Giraudo C G, Daniotti J L, Maccioni H J
Centro de Investigaciones en Quimica Biológica de Córdoba, Departamento de Quimica Biológica, Facultad de Ciencias Quimicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina.
Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1625-30. doi: 10.1073/pnas.98.4.1625. Epub 2001 Jan 23.
Glycolipid glycosyltransferases catalyze the stepwise transfer of monosaccharides from sugar nucleotides to proper glycolipid acceptors. They are Golgi resident proteins that colocalize functionally in the organelle, but their intimate relationships are not known. Here, we show that the sequentially acting UDP-GalNAc:lactosylceramide/GM3/GD3 beta-1,4-N-acetyl-galactosaminyltransferase and the UDP-Gal:GA2/GM2/GD2 beta-1,3-galactosyltransferase associate physically in the distal Golgi. Immunoprecipitation of the respective epitope-tagged versions expressed in transfected CHO-K1 cells resulted in their mutual coimmunoprecipitation. The immunocomplexes efficiently catalyze the two transfer steps leading to the synthesis of GM1 from exogenous GM3 in the presence of UDP-GalNAc and UDP-Gal. The N-terminal domains (cytosolic tail, transmembrane domain, and few amino acids of the stem region) of both enzymes are involved in the interaction because (i) they reproduce the coimmunoprecipitation behavior of the full-length enzymes, (ii) they compete with the full-length counterpart in both coimmunoprecipitation and GM1 synthesis experiments, and (iii) fused to the cyan and yellow fluorescent proteins, they localize these proteins to the Golgi membranes in an association close enough as to allow fluorescence resonance energy transfer between them. We suggest that these associations may improve the efficiency of glycolipid synthesis by channeling the intermediates from the position of product to the position of acceptor along the transfer steps.
糖脂糖基转移酶催化单糖从糖核苷酸逐步转移至合适的糖脂受体。它们是驻留在高尔基体中的蛋白质，在该细胞器中功能上共定位，但它们之间的密切关系尚不清楚。在此，我们表明顺序作用的UDP-GalNAc：乳糖基神经酰胺/GM3/GD3β-1,4-N-乙酰半乳糖胺基转移酶和UDP-Gal：GA2/GM2/GD2β-1,3-半乳糖基转移酶在高尔基体远端物理结合。对转染的CHO-K1细胞中表达的各自表位标记版本进行免疫沉淀，导致它们相互共免疫沉淀。在UDP-GalNAc和UDP-Gal存在的情况下，免疫复合物有效地催化两个转移步骤，从而从外源性GM3合成GM1。两种酶的N端结构域（胞质尾、跨膜结构域和茎区的少数氨基酸）参与相互作用，因为（i）它们重现了全长酶的共免疫沉淀行为，（ii）它们在共免疫沉淀和GM1合成实验中与全长对应物竞争，并且（iii）与青色和黄色荧光蛋白融合后，它们将这些蛋白质定位到高尔基体膜上，其结合紧密到足以允许它们之间的荧光共振能量转移。我们认为这些结合可能通过在转移步骤中将中间体从产物位置引导至受体位置来提高糖脂合成的效率。
