Endoplasmic reticulum-through-Golgi transport assay based on O-glycosylation of native glycophorin in permeabilized erythroleukemia cells: role for Gi3.

Abstract

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.

• Authors: B S Wilson, G E Palade, M G Farquhar
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 90 Issue 5 Pg. 1681-5 (Mar 01 1993) ISSN: 0027-8424 [Print] United States
• PMID: 8446582 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Glycophorins
• Streptolysins
• Adenosine Triphosphate
• GTP-Binding Proteins

Topics

• Adenosine Triphosphate (metabolism)
• Animals
• Biological Transport
• Cell Membrane Permeability
• Cytoskeleton (metabolism)
• Endoplasmic Reticulum (metabolism)
• GTP-Binding Proteins (metabolism)
• Glycophorins (metabolism)
• Glycosylation
• Golgi Apparatus (metabolism, ultrastructure)
• In Vitro Techniques
• Leukemia, Erythroblastic, Acute
• Mice
• Protein Processing, Post-Translational
• Streptolysins (chemistry)
• Tumor Cells, Cultured