Uliana Andrea S, Crespo Pilar M, Martina Jose A, Daniotti Jose L, Maccioni Hugo J F
Centro de Investigaciones en Química Biológica de Córdoba, UNC-CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina.
J Biol Chem. 2006 Oct 27;281(43):32852-60. doi: 10.1074/jbc.M605805200. Epub 2006 Sep 1.
Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2(+) cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2(+) cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2(+) cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.
神经节苷脂糖基转移酶组装成多酶复合物，定位于不同的高尔基体亚区室。在此，我们研究了在缺乏CMP - NeuAc:GM3唾液酸转移酶（SialT2）的CHO - K1细胞中，当该复合物的另一个成员SialT2共表达时，UDP - Gal:葡萄糖神经酰胺β - 1,4 - 半乳糖基转移酶（GalT1）和CMP - NeuAc:乳糖基神经酰胺唾液酸转移酶（SialT1）复合物的高尔基体亚区室定位是否受到影响。通过研究布雷菲德菌素A（BFA）和莫能菌素对糖脂合成的影响以及通过单细胞荧光显微镜和等密度亚分级分离法研究GalT1(1 - 52)-CFP（青色荧光蛋白）和SialT1(1 - 54)-YFP（黄色荧光蛋白）嵌合体的高尔基体亚区室定位，来确定GalT1和SialT1的高尔基体亚区室定位。我们发现，BFA以及莫能菌素在野生型（WT）细胞中会损害GM3神经节苷脂以上糖脂的合成，但在SialT2(+)细胞中会损害葡萄糖神经酰胺以上糖脂的合成。虽然BFA在WT细胞中将GalT1 - CFP和SialT1 - YFP重新分布到内质网，但在SialT2(+)细胞中，这些嵌合体的一部分仍与富含反式高尔基体网络和回收内体标志物的远端高尔基体区室相关。在BFA处理的细胞中，通过等密度亚分级分离法分离的与类高尔基体膜组分相关的GalT1 - CFP和SialT1 - YFP的百分比在SialT2(+)细胞中高于WT细胞。用特异性siRNA敲低SialT2的表达可逆转这些效应。结果表明，糖基转移酶复合物的高尔基体亚区室定位可能会根据参与酶的相对表达水平而改变，并揭示了细胞器使糖脂合成机制的拓扑结构适应细胞功能状态的能力。
