Brefeldin A induces a microtubule-dependent fusion of galactosyltransferase-containing vesicles with the rough endoplasmic reticulum.
The fungal drug brefeldin A (BFA) has recently been found to induce a redistribution of medial- and cis-Golgi components to the endoplasmic reticulum (ER), raising the possibility of the existence of a retrograde pathway from the Golgi complex to the ER. Here, we demonstrate a BFA-induced reversible rearrangement of the trans-Golgi membrane protein galactosyltransferase (Gal-T) to the ER in HeLa cells. With immunofluorescence microscopy we have shown that BFA first caused a rapid change of Gal-T immunolabelling from a normal Golgi complex pattern to long and slender structures emanating from the cell centre and co-localizing with tubulin. Then immunofluorescence became ER-like. This effect was not dependent on ongoing protein synthesis and was reversed to normal within 120 min after removal of the drug. Restoration of the Golgi complex after removal of brefeldin A was energy-dependent but not mediated by microtubules nor dependent on protein synthesis. BFA-induced backflow of Gal-T was inhibited by nocodazole, a microtubule-disrupting agent. Immunoelectron microscopy showed that BFA treatment resulted in the fusion of Gal-T-containing vesicles with the ER. Furthermore, sucrose gradient centrifugation showed a significant shift in density of mature Gal-T polypeptides upon BFA treatment: about 40% of the enzyme migrated from its original density (1.13 g/ml) to the density of rough ER (1.19 g/ml). Thus, BFA caused microtubule-dependent vesicular backflow from a trans-Golgi component to the ER followed by fusion of the Golgi-derived vesicles with the ER.
Strous
G J
GJ
Laboratory of Cell Biology, University of Utrecht, The Netherlands.
Berger
E G
EG
van Kerkhof
P
P
Bosshart
H
H
Berger
B
B
Geuze
H J
HJ
D000935:Antifungal Agents / Q000494:pharmacology*; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005700:Galactosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000187:drug effects / Q000201:enzymology*
D000935:Antifungal Agents; D003517:Cyclopentanes; D020126:Brefeldin A; D005700:Galactosyltransferases