Incorporation of Ceramides into Saccharomyces cerevisiae Glycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro
Incorporation of Ceramides into Saccharomyces cerevisiae Glycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro
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DOI：
10.1128/ec.00257-08
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发表时间：
2009-03-01
期刊：
影响因子：
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通讯作者：
Conzelmann, Andreas
中科院分区：
文献类型：
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作者：
Bosson, Regine;Guillas, Isabelle;Conzelmann, Andreas
将糖基磷脂酰肌醇（GPIs）添加到酿酒酵母的GPI蛋白中后，二酰基甘油部分的脂肪酸通过Per 1和Gup 1的后续作用交换为C-26：0脂肪酸。在大多数GPI锚中，这种基于二酰基甘油的改性锚随后转化为含神经酰胺的锚，这是一种需要Cwh 43的反应。在这里，我们表明，GPI锚脂质重塑的最后一步可以在微粒体中监测。该测定使用来自在多球壳菌素存在下生长的细胞的微粒体，多球壳菌素是一种阻断二氢鞘氨醇（DHS）的生物合成从而抑制基于神经酰胺的锚的生物合成的化合物。当与[H-3]DHS孵育时，这些微粒体产生与完整细胞相同结构的放射性标记的含神经酰胺的锚脂质。来自cwh 43 Delta或mcd 4 Delta突变体的微粒体不能在体内制造基于神经酰胺的锚钉，在体外不会将[H-3]DHS掺入锚钉中。此外，gup 1 δ微粒体将[H-3]DHS掺入到与gup 1 δ细胞体内合成的相同的异常锚脂质中。因此，神经酰胺掺入GPI锚的体外测定忠实地再现了突变细胞中发生的事件。在单独的微粒体中观察到[H-3]DHS掺入GPI蛋白，但该反应受到细胞质或牛血清白蛋白、ATP加辅酶A（CoA）或C-26：0-CoA的刺激，特别是如果微粒体耗尽酰基-CoA。因此，[H-3]DHS在不存在酰基-CoA的情况下不能掺入蛋白质中。
After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C-26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. Such microsomes, when incubated with [H-3]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43 Delta or mcd4 Delta mutants, which are unable to make ceramide-based anchors in vivo, do not incorporate [H-3]DHS into anchors in vitro. Moreover, gup1 Delta microsomes incorporate [H-3]DHS into the same abnormal anchor lipids as gup1 Delta cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [H-3]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A (CoA), or C-26:0-CoA, particularly if microsomes are depleted of acyl-CoA. Thus, [H-3]DHS cannot be incorporated into proteins in the absence of acyl-CoA.