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

Tabitha A Peterson, Robert C Piper

Methods in molecular biology (Clifton, N.J.) 2021

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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. © 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Tabitha A Peterson, Robert C Piper. Deconvolution of Multiple Rab Binding Domains Using the Batch Yeast 2-Hybrid Method DEEPN. Methods in molecular biology (Clifton, N.J.). 2021;2293:117-141