Heterogeneous behavior of cells with respect to induction of retrograde transport from the trans-Golgi network to the Golgi upon inhibition of the vacuolar proton pump.
We investigated whether the vacuolar proton pump is required for proper vesicular trafficking through the trans-Golgi network. Previously, we provided evidence that endocytic transport to early and late endosomes, as well as recycling from endosomes to the plasma membrane, but not transport to lysosomes, continued in the presence of bafilomycin A1, a specific inhibitor of the vacuolar proton pump (Van Weert et al., J. Cell Biol. 130, 821-834 (1995)). Following endocytosis, recycling of cell surface glycoproteins occurs directly from endosomes to the plasma membrane, but also involves trafficking via the trans-Golgi network. We now used transferrin conjugated to horseradish peroxidase (Tf/HRP) to label and monitor the itinerary of the transferrin receptor using electron microscopy. In the presence of bafilomycin A1, transport of Tf/HRP to the trans-Golgi network continued. However, in a limited percentage of cells bafilomycin A1 induced transport of Tf/HRP into the Golgi stack. Retrograde transport into the Golgi stack was analyzed further in cells that were additionally treated with Brefeldin A, a drug that induces a redistribution of Golgi, but not trans-Golgi network markers, to the endoplasmic reticulum and the nuclear envelope. Sequential addition of bafilomycin A1 and Brefeldin A to exponentially growing A431 cells resulted in heavy Tf/HRP-labeling of the endoplasmic reticulum and the nuclear envelope in about 3% of the cells. No Tf/HRP was detected in endoplasmic reticulum or nuclear envelope of the other cells. the same observation was made for HepG2, CHO, and HeLa cells or when HRP was used as a fluid phase-endocytosed marker. Experiments performed with cell cycle-synchronized A431 cells revealed that retrograde transport was not linked to any specific phase of the cell cycle. The presence of epidermal growth factor, however, increased the amount of positive nuclear envelopes in A431 cells to 8%. In conclusion, our results show that inhibition of the vacuolar proton pump by bafilomycin A1 induces retrograde transport from the trans-Golgi network into the Golgi, and indicate that clonal cell lines display heterogeneity in this respect.
van Weert
A W
AW
Department of Cell Biology, Faculty of Medicine and Institute of Biomembranes, Utrecht University, The Netherlands.
Geuze
H J
HJ
Stoorvogel
W
W
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport; D002453:Cell Cycle; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D018942:Macrolides; D017494:Proton Pumps; D014407:Tumor Cells, Cultured; D014617:Vacuoles
D000900:Anti-Bacterial Agents; D018942:Macrolides; D017494:Proton Pumps; C040929:bafilomycin A1