Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes |
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Authors: | Ioana Grigoras Pierre Plateau Sylvain Blanquet |
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Affiliation: | Laboratoire de Biochimie, Ecole Polytechnique, CNRS, F-91128 Palaiseau cedex, France |
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Abstract: | Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. Vmax and Km values of ∼ 0.8 s− 1 and 50 ± 8 μM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Δyor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[α-32P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation. |
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Keywords: | ABC, ATP binding cassette TMD, Transmembrane domain NBD, Nucleotide binding domain P-gp, P-glycoprotein CFTR, Cystic fibrosis transmembrane conductance regulator MRP, Multidrug resistance associated protein p-CMPS, p-chloromercuriphenylsulfonate DCC, N,N'-di-cyclohexylcarbodiimide NEM, N-ethylmaleimide DTT, 1-4 dithiothreitol BSA, Bovine serum albumine EDTA, Ethylenediaminetetraacetic acid PNK, Polynucleotide 5&prime -hydroxyl-kinase MIC, Minimal inhibitory concentration |
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