ENDOPLASMIC RETICULUM-THROUGH-GOLGI TRANSPORT ASSAY BASED ON O-GLYCOSYLATION OF NATIVE GLYCOPHORIN IN PERMEABILIZED ERYTHROLEUKEMIA-CELLS - ROLE FOR G(I3)

被引:40
作者
WILSON, BS [1 ]
PALADE, GE [1 ]
FARQUHAR, MG [1 ]
机构
[1] UNIV CALIF SAN DIEGO,CTR MOLEC GENET,LA JOLLA,CA 92093
关键词
D O I
10.1073/pnas.90.5.1681
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
An assay for endoplasmic reticulum (ER)-through-Golgi transport has been developed in streptolysin O-permeabilized murine erythroleukemia (MEL) cells. The reporter proteins are metabolically labeled native murine glycophorins, which display a distinctive shift in electrophoretic mobility after acquisition of O-linked oligosaccharides. The O-linked sugars are acquired at a site distal to a brefeldin A block, presumably in a cis Golgi compartment, and sialylation occurs in middle and/or trans Golgi compartments. In permeabilized cells supplemented with cytosolic proteins and an ATP-generating system, 20-50% of the radiolabeled precursor glycophorins can be converted to the mature, sialylated form. This maturation process is ATP- and cytosol-dependent and is blocked by guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]). Electron microscopy of permeabilized MEL cells shows retention of ER elements, stacked Golgi cisternae, free polysomes, and other subcellular components. In the presence of GTP[gammaS], dilated vesicles accumulate around the Golgi stacks. Antisera to the carboxyl terminus of the Golgi resident alpha subunit of G(i3) inhibit maturation of glycophorin. To our knowledge, a transport assay utilizing O-glycosylation of an endogenous protein as a monitor of ER-through-Golgi traffic in permeabilized cells has not been reported previously. Furthermore, the data provide evidence for heterotrimeric GTP-binding protein involvement in Golgi function.
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页码:1681 / 1685
页数:5
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