Deconvolution of Multiple Rab Binding Domains Using the Batch Yeast 2-Hybrid Method DEEPN.

A hallmark of functionally significant interactions between Rab proteins and their targets is whether that binding depends on the type of nucleotide bound to the Rab GTPase. A system that can directly compare those sets of interactions mediated by a Rab in its GTP-bound conformation versus its GDP bound conformation ...
would provide a direct route to finding biologically relevant partners. Comprehensive large-scale yeast 2-hybrid assays allow a potential method to compare one interactome against another provided that the same set of potentially interacting partners is interrogated between samples. Here we describe the use of such a yeast 2-hybrid system that lends itself toward comparing pairs of Rab mutants, locked in either their GTP or GDP conformation. Importantly, using a complex library of protein fragments as potential binding ("prey") partners, identification of interacting proteins as well as the domain(s) mediating those interactions can be determined using a series of sequence analyses and binary validation experiments.
Mesh Terms:
Gene Library, Guanosine Triphosphate, Protein Binding, Saccharomyces cerevisiae, Two-Hybrid System Techniques, rab GTP-Binding Proteins
Methods Mol Biol
Date: Aug. 29, 2021
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