Abstract
Radioactively labeled brefeldin A was used to probe for proteins that interact with this metabolite. The most prominent protein labeled after in vivo incubation of Chinese hamster ovary cells with [3H]brefeldin A turned out to have an apparent molecular mass of 26 kDa. Radioactive peptides derived from the [3H]brefeldin A-labeled protein showed sequence identity with glutathione S-transferases, and immunoblotting after two-dimensional gel electrophoresis confirmed this result. In addition, Chinese hamster ovary cells convert the antibiotic to its glutathionyl and cysteinyl derivatives and secrete them rapidly into the medium. From these findings we conclude that detoxification of the antibiotic in mammalian cells occurs via the glutathion S-transferase system. This may explain the often observed reversibility of brefeldin A's action on the steady state of organelles in mammalian cells.
Highlights
From the Slnstitut fur BiochemieI, Universitat Heidelberg, Im Neuenheimer Feld 328, W-6900 Heidelberg, the SAbteilung Tumorbiochemie,Deutsches Krebsforschungszentrum,Im Neuenheimer FeM 280, W-6900Heidelberg, the IIZnstitutfur Biologie II, Universitat Freiburg, Schunzlestrasse 1, W-7800 Freiburg, and the **Max-Planck-Znstitutfur Biochemie, Am Klopferspitz 18A, W-8033Martinsried, Germany
Radioactive peptides derived from the t3H]brefeldin A-labeledprotein showed sequence identity with glutathione S-transferases, and immunoblotting after in mammalian cells and have characterized a component of the non-clathrin coat of coated Golgi transport vesicles (21) which dissociates from the Golgi immediately after administration of Brefeldin A (BFA) (22, 23)
The present study addresses a possible pathway for deactivation of the antibiotic BFAvia conjugation with GSH catalyzed by glutathione S-transferase (GST)
Summary
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 267, No 11, Issue of April 15, pp. 77267732, 1992 Printed in U.S.A. Radioactive peptides derived from the t3H]brefeldin A-labeledprotein showed sequence identity with glutathione S-transferases, and immunoblotting after in mammalian cells and have characterized a component of the non-clathrin coat of coated Golgi transport vesicles (21) which dissociates from the Golgi immediately after administration of BFA (22, 23) This observation, together with the two-dimensional gel electrophoresis confirmed this re- mentioned effects on membrane transport and organelle insult. The abbreviations used are: BFA, brefeldin A; CHO, Chinese hamster ovary;FAB-MS, fast atom bombardment-mass spectroscopy; GST, glutathione S-transferase;HPLC, high performance liquid chromatography; a-MEM, a-minimal essentialmedium; PAGE, poly-. Samples of 1 x lo6CHO cells were incubated with 5 pCi of ['HJBFA in 50 pi ofor-MEM at 37 "C, centrifuged, and the pellet extracted with 50 pl of methanol/ water (1:1), 5-pl aliquots of the cell extracts and the corresponding media were analyzed by TLC. Isoiation of GST from the cytosol fraction (l00,OOO X g supernatant) of CHO cells was performed by affinity chromatography using an S-hexylglut,athione-linked Sepharose 6B column accordingto (34)
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