title: Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.
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 Deltaycf1 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.
authors: Department of Cell Biology, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
authors: Christian M
authors: CM
authors: Paumi
authors: Javier
authors: J
authors: Menendez
authors: Anthony
authors: A
authors: Arnoldo
authors: Kim
authors: K
authors: Engels
authors: Kavitha Ravee
authors: KR
authors: Iyer
authors: Safia
authors: S
authors: Thaminy
authors: Oleg
authors: O
authors: Georgiev
authors: Yves
authors: Y
authors: Barral
authors: Susan
authors: S
authors: Michaelis
authors: Igor
authors: I
authors: Stagljar
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism / Q000502:physiology; D019216:Metals, Heavy / Q000494:pharmacology; D008954:Models, Biological; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D020662:Guanine Nucleotide Exchange Factors; D019216:Metals, Heavy; D029701:Saccharomyces cerevisiae Proteins; C448998:TUS1 protein, S cerevisiae; D025801:Ubiquitin; C089179:YCF1 protein, S cerevisiae; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins