We have investigated whether ARF-GAPs may play a role in recruiting v-SNAREs into vesicles. Our results indicate that the ARF-GAPs, Glo3p, and Gcs1p may play a pivotal role in this process. They are necessary and sufficient to allow the interaction of the small GTPase Arf1p where the NH2
-terminal 17 amino acids were deleted (Arf1ΔN17p) with v-SNARE–GST fusion proteins. Arf1ΔN17p binding to the v-SNAREs did not require the continued presence of ARF-GAP. Instead, a prior contact between the v-SNAREs and the ARF-GAP was essential and sufficient for subsequent binding of Arf1ΔN17p to SNAREs. ARF-GAP (Glo3p or Gcs1p) would bind to v-SNAREs present on the Golgi membrane and would induce a conformational change (). This could lead to two different possibilities. The altered conformation of the SNAREs might lower the affinity of the ARF-GAPs for the v-SNAREs, allowing the complex to dissociate ( A). In the next step, Arf1p would be able to interact with v-SNAREs. ARF-GAP might start recruiting cargo proteins like the loaded HDEL receptor. If there is no cargo to be included into vesicles, ARF-GAP might bind to Arf1p, which could result in an abortive complex. Alternatively, in vivo SNAREs, ARF-GAP, and Arf1p may form a complex ( B). Only after coatomer binds to this activated complex could GTP hydrolysis on Arf1p occur. We favor the first hypothesis because GTP hydrolysis could not take place prematurely, thus leading to an efficient budding process. Although we did not detect a difference between dominant-activated and dominant-inactivated Arf1ΔN17p in vitro, this step might still be dependent on a prior immobilization of Arf1p to the Golgi membrane, since the binding domain in Bet1p is close to the membrane. This process may allow the selection of the site where the next vesicle should emerge. Thus, SNAREs, Arf1p, ARF-GAP, and coatomer could form a primer that would subsequently lead to diffusion of cargo into the bud formation area and finally to vesicle emergence as suggested by Springer et al. (1999)
. In our view, the conformational change in the SNAREs that is brought about by the interaction with ARF-GAP is necessary for efficient uptake in COPI vesicles. This mechanism would ensure the enclosure of v-SNAREs with high fidelity. Recently, Lanoix et al. (2001)
have shown that ARF-GAP plays a role in sorting Golgi resident proteins into different subpopulations of COPI vesicles. This is in very good agreement with our data. However, they postulate that ARF-GAP exists in a high affinity and a low affinity state for Arf1p. The high affinity state stimulates GTP hydrolysis by Arf1p in the presence of coatomer but in the absence of cargo. Thus, Arf1p should undergo futile GDP-GTP cycles. ARF-GAP reaches the low affinity state by interaction with cargo, which slows down the Arf1p GTPase activity, allowing a COPI vesicle to form. Since we do not have any data for different affinities of ARF-GAP for Arf1p, we do not include this view in our model. Nor do we think that coatomer must be present for the abortive ARF-GAP–Arf1p complex to form in the absence of cargo. Nevertheless, the work of Lanoix et al. (2001)
and our own data suggest a role of ARF-GAP in early events of COPI vesicle formation.
Figure 9. Schematic drawing of possible mechanisms for the “priming step” of ER-Golgi v-SNAREs. In A, ARF-GAP only interacts temporally with the SNAREs and reenters the complex after Arf1p and coatomer have been recruited to the SNAREs. The mechanism (more ...)
The COPI-dependent priming event is different from COPII budding where it seems that the small GTPase Sar1p may play a crucial role in bud site selection (Springer and Schekman, 1998
). The GAP for Sar1p is Sec23p, a subunit of the COPII coat. However, interestingly the conformational change on the SNAREs that might be induced by Glo3p is sufficient to recruit the Sec23/24p complex. Thus, this first interaction does not determine high specificity rather than preparing a platform. COPII vesicle budding requires Sec12p, a resident ER membrane protein, to promote Sar1p nucleotide exchange. Thus, this event is precluded at the cis-Golgi membrane. Furthermore, it seems unlikely that ARF-GAP would prime the v-SNAREs for uptake in COPII vesicles. Therefore, in vivo this conformational change at the ER exit sites might be brought about by another factor, most likely Sar1p.
Springer and Schekman (1998)
have reported a preference of Sar1p for Bet1p and Bos1p, although interactions with Sec22p were not detectable. Here again the situation is different for the retrograde transport from the Golgi to the ER. We observed interactions between Arf1ΔN17p and all three v-SNAREs, although there was a clear preference for Bet1p. A likely explanation would be that for the anterograde vesicle fusion Sec22p is dispensable and thus does not have to be present. Although Bos1p is not required for the consumption of retrograde transport vesicles, it has to be retrieved with high efficiency in order to undergo another round of transport.
Do v-SNAREs act as the elusive ARF receptors on membranes? Our results do not allow any conclusion in this respect. The microsome experiments with Arf1ΔN17p do point in this direction. However, we have not yet been able to obtain the same result with full-length myristoylated Arf1p. Furthermore, there might be more than one way to recruit Arf1p to membranes.
Our data suggests that for the inclusion of v-SNAREs in a COPI vesicle an ARF-GAP needs to interact first with the v-SNAREs. This initial interaction may serve to recruit Arf1ΔN17p to an exit site. This model would predict that ARF-GAP might have two functions. The first function would be a chaperone-like activity that induces a conformational change on the SNAREs (). One possibility would be that ARF-GAP mediates or facilitates the formation of helix bundles as has been described for the synaptic exocytotic SNARE complex (Sutton et al., 1998
). This helix bundle formation may be artificially facilitated in our system due to the dimerization abilities of GST. Thus, two SNARE molecules would already be in close proximity to interact. SNAREs exist as oligomeric protein complexes in vitro and most likely also in vivo (Swanton et al., 1998
; Ungermann et al., 1999
; Xu et al., 2000
). Although the SNARE–GST fusion proteins might represent homodimers, in vivo SNAREs probably form heteromeric complexes. The other possibility is a conformational change within one SNARE molecule that would render it more compact and thus less susceptible to protease digestion.
The second function of the ARF-GAP would then be the GAP activity itself. These two activities should be separated in order to allow the formation of a productive budding complex and marking the budding area into which additional cargo could diffuse. Recently, Goldberg (1998)
) has shown that coatomer stimulates the activity of ARF-GAP and that certain cargo molecules retard the coatomer-stimulated rate of GTP hydrolysis. Based on these data, Goldberg concluded that ARF-GAPs possess a proof reading activity. However this model is limited to proteins containing a coatomer-binding site for which cargo recognition may be coupled to GTP hydrolysis on Arf1ΔN17p. In contrast, the interaction between v-SNAREs, Glo3p, and Arf1ΔN17p probably does not require GTP hydrolysis. Thus, this interaction should be of a different nature. In addition, ER-Golgi v-SNAREs bind very weakly to coatomer if at all. However, this interaction could be mediated by ARF-GAP. The coatomer binding to the v-SNAREs was increased in the presence of GTP-bound Arf1ΔN17p, indicating that GTP-dependent interaction of Arf1p and coatomer occurs also in a SNARE–Arf1p–coatomer complex. It should be pointed out that Szafer et al. (2000)
) have reported a stimulatory role of ARF-GAP in the absence of coatomer. Thus, without the crystal structure of full-length Arf1p with full-length ARF-GAP the nucleotide requirements and staging of hydrolysis will remain open for a wide range of speculations. In summary, in this study we emphasize a novel additional role for ARF-GAPs that is required before the proofreading activity. Both activities could be linked in vivo.
A role of ARF-GAP in cargo uptake into COPI vesicles has been suggested by Aoe et al. (1998)
and more recently by Lanoix et al. (2001)
. Aoe et al. (1999)
showed that the noncatalytical domain of ARF-GAP is responsible for the interaction with the KDEL receptor. The authors concluded that the KDEL receptor and ARF-GAP regulate retrograde transport. These data are not in conflict with our results, since we invoke an additional function for ARF-GAP. ARF-GAP may regulate budding from the cis-Golgi in a spatially and temporally regulated manner. An initial interaction with the v-SNAREs could determine the site of vesicle emergence. Subsequent interaction with the ligand-occupied KDEL receptor would ensure that cargo is included into the vesicle or alternatively may sense if there is a need for vesicle formation. This would allow the establishment of equilibrium of vesicle and membrane flow between the Golgi and ER.