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 reconstitute d this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the forma tion of an intricate, interconnected tubular membrane network, a process th at, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5'-O-(3-thiotriphosph ate). Surprisingly, membrane tubule formation is not due to the action of c onventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of similar to 100 and similar to 130 kDa ha ve been identified using the H1 antibody, both of which exhibit motor prope rties in a biochemical assay. Finally, BFA-induced membrane tubule formatio n does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared wit h interphase cytosol, these results suggest that either the ATPase or micro tubule-binding activity of the relevant motor is cell cycle regulated.