Mapping Protein-Protein Interactions for the Yeast ABC Transporter Ycf1p by Integrated Split-Ubiquitin Membrane Yeast Two-Hybrid Analysis

Christian M. Paumi, Javier Menendez, Anthony Arnoldo, Kim Engels, Kavitha Ravee Iyer, Safia Thaminy, Oleg Georgiev, Yves Barral, Susan Michaelis, Igor Stagljar

Research output: Contribution to journalArticle

Abstract

The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p. Strains deleted for several of these genes result in arsenite sensitivity similar to a Δycf1 strain. Transport assays show that one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for the small GTPase Rho1p, is a Rho1p-dependent-positive regulator of Ycf1p. Our study provides proof of principle that iMYTH is an ideal methodology to identify physiological interactors and regulators of ABC transporters and other yeast transmembrane proteins.

Original languageEnglish (US)
Pages (from-to)15-25
Number of pages11
JournalMolecular cell
Volume26
Issue number1
DOIs
StatePublished - Apr 13 2007

Keywords

  • PROTEINS

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    Paumi, C. M., Menendez, J., Arnoldo, A., Engels, K., Iyer, K. R., Thaminy, S., Georgiev, O., Barral, Y., Michaelis, S., & Stagljar, I. (2007). Mapping Protein-Protein Interactions for the Yeast ABC Transporter Ycf1p by Integrated Split-Ubiquitin Membrane Yeast Two-Hybrid Analysis. Molecular cell, 26(1), 15-25. https://doi.org/10.1016/j.molcel.2007.03.011