Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells
Ganglioside glycosyltransferases organize in distinct multienzyme complexes in CHO-K1 cells
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DOI：
10.1074/jbc.m305455200
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发表时间：
2003-10-10
影响因子：
4.8
通讯作者：
Maccioni, HJF
中科院分区：
文献类型：
--
作者：
Giraudo, CG;Maccioni, HJF
神经节苷脂的合成在高尔基复合体中是分隔的。在大多数细胞中，LacCer、GM3和GD3的糖基化形成更高阶物种(GA2、GM2、GD2、GM1、GD1b)被移位到高尔基体和反高尔基体网络的最远端，在那里涉及的转移酶(GalNAcT和GalT2)形成物理和功能联系。另一方面，简单物种LacCer、GM3和GD3的糖基化被转移到更近端的高尔基间隔，我们在这里调查涉及的转移酶(GalT1、SialT1和SialT2)是否具有形成物理关联的特性。表达这些酶的表位标记版本的CHO-K1细胞膜的免疫共沉淀实验表明，GalT1、SialT1和SialT2以SialT1依赖的方式物理结合，它们的N-末端结构域参与了这些相互作用。活细胞中的显微荧光共振能量转移和光漂白后的荧光恢复证实了这种相互作用，除了显示高尔基体定位的复合体外，还将它们的形成映射到内质网。免疫共沉淀和荧光共振能量转移均未检测到GalT2或GalNAcT与GalT1或SialT1或SialT2之间的相互作用。这些结果以及诺康唑处理的细胞中高尔基体衍生的微囊的三色成像表明，神经节苷脂的合成是以不同的单位组织的，每个单位都由特定的糖基转移酶组成，这些糖基转移酶集中在不同的高尔基亚区。
The synthesis of gangliosides is compartmentalized in the Golgi complex. In most cells, glycosylation of LacCer, GM3, and GD3 to form higher order species (GA2, GM2, GD2, GM1, GD1b) is displaced toward the most distal aspects of the Golgi and the trans-Golgi network, where the involved transferases (GalNAcT and GalT2) form physical and functional associations. Glycosylation of the simple species LacCer, GM3, and GD3, on the other hand, is displaced toward more proximal Golgi compartments, and we investigate here whether the involved transferases (GalT1, SialT1, and SialT2) share the property of forming physical associations. Co-immunoprecipitation experiments from membranes of CHO-K1 cells expressing epitope-tagged versions of these enzymes indicate that GalT1, SialT1, and SialT2 associate physically in a SialT1-dependent manner and that their N-terminal domains participate in these interactions. Microscopic fluorescence resonance energy transfer and fluorescence recovery after photobleaching in living cells confirmed the interactions, and in addition to showing a Golgi apparatus localization of the complexes, mapped their formation to the endoplasmic reticulum. Neither co-immunoprecipitation nor fluorescence resonance energy transfer detected interactions between either GalT2 or GalNAcT and GalT1 or SialT1 or SialT2. These results, and triple color imaging of Golgi-derived microvesicles in nocodazole-treated cells, suggest that ganglioside synthesis is organized in distinct units each formed by associations of particular glycosyltransferases, which concentrate in different sub-Golgi compartments.