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

Tabitha A Peterson; Robert C Piper

doi:10.1007/978-1-0716-1346-7_9

Back

Journal article

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

Tabitha A Peterson and Robert C Piper

Methods in molecular biology (Clifton, N.J.), Vol.2293, pp.117-141

2021

DOI: 10.1007/978-1-0716-1346-7_9

PMCID: PMC8524840

PMID: 34453714

Files and links (1)

url

https://www.ncbi.nlm.nih.gov/pmc/articles/8524840View

Open Access

Abstract

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.

Gene Library

Guanosine Triphosphate - metabolism

Protein Binding

rab GTP-Binding Proteins - genetics

rab GTP-Binding Proteins - metabolism

Saccharomyces cerevisiae - genetics

Saccharomyces cerevisiae - metabolism

Two-Hybrid System Techniques

Details

Title: Subtitle: Deconvolution of Multiple Rab Binding Domains Using the Batch Yeast 2-Hybrid Method DEEPN
Creators: Tabitha A Peterson - University of Iowa
Robert C Piper - Roy J. and Lucille A. Carver College of Medicine
Resource Type: Journal article
Publication Details: Methods in molecular biology (Clifton, N.J.), Vol.2293, pp.117-141
DOI: 10.1007/978-1-0716-1346-7_9
PMID: 34453714
PMCID: PMC8524840
NLM abbreviation: Methods Mol Biol
ISSN: 1064-3745
eISSN: 1940-6029
Grant note: R01 GM058202 / NIGMS NIH HHS
Language: English
Date published: 2021
Academic Unit: Molecular Physiology and Biophysics; Medicine Administration; Internal Medicine
Record Identifier: 9984297605702771

Metrics

40 Record Views