Anomalously slow mobility of fluorescent lipid probes in the plasma membrane of the yeastSaccharomyces cerevisiae

Summary We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (dil) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma mem branesof Saccharomyces cerevisiae inol andopi 3 spheroplasts. These are well-characterized strains with mutations in the inositol and phosphatidylcholine biosynthetic pathways. Membrane phospholipid composition was altered by growing these mutants in the presence or absence of inositol and choline. Lateral mobil ity was measured by fluorescence recovery after photobleaching (FRAP). Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe dil, confirming that at least one of the probes was largely incorporated into the bilayer membrane. Our results demonstrated anomalously slow mobility of both lipid probes for both mutants, regardless of whether the lipid composition was near normal or dramatically altered in relative composition of phosphatidylinositol and phosphatidylcholine. Trypsinization of the spheroplasts to remove surface proteins resulted in markedly increased lateral mobility. However, even in trypsinized sphero plasts, mobility was still somewhat lower than the mobility ob served in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.