Isolation of Yeast Plasma Membranes

Plasma membranes are often prepared from yeast by initially spheroplasting the cells (1 –3 ). Procedure 1 following uses spheroplasting, gives high yields, and is ideally suited to the large-scale isolation of plasma membranes. Modified after Schmidt et al. (2 ) and Cartwright et al. (4 ) it coats the negatively charged surface of the spheroplasts with dense cationic silica beads, so as to make the plasma membrane much denser than any other membranous organelle of the cell. A washing procedure then removes the excess cationic beads, followed by addition of polyacrylic acid to block the free cationic groups on these beads. The coated spheroplasts are subsequently lysed by hand homogenization in an EGTA-containing lysis buffer (the EGTA binding divalent cations, thereby preventing aggregation of membrane components). Centrifugation of the spheroplast lysate then pellets the heavy plasma membrane-microbead assemblies, leaving intracellular membranous organelles in the supernatant.