Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system

Rat liver Golgi stacks were incubated with mitotic cytosol for 30 min at 37°C to generate mitotic Golgi fragments comprising vesicles, tubules, and cisternal remnants. These were isolated and further incubated with rat liver cytosol for 60 min. The earliest intermediate observed by electron microscopy was a single, curved cisterna with tubular networks fused to the cisternal rims. Elongation of this cisterna was accompanied by stacking and further growth at the cisternal rims. Stacks also fused laterally so that the typical end product was a highly curved stack of 2-3 cisternae mostly enclosing an electron-lucent space. Reassembly occurred in the presence of nocodazole or cytochalasin B but not at 4°C or in the absence of energy supplied in the form of ATP and GTP. Pretreatment of the mitotic fragments and cytosol with N-ethylmaleimide (NEM) also prevented reassembly. GTPγS and A1F prevented reassembly when added during fragmentation but not when added to the reassembly mixture. In fact, GTPγS stimulated reassembly such that all cisternae were stacked at the end of the incubation and comprised 40% of the total membrane. In contrast, microcystin inhibited stacking so that only single cisternae accumulated. Together these results provide a detailed picture of the reassembly process and open up the study of the architecture of the Golgi apparatus to a combined morphological and biochemical analysis.