Greenberg M L, Axelrod D
Department of Biological Sciences, Wayne State University, Detroit, Michigan.
J Membr Biol. 1993 Jan;131(2):115-27. doi: 10.1007/BF02791320.
We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (diI) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma membranes of Saccharomyces cerevisiae ino1 and opi3 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 mobility was measured by fluorescence recovery after photobleaching (FRAP). Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe diI, 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 spheroplasts, mobility was still somewhat lower than the mobility observed in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.
我们测量了酿酒酵母ino1和opi3原生质球质膜中两种荧光脂质探针——二辛基吲哚碳菁（diI）和四甲基罗丹明磷脂酰乙醇胺（R-PE）的侧向流动性。这些是在肌醇和磷脂酰胆碱生物合成途径中具有突变的特征明确的菌株。通过在有或没有肌醇和胆碱的情况下培养这些突变体，膜磷脂组成发生了改变。侧向流动性通过光漂白后荧光恢复（FRAP）进行测量。采用CCD数字成像的显微荧光偏振产生了脂质探针diI的有序取向分布，证实至少其中一种探针大量掺入了双层膜。我们的结果表明，无论磷脂酰肌醇和磷脂酰胆碱的相对组成是接近正常还是显著改变，两种突变体的两种脂质探针的流动性都异常缓慢。对原生质球进行胰蛋白酶处理以去除表面蛋白，导致侧向流动性显著增加。然而，即使在经胰蛋白酶处理的原生质球中，流动性仍略低于在此作为对照测试的哺乳动物细胞（如大鼠平滑肌培养细胞）膜中观察到的流动性。
