improves false positive detection (Grefen et al, 2007(Grefen et al, , 2009Horaruang and Zhang, 2017).…”

“…Compared with the conventional yeast two-hybrid system, commonly used for screening interactors from a prey library, the yeast mating-based split-ubiquitin system (mbSUS;Grefen et al, 2007Grefen et al, , 2009Horaruang and Zhang, 2017) has proven effective, especially for studying interactions between membrane proteins. The technology incorporates several advantages over conventional yeast two-hybrid approaches, including mating-based recombination, which greatly enhances transformation efficiencies, and bait suppression by Met, which improves false positive detection (Grefen et al, 2007(Grefen et al, , 2009Horaruang and Zhang, 2017). mbSUS is particularly stringent and has proven effective even in identifying the individual amino acid residues that define interaction motifs essential for binding with the prey (Honsbein et al, 2009;Grefen et al, 2010Hachez et al, 2014;Zhang et al, 2015Zhang et al, , 2017.…”