Cell-free Transport to Distinct Golgi Cisternae Is Compartment Specific and ARF Independent

Abstract

The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide–sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5′-O-(3-thiotriphosphate) (GTPγS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPγS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPγS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor 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-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

Coatomer Vesicles are not Required for Inhibition of Golgi Transport by G‐Protein Activators

“…We have previously reported that incubation of Golgi membranes with ARF-depleted cytosol causes an increase in the average size of cisternal fenestrae and a modest increase in their number (23). In the current study, this observation has been confirmed and extended.…”

“…From these data, we estimate that the concentration of coatomer in a standard transport reaction containing 5 ml of Golgi and 2.5-10 ml of cytosol is 1 -3.6 mg/ml in reactions containing mockdepleted cytosol, and is B0.08 -0.24 mg/ml in reactions containing coatomer-depleted cytosol. For comparison, cytosol depleted of \ 95% of the endogenous ARF was also prepared by ion-exchange chromatography, as previously described (23).…”

“…Bovine brain cytosol was prepared according the method of Clary and Rothman (72). ARF-depleted cytosol was prepared by ion-exchange chromatography, as previously described (23). Coatomer-depleted cytosol was prepared from CHO cytosol by immunodepletion with the monoclonal anti-coatomer antibody CM1A10 (kindly provided by J. Ostermann, Vanderbilt University) using the method of Palmer et al (4), with minor modification.…”

“…Intra-Golgi transport assays were performed as previously described (23). Briefly, each 25 ml reaction contained buffer salts, an ATP-regeneration system, palmitoyl-CoA, 2.5 ml acceptor membranes (approximately 60 mg/ml final), and 2.5 -10 ml CHO cytosol (0.5 -2 mg/ml final).…”

“…The micrographs were numbered and randomized for the quantification of buds and vesicles with or without a punctate coating (e.g. coated or uncoated) on each cisterna, as previously described (23). The micrographs were also scored for the number and estimated average size of fenestrae in each cisternae.…”