ESR determinations of membrane permeability in a yeast sterol mutant

Abstract

Yeast sterol mutants were subjected to ESR analysis in an attempt to elucidate how altered sterol composition correlates with membrane permeability. The technique requires spin labeling the intact yeast cells with a small, water-soluble nitroxide probe (2,2,5,5 tetramethyl-3-pyrrolin-1-oxyl-3-carboxylic acid, PCA), suspending cells in a NiCl2 solution, and measuring the extent of Ni2+ entry through the membrane by its magnetic dipolar line broadening effect on the PCA signal. The wild type, A184D, was found to be impermeable to Ni2+ during all growth phases while the sterol mutant erg 6/2 was readily permeable to Ni2+. Other sources of line broadening such as increased rotational correlation time and cell nonviability are shown to be neglibible. Internal Ni2+ concentrations for erg 6/2 and kinetics of Ni2+ entry were determined.