Sipos G, Puoti A, Conzelmann A
Institute of Biochemistry, University of Fribourg, Switzerland.
J Biol Chem. 1995 Aug 25;270(34):19709-15. doi: 10.1074/jbc.270.34.19709.
Glycosylphosphatidylinositol (GPI) anchors of the yeast Saccharomyces cerevisiae have been reported to contain three different types of side chains attached to contain three different types of side chains attached to the alpha 1,2-linked mannose of the conserved protein-ethanolamine-PO4-Man alpha 1,2Man alpha 1,6Man alpha 1,4GlcNH2-inositol glycan core. The possible side chains are Man alpha 1,2- or Man alpha 1,2Man alpha 1,2- or Man alpha 1,3Man alpha 1,2- (Fankhauser, C., Homan, S. W., Thomas Oates, J. E., McConville, M. J., Desponds, C., Conzelmann, A., and Ferguson, M. A. (1993) J. Biol. Chem. 268, 26365-26374). To determine in what subcellular compartment these side chains are made, we metabolically labeled GPI-anchored membrane proteins with myo-[2-3H]inositol in secretion mutants blocked at various stages of the secretory pathway and analyzed the anchor structure of the labeled glycoproteins. When the exit of vesicles from the endoplasmic reticulum or entry into the cis-Golgi were blocked in sec12 or sec18 cells, all anchors contained a side chain consisting of a single alpha 1,2-linked mannose. GPI proteins trapped in the cis-Golgi of sec7 contained Man alpha 1,3Man alpha 1,2- but no Man alpha 1,2Man alpha 1,2-side chains. Mutants blocked at later stages of the secretory pathway made increased amounts of side chains containing two mannoses. Man alpha 1,2Man alpha 1,2- and Man alpha 1,3Man alpha 1,2- side chains were preferentially associated with ceramide- and diacylglycerol-containing GPI anchors, respectively. Mnn1, mnn2, mnn3, mnn5, and mnt1(=kre2), i.e. mutants which lack or down-regulate 1,2- and 1,3- mannosyltransferases used in the elongation of N- and O-glycans in the Golgi, add the fifth mannose to GPI anchors normally. The same conclusion was reached through the analysis of deletion mutants in KTR1, KTR2, KTR3, KTR4, and YUR1 which all are open reading frames with high homology to MNT1. Mutants deficient in the Golgi elongation of N-glycans such as anp1, van1, mnn9 are deficient in the maturation of the N-glycans of GPI-anchored glycoproteins, but process the GPI anchor side chain normally. Data are consistent with the idea that the fourth mannose is added to proteins as part of the anchor precursor glycolipid in the endoplasmic reticulum, whereas the fifth mannose is added by not yet identified alpha 1,3- and alpha 1,2-mannosyltransferases located in the Golgi apparatus.
据报道，酿酒酵母的糖基磷脂酰肌醇（GPI）锚定物含有三种不同类型的侧链，这些侧链连接在保守的蛋白 - 乙醇胺 - PO4 - Manα1,2Manα1,6Manα1,4GlcNH2 - 肌醇聚糖核心的α1,2 - 连接的甘露糖上。可能的侧链为Manα1,2 - 或Manα1,2Manα1,2 - 或Manα1,3Manα1,2 - （Fankhauser, C., Homan, S. W., Thomas Oates, J. E., McConville, M. J., Desponds, C., Conzelmann, A., and Ferguson, M. A. (1993) J. Biol. Chem. 268, 26365 - 26374）。为了确定这些侧链是在哪个亚细胞区室中合成的，我们用肌醇 - [2 - 3H]肌醇对分泌途径不同阶段受阻的分泌突变体中的GPI锚定膜蛋白进行代谢标记，并分析标记糖蛋白的锚定结构。当sec12或sec18细胞中内质网的囊泡输出或进入顺式高尔基体受阻时，所有锚定物都含有由单个α1,2 - 连接的甘露糖组成的侧链。被困在sec7顺式高尔基体中的GPI蛋白含有Manα1,3Manα1,2 - 但没有Manα1,2Manα1,2 - 侧链。在分泌途径后期受阻的突变体产生了更多含两个甘露糖的侧链。Manα1,2Manα1,2 - 和Manα1,3Manα1,2 - 侧链分别优先与含神经酰胺和二酰基甘油的GPI锚定物相关。Mnn1、Mnn2、Mnn3、Mnn5和Mnt1（= Kre2），即缺乏或下调高尔基体中用于N - 和O - 聚糖延长的1,2 - 和1,3 - 甘露糖基转移酶的突变体，能正常地将第五个甘露糖添加到GPI锚定物上。通过对KTR1、KTR2、KTR3、KTR4和YUR1中的缺失突变体进行分析也得出了相同的结论，这些基因都是与MNT1具有高度同源性的开放阅读框。缺乏高尔基体中N - 聚糖延长功能的突变体，如anp1、van1、mnn9，在GPI锚定糖蛋白的N - 聚糖成熟方面存在缺陷，但GPI锚定侧链的加工正常。数据与以下观点一致，即第四个甘露糖作为锚定前体糖脂的一部分在内质网中添加到蛋白质上，而第五个甘露糖则由高尔基体中尚未确定的α1,3 - 和α1,2 - 甘露糖基转移酶添加。
