Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species

Abstract

Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter <i>Kl</i>Pdr5p confers resistance to several unrelated drugs in <i>Kluyveromyces lactis. Kl</i>Pdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an energy-dependent manner. Incubation of yeast cells in the presence of 2-deoxy-D-glucose (inhibitor of glycolysis) and R6G (mitochondrial ATPase inhibitor) leads to marked depletion of intracellular ATP pool ( Kolaczkowski <i>et al.</i>, 1996 ). An active <i>Kl</i>Pdr5p mediated extrusion of R6G from intact yeast cells can be followed by direct measurement of the fluorescence of extruded R6G in the assay buffer.