COP-COATED VESICLES ARE INVOLVED IN THE MITOTIC FRAGMENTATION OF GOLGI STACKS IN A CELL-FREE SYSTEM

Rat liver Golgi stacks fragmented when incubated with mitotic but not interphase cytosol in a process dependent on time, temperature, energy (added in the form of ATP) and cdc2 kinase. The cross-sectional lengt h of Golgi stacks fell in the presence of mitotic cytosol by approxima tely 50% over 30 min without a corresponding decrease in the number of cisternae in the stack. The loss of membrane from stacked and single cisternae occurred with a half-time of approximately 20 min, and was m atched by the appearance of both small (50-100 nm in diameter) and lar ge (100-200 nm in diameter) vesicular profiles. Small vesicular profil es constituted more than 50% of the total membrane after 60 min of inc ubation and they were shown to be vesicles or very short tubules by se rial sectioning. In the presence of GTPgammaS all of the small vesicle s were COP-coated and both the extent and the rate at which they forme d were sufficient to account for the production of small vesicles duri ng mitotic incubation. The involvement of the COP-mediated budding mec hanism was confirmed by immunodepletion of one of the subunits of COP coats (the coatomer) from mitotic cytosol. Vesicles were no longer for med but highly fenestrated networks appeared, an effect reversed by th e readdition of purified coatomer. Together these experiments provide strong support for our hypothesis that the observed vesiculation of th e Golgi apparatus during mitosis in animal cells is caused by continue d budding of COP-coated transport vesicles but an inhibition of their fusion with their target membranes.