Kok J W, Babia T, Klappe K, Egea G, Hoekstra D
University of Groningen, Department of Physiological Chemistry, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Biochem J. 1998 Aug 1;333 ( Pt 3)(Pt 3):779-86. doi: 10.1042/bj3330779.
Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degreesC, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.
在内质网(ER)到高尔基体的神经酰胺(Cer)转运是在阻断囊泡介导的蛋白质转运的条件下进行测量的。这通过两种方式实现,一是在完整细胞中将孵育温度降至15℃,二是在经链球菌溶血素O通透处理的细胞中通过操纵细胞内环境来实现。在这两种情况下,神经酰胺转运均未受到抑制,从代谢性(用[14C]丝氨酸)标记的神经酰胺从头合成神经酰胺单己糖苷和鞘磷脂(SM)即可证明这一点。该检测基于这样的知识,即神经酰胺从丝氨酸和棕榈酰辅酶A开始在ER合成,而糖鞘脂和SM在(早期)高尔基体中合成。在阻断囊泡介导的水疱性口炎病毒糖蛋白转运的条件下观察到了[14C]糖鞘脂和[14C]SM的形成。这些结果表明,[14C]神经酰胺通过非囊泡机制从内质网转移到高尔基体。