Ktistakis N T, Kao C Y, Wang R H, Roth M G
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 72935-9038, USA.
Mol Biol Cell. 1995 Feb;6(2):135-50. doi: 10.1091/mbc.6.2.135.
The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.
利用报告蛋白来研究分泌调节常常因翻译后加工事件而变得复杂，这些事件会影响某些蛋白质的分泌，但并非特异性调节分泌的细胞机制的一部分。这对于分离哺乳动物分泌突变体来说一直是个特别的限制，因为该过程通常很缓慢。为了提供一种独立于蛋白质加工事件的分泌活性报告分子，用荧光脂质类似物C5-DMB-神经酰胺（与荧光团硼二吡咯亚甲基二氟化物偶联的神经酰胺）标记细胞，并通过荧光显微镜和荧光激活细胞分选追踪其分泌情况。布雷菲德菌素A能严重抑制中国仓鼠卵巢细胞的分泌，它阻断了C5-DMB-神经酰胺的分泌。在高温下，C5-DMB-神经酰胺的输出在HRP-1细胞中受到抑制，HRP-1细胞在分泌方面存在条件性缺陷。以C5-DMB-神经酰胺作为分泌活性的报告分子，设计了几种不同的脉冲追踪方案，筛选出对布雷菲德菌素A有抗性的突变中国仓鼠卵巢细胞以及糖蛋白向细胞表面转运存在缺陷的其他细胞。在诱变群体中，这两种类型的突变细胞的鉴定频率均为10^(-6)。因此，荧光脂质C5-DMB-神经酰胺可作为分泌活性的特异性标记物，为分离分泌途径存在缺陷的哺乳动物细胞提供一种高效、通用的方法。
