Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes.
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 approximately 0.8 s(-1) and 50+/-8 microM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Deltayor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[alpha-(32)P]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.
Grigoras
Ioana
I
Laboratoire de Biochimie, Ecole Polytechnique, CNRS, F-91128 Palaiseau cedex, France.
Lazard
Myriam
M
Plateau
Pierre
P
Blanquet
Sylvain
S
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D025141:Drug Resistance, Fungal / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D013379:Substrate Specificity / Q000187:drug effects
D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D029701:Saccharomyces cerevisiae Proteins; C097077:YOR1 protein, S cerevisiae; D000251:Adenosine Triphosphatases