Brefeldin A-dependent membrane tubule formation reconstituted in vitro is driven by a cell cycle-regulated microtubule motor.
Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus, trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5'-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of approximately 100 and approximately 130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.
Robertson
A M
AM
School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
Allan
V J
VJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D020126:Brefeldin A / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002417:Cattle; D002453:Cell Cycle / Q000502:physiology; D003600:Cytosol / Q000187:drug effects / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D066298:In Vitro Techniques; D016547:Kinesins / Q000276:immunology / Q000502:physiology*; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008677:Metaphase / Q000187:drug effects / Q000502:physiology; D008870:Microtubules / Q000502:physiology*; D020409:Molecular Motor Proteins / Q000502:physiology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D014452:Ubiquitins / Q000378:metabolism; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D020409:Molecular Motor Proteins; D011500:Protein Synthesis Inhibitors; D014452:Ubiquitins; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D016547:Kinesins