Mapping protein-protein interactions for the yeast ABC transporter Ycf1p by integrated split-ubiquitin membrane yeast two-hybrid analysis.
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.
Paumi
Christian M
CM
Department of Cell Biology, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
Menendez
Javier
J
Arnoldo
Anthony
A
Engels
Kim
K
Iyer
Kavitha Ravee
KR
Thaminy
Safia
S
Georgiev
Oleg
O
Barral
Yves
Y
Michaelis
Susan
S
Stagljar
Igor
I
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
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