A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer.
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机译：开发了一种基于Förster共振能量转移的融合和转移测定方法，以在模型膜中研究蛋白质介导的膜融合和荧光鞘脂类似物的膜间脂质转移。对于该测定，变得有必要施加对自发的以及蛋白质介导的膜间转移具有抗性的标记的报告分子。该测定法的新颖之处在于不可跨越荧光膜的双极性脂质的使用。从四醚脂质钙降丁醇开始，我们合成了在两个极性末端都带有荧光团的荧光类似物。此外，我们合成了放射性糖基化的降钙素醇。这些标记的脂质显示可以延伸穿过双层膜，而不是在脂质体的单个脂质层中环化。更重要的是，与磷酸甘油酯相比，跨膜脂质（MSL）不能被蛋白质提取。我们可以证明GM2激活蛋白（GM2AP）在甘油和鞘脂转移方面是混杂的。 Saposin（Sap）B也转移了鞘脂，尽管其动力学不同于GM2AP。此外，我们可以明确显示重组激活蛋白Sap C x His6诱导膜融合而不是膜间脂质转移。这些发现表明，与荧光磷酸甘油脂相比，这些新型MSL非常适合用于膜融合和膜间脂质转移的无损监测。
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