Endoplasmic reticulum-through-Golgi transport assay based on O-glycosylation of native glycophorin in permeabilized erythroleukemia cells: role for Gi3.
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.
Wilson
B S
BS
Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla 92093.
Palade
G E
GE
Farquhar
M G
MG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002463:Cell Membrane Permeability; D003599:Cytoskeleton / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006021:Glycophorins / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D066298:In Vitro Techniques; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D011499:Protein Processing, Post-Translational; D013301:Streptolysins / Q000737:chemistry; D014407:Tumor Cells, Cultured
D006021:Glycophorins; D013301:Streptolysins; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins