Isolation and Characterization of Membrane Vesicles ofSaccharomyces cerevisiaeHarboring the High-Affinity Phosphate Transporter

Abstract

Membrane vesicles with an inside-out orientation were isolated from the plasma membrane ofSaccharomyces cerevisiaeby an improved aqueous two-phase partitioning technique. The activity of the orthovanadate-sensitive H+-pumping ATPase, the plasma membrane marker, was highly enriched by the partitioning technique. The obtained results suggest that the membrane vesicles produced were predominantly oriented inside-out. The isolated plasma membrane vesicles displayed cross-reactions with antibodies raised against synthetic peptide corresponding to the N-terminal (residues 1–10) and the C-terminal (residues 578–597) regions of the plasma membrane phosphate transporter encoded by thePHO84gene and the H+-pumping ATPase ofS. cerevisiae.The purified membrane vesicles catalyzed a derepressible inhibitor-sensitive phosphate uptake at levels comparable with the situation in intact cells ofS. cerevisiaeindicating that transport of phosphate across the membrane is both functional and bidirectional. The PHO84 transporter harbored in isolated plasma membranes could moreover be enriched in a high state of purity by immunoaffinity chromatography using immobilized anti-PHO84 antibodies.