The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A.
Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10-15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.
Roa
M
M
Unité de Génétique Somatique (URA CNRS 361), Institut Pasteur, Paris, France.
Cornet
V
V
Yang
C Z
CZ
Goud
B
B
D016475:3T3 Cells; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D002458:Cell Fractionation; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D051379:Mice
D017607:Aluminum Compounds; D003517:Cyclopentanes; D020126:Brefeldin A; D019204:GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride