Wattenberg B W
Cell Biology Unit, Upjohn Company, Kalamazoo, Michigan 49007.
J Cell Biol. 1990 Aug;111(2):421-8. doi: 10.1083/jcb.111.2.421.
Glycolipid transport between compartments of the Golgi apparatus has been reconstituted in a cell free system. Transport of lactosylceramide (galactose beta 1-4-glucose-ceramide) was followed from a donor to an acceptor Golgi population. The major glycolipid in CHO cells is GM3 (sialic acid alpha 2-3 galactose beta 1-4-glucose-ceramide). Donor membranes were derived from a Chinese hamster ovary (CHO) cell mutant (Lec2) deficient in the Golgi CMP-sialic acid transporter, and therefore contained lactosylceramide as the predominant glycolipid. Acceptor Golgi apparatus was prepared from another mutant, Lec8, which is defective in UDP-Gal transport. Thus, glucosylceramide is the major glycolipid in Lec8 cells. Transport was measured by the incorporation of labeled sialic acid into lactosylceramide (present originally in the donor) by transport to acceptor membranes, forming GM3. This incorporation was dependent on ATP, cytosolic components, intact membranes, and elevated temperature. Donor membranes were prepared from Lec2 cells infected with vesicular stomatitus virus (VSV). These membranes therefore contain the VSV membrane glycoprotein, G protein. Donor membranes derived from VSV-infected cells could then be used to monitor both glycolipid and glycoprotein transport. Transport of these two types of molecules between Golgi compartments was compared biochemically and kinetically. Glycolipid transport required the N-ethylmaleimide sensitive factor previously shown to act in glycoprotein transport (Glick, B. S., and J. E. Rothman. 1987. Nature [Lond.]. 326:309-312; Rothman, J. E. 1987. J. Biol. Chem. 262:12502-12510). GTP gamma S inhibited glycolipid and glycoprotein transport similarly. The kinetics of transport of glycolipid and glycoprotein were also compared. The kinetics of transport to the end of the pathway were similar, as were the kinetics of movement into a defined transport intermediate. It is concluded that glycolipid and glycoprotein transport through the Golgi occur by similar if not identical mechanisms.
糖脂在高尔基体各间隔之间的转运已在无细胞体系中实现了重建。乳糖基神经酰胺（半乳糖β1-4-葡萄糖-神经酰胺）从供体高尔基体群体转运至受体高尔基体群体。中国仓鼠卵巢（CHO）细胞中的主要糖脂是GM3（唾液酸α2-3半乳糖β1-4-葡萄糖-神经酰胺）。供体膜来自一种缺乏高尔基体CMP-唾液酸转运蛋白的中国仓鼠卵巢（CHO）细胞突变体（Lec2），因此以乳糖基神经酰胺作为主要糖脂。受体高尔基体是从另一种在UDP-半乳糖转运方面有缺陷的突变体Lec8制备而来。因此，葡萄糖基神经酰胺是Lec8细胞中的主要糖脂。通过将标记的唾液酸转运至受体膜并掺入乳糖基神经酰胺（最初存在于供体中）形成GM3来测定转运情况。这种掺入依赖于ATP、胞质成分、完整的膜以及升高的温度。供体膜由感染水疱性口炎病毒（VSV）的Lec2细胞制备。因此，这些膜含有VSV膜糖蛋白G蛋白。源自VSV感染细胞的供体膜随后可用于监测糖脂和糖蛋白的转运。对这两种类型分子在高尔基体间隔之间的转运进行了生化和动力学比较。糖脂转运需要先前已证明在糖蛋白转运中起作用的N-乙基马来酰亚胺敏感因子（Glick，B.S.，和J.E.Rothman.1987.《自然》[伦敦].326:309-312；Rothman，J.E.1987.《生物化学杂志》262:12502-12510）。GTPγS对糖脂和糖蛋白转运的抑制作用相似。还比较了糖脂和糖蛋白的转运动力学。转运至途径终点的动力学相似，进入特定转运中间体的动力学也相似。得出的结论是，糖脂和糖蛋白通过高尔基体的转运即使不是完全相同的机制，也是相似的。
