Transport of newly synthesized glucosylceramide to the plasma membrane by a non-Golgi pathway.
Transport of newly synthesized glucosylceramide to the plasma membrane by a non-Golgi pathway.
复制标题
通过非高尔基体途径将新合成的葡萄糖神经酰胺转运至质膜。
DOI：
10.1073/pnas.91.7.2708
复制
发表时间：
1994
影响因子：
11.1
通讯作者：
Baenziger,JU
中科院分区：
文献类型：
--
作者：
Warnock,DE;Lutz,MS;Blackburn,WA;YoungJr,WW;Baenziger,JU
高梯度磁亲和层析（HIMAC）已被用于从培养的中国仓鼠卵巢（CHO）细胞中获得不含细胞内膜污染物的高度富集质膜，产率≥ 80%。使用这个程序，我们的特点是运输的葡萄糖神经酰胺（GlcCer）和神经节苷脂GM3的质膜。新合成的GlcCer在7.2分钟内到达质膜，而GM3需要21.5分钟到达质膜。布雷菲德菌素A阻止新合成的GM 3和鞘磷脂转运至质膜，但对GlcCer的转运没有影响。类似地，在15 ° C下孵育CHO细胞阻断GM3和鞘磷脂向质膜的转运，但对GlcCer运动没有影响。我们建议，载体介导的运输占质膜GlcCer的主要部分。用[3H]葡萄糖或[3H]棕榈酸酯的脉冲追踪研究表明，已经到达质膜的新合成的GlcCer不用于合成GM3，而是快速降解或转化为尚未鉴定的产物。我们的研究结果表明，除了作为前体的高尔基体中的更高的糖基化，一个新合成的GlcCer的主要部分被迅速运输到质膜的非高尔基体途径，然后迅速翻转。
High-gradient magnetic affinity chromatography (HIMAC) has been used to obtain highly enriched plasma membranes, free of intracellular membrane contaminants, from cultured Chinese hamster ovary (CHO) cells in yields of > or = 80%. Using this procedure we have characterized the transport of glucosylceramide (GlcCer) and the ganglioside GM3 to the plasma membrane. Newly synthesized GlcCer reaches the plasma membrane in 7.2 min, whereas GM3 requires 21.5 min to reach the plasma membrane. Brefeldin A prevents transport of newly synthesized GM3 and sphingomyelin to the plasma membrane but has no effect on the transport of GlcCer. Similarly, incubation of CHO cells at 15 degrees C blocks transport of GM3 and sphingomyelin to the plasma membrane but has no effect on GlcCer movement. We propose that carrier-mediated transport accounts for a major fraction of the plasma membrane GlcCer. Pulse-chase studies with either [3H]glucose or [3H]palmitate indicate that newly synthesized GlcCer which has reached the plasma membrane is not utilized for the synthesis of GM3 but is instead rapidly either degraded or converted into an as yet unidentified product. Our results indicate that in addition to serving as a precursor for higher glycosylation in the Golgi, a major fraction of newly synthesized GlcCer is rapidly transported to the plasma membrane by a non-Golgi pathway and then rapidly turned over.