CELL-FREE TRANSPORT TO DISTINCT GOLGI CISTERNAE IS COMPARTMENT-SPECIFIC AND ARF INDEPENDENT

The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterogr ade transport to the medial-Golgi in a mammalian in vitro transport sy stem, This might indicate that the in vivo mechanism of intra-Golgi tr ansport is not faithfully reproduced in vitro, or that intra-Golgi tra nsport occurs by a nonvesicular mechanism, As one approach to distingu ishing between these possibilities, we have characterized two addition al cell-free systems that reconstitute transport to the trans-Golgi (t rans assay) and trans-Golgi network (TGN assay). Like in vitro transpo rt to the medial-Golgi (medial assay), transport to the trans-Golgi an d TGN requires cytosol, ATP, and N-ethylmaleimide-sensitive fusion pro tein (NSF). However, each assay has its own distinct characteristics o f transport. The kinetics of transport to late compartments are slower , and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) ( GTP gamma S) to inhibit transport, suggesting that each assay reconsti tutes a distinct transport event. Depletion of ARF from cytosol abolis hes vesicle formation and inhibition by GTP gamma S, but transport in all assays is otherwise unaffected, Purified recombinant myristoylated ARF1 restores inhibition by GTP gamma S, indicating that the GTP-sens itive component in all assays is ARF, We also show that asymmetry in d onor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles, These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-fr ee system and that vesicle formation is not required for in vitro tran sport at any level of the stack.