ESR determination of membrane order parameter in yeast sterol mutants

ESR investigations designed to determine membrane order parameter in sterol mutants of Saccharomyces cerevisiae were conducted using the membrane probe, 5-doxyl stearic acid. These mutants are blocked in the ergosterol biosynthetic pathway and thus do not synthesize ergosterol, the end product sterol. They do not require exogenous ergosterol for growth and, therefore, incorporate ergosterol biosynthetic intermediates in their membrane. Increasing order parameter is reflective of an increase in membrane rigidity. Single mutants involving B-ring delta 8 leads to delta 7 isomerization (erg 2) and C-24 methylation (erg 6) showed greater membrane rigidity than wild-type during exponential growth. A double mutant containing both lesions (erg 6/2) showed an even greater degree of membrane rigidity. During stationary phase the order of decreasing membrane rigidity was erg 6 greater than erg 6/2 greater than erg 2 = wild-type. The increased membrane order parameter was attributed to the presence of substituted sterols rather than increased sterol content or altered fatty acid synthesis.