Abstract #73

Identification of Proteins that Interact with the ABC Transporter Ycf1p using the Membrane Yeast Two-Hybrid (MbYTH) Split-Ubiquitin System. Christian M Paumi¹, Kim Engels², Kavitha Iyer², Oleg Georgiev², Igor Stagljar², Susan Michaelis¹. 1) Cell Biology, Johns Hopkins University, Baltimore, MD; 2) Donnelly Centre for Cellular and Biomolecular Research (CCBR), Department of Biochemistry & Department of Medical Genetics and Microbiology, University of Toronto, Toronto, ON, Canada.
   The multidrug resistance-associated (MRP) subfamily of ATP-binding cassette transporters play a key role in protecting cells from environmental and endogenous toxins. The MRPs contain an N-terminal extension (5 membrane spans) in addition to their ABC “core” (12 membrane spans), and they transport substrates as glutathione conjugates. We are studying Ycf1p, a prototypical representative of the six-membered MRP subfamily in yeast. Ycf1p resides in the vacuolar membrane and mediates glutathione-dependent resistance to heavy metals (e.g. cadmium) and production of red pigment in an ade2 yeast strain. To better understand cellular components involved in Ycf1p function we have embarked on a search for Ycf1p protein interactors. Identification of membrane protein interactors is challenging because their hydrophobic nature precludes the use of standard approaches. Instead, we used the membrane protein yeast two hybrid (MbYTH) system, which is specifically designed circumvent this problem. A MbYTH screen identified six potential Ycf1p interactors. Strains deleted for several of these genes result in cadmium sensitivity and ade2 pigmentation defects similar to a ycf1D strain. We have focused further analysis on one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for Rho1p. In the absence of Tus1p, neither the expression nor trafficking of Ycf1p is altered. Strikingly, in an in vitro transport assay using purified Ycf1p-containing vacuoles, we find that addition of a WT (TUS1) cytosolic extract and not Dtus1 cytosolic extract stimulates transport. Thus, Tus1p plays a novel and unanticipated role in regulating the activity of Ycf1p. Our studies suggest that MbYTH represents a robust methodology to identify key physiological regulators for all classes of ABC proteins.

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