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[gamma S]). 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[gamma S], dilated vesicles accumulate around the Golgi stacks.
Antisera to the carboxyl terminus of the Golgi resident alpha subunit of Gi3 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.