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

Abstract

The G-protein activators guanosine 5%-O-(3-thiodiphosphate) (GTPgS) and aluminum fluoride (AlF) are thought to inhibit transport between Golgi cisternae by causing the accumulation of nonfunctional coatomer-coated transport vesicles on the Golgi. Although GTPgS and AlF inhibit transport in cell-free intra-Golgi transport systems, blocking coatomer vesicle formation does not. We therefore determined whether inhibition of in vitro Golgi transport by these agents requires coatomer vesicle formation. Depletion of coatomer was found to completely block coated vesicle formation on Golgi cisternae without affecting inhibition of in vitro transport by either GTPgS or AlF. Depletion of ADP-ribosylation factor (ARF) prevented inhibition of transport by GTPgS, but not by AlF, suggesting that the AlF-sensitive component in transport may not be a GTP-binding protein. Surprisingly, depletion of cytosolic ARF did not prevent the GTPgS-induced formation of Golgi-coated vesicles, whereas ARF was required for AlF-induced vesicle formation. Although ARF or coatomer depletion caused an increase in the fenestration of cisternae, no other utrastructural changes were observed that might explain the inhibition of transport by GTPgS or AlF. These findings suggest that ARF-GTPgS and AlF act by distinct and coatomer-independent mechanisms to inhibit membrane fusion in cell-free intra-Golgi transport.

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“…This apparent discrepancy may arise from the fact that GTP analogs and AlF 4 inhibit different cellular targets. For example, it has been suggested that the AIF-sensitive component in vesicle transport may not be a GTP-binding protein (21). Indeed, AlF 4 is known to inhibit other phosphoryl transfer reactions involved in vesicle transport, such as phospholipase D activity (27,34).…”

In Vitro Transport on Cis and Trans Sides of the Golgi Involves Two Distinct Types of Coatomer and ADP-ribosylation Factor-independent Transport Intermediates

“…Unfractionated and reconstituted cytosols had identical capacity to inhibit transport in the presence of the non-hydrolysable analog of GTP, GTP␥S, indicating that ARF function was reconstituted (27). COPI-depleted cytosol was prepared by immunodepletion with the COPI monoclonal antibody, CM1A10 (26). Cytosol was incubated twice with either CM1A10 protein G beads to produce COPI-depleted cytosol or with c-Myc monoclonal antibody (9E10) protein G beads to produce mock-depleted cytosol.…”

“…A straightforward explanation for this cytosol requirement might be that the steady-state level of free intermediates is dependent on the amount of Golgi coat proteins available to produce them. COPI is the dominant coat protein associated with Golgi vesicles, and depletion of COPI from cytosol essentially eliminates the production of coated vesicles on cisternae during an in vitro incubation (26). Paradoxically, COPI depletion does not inhibit transport in these in vitro assays (26).…”

“…COPI is the dominant coat protein associated with Golgi vesicles, and depletion of COPI from cytosol essentially eliminates the production of coated vesicles on cisternae during an in vitro incubation (26). Paradoxically, COPI depletion does not inhibit transport in these in vitro assays (26). It was therefore important to determine whether COPI depletion would suppress the formation of Golgi enzyme-containing intermediates during a stage I incubation.…”

“…This in vitro reconstitution system has been a powerful tool for the identification of protein components involved in transport, including COPI and its regulator ARF (20,21). Nevertheless, even though COPI vesicles form during an in vitro transport incubation (22)(23)(24), it has been impossible to unambiguously demonstrate that COPI vesicles are required for intra-Golgi transport in vitro (25)(26)(27)(28).…”