Abstract
The fungal metabolite brefeldin A (BFA) induces profound alterations in the morphology of intracellular organelles. Although BFA promotes the formation of extensive tubular endosomal domains, our understanding of the effects of the antibiotic on vesicle traffic events associated with endocytosis is limited. Thus, alterations in the transferrin (Tf) receptor's endocytic/recycling pathway upon treatment of human erythroleukemia K562 cells with BFA were studied as a pharmacological response. Treatment of K562 cells with BFA caused a down-regulation in the number of cell surface Tf receptors. This effect is highly reminiscent of the well-known action of phorbol 12-myristate 13-acetate (PMA) on Tf receptor traffic in K562 cells. However, our results demonstrate that these two agents down-regulate the Tf receptor via different mechanisms. The effects of BFA and PMA were additive when K562 cells were incubated with both together. Using the In/Sur method, the endocytic rate constant for Tf internalization was determined and PMA was found to greatly enhance ke, from 0.28 min−1 to 0.43 min−1, while BFA had little effect (Ke=0.20 min−1). In contrast, BFA-treatment alters the exocytic rate constant for return of internalized receptors to the cell surface, with the largest effect exerted on a ‘slow-release’, monensin-sensitive, compartment. The sum of the endocytic and exocytic kinetic data support a model in which BFA and PMA down-regulate the Tf receptor in K562 cells by mechanistically distinct actions, with BFA targeting exocytic monensin-sensitive intracellular compartments and PMA acting to exert a profound influence on elements of receptor internalization.
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Abbreviations
- BFA:
-
brefeldin A
- ARF:
-
ADP-ribosylation factor
- HRP:
-
horseradish peroxidase
- Tf:
-
transferrin
- PMA:
-
phorbol 12-myristate 13-acetate
- DMSO:
-
dimethyl sulfoxide
- PBS:
-
phosphate-buffered saline
- HEPES:
-
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- BSA:
-
bovine serum albumin
- FITC-Tf:
-
fluorescein isothiocyanate-labelled transferrin
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Schonhorn, J.E., Wessling-Resnick, M. Brefeldin a down-regulates the transferrin receptor in K562 cells. Mol Cell Biochem 135, 159–169 (1994). https://doi.org/10.1007/BF00926519
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DOI: https://doi.org/10.1007/BF00926519