Interaction of SNAREs with ArfGAPs Precedes Recruitment of Sec18p/NSF

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key components of the fusion machinery in vesicular transport and in homotypic membrane fusion. We previously found that ADP-ribosylation factor GTPase activating proteins (ArfGAPs) promoted a conformational change on SNAREs that allowed recruitment of the small GTPase Arf1p in stoichiometric amounts. Here, we show that the ArfGAP Gcs1p accelerates vesicle (v)-target membrane (t)-SNARE complex formation in vitro, indicating that ArfGAPs may act as folding chaperones. These SNARE complexes were resolved in the presence of ATP by the yeast homologues of ␣-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the "activated" SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred.

The GAP Domain and the SNARE, Coatomer and Cargo Interaction Region of the ArfGAP2/3 Glo3 are Sufficient for Glo3 Function

“…In addition, the interaction between coatomer and mammalian ArfGAP3 has been recently reported (6). Furthermore, Glo3 is capable of interacting with and inducing a conformational change in a wide variety of SNARE proteins (15,22). Moreover, recombinant Glo3 bound directly to glutathione S‐transferase (GST)–SNARE fusion proteins (Figure 3C).…”

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

“…Further complicating the problem, is evidence that implicate ArfGAP proteins as positive regulators of the COPI coat rather than negative regulators: overexpression of any of the four yeast ArfGAPs suppressed the lethality of an arf1 mutant (Zhang et al 1998(Zhang et al , 2003. Further yeast experiments also support an active role for Gcs1 and Glo3 in cargo selection, acting on SNARE proteins prior to incorporation into vesicles to promote Arf1 and coatomer interaction (Rein et al 2002;Schindler and Spang 2007;Schindler et al 2009). Clearly, the precise role of the GAP in the COPI system remains to be fully understood, complicated by conflicting results from different labs and/or systems and may in fact be multifaceted by serving both positive and negative roles at different stages during the vesicle formation process (Spang et al 2010).…”

Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

“…Bre5 complex to maintain the heptameric coatomer complex (Cohen et al 2003). Removal of at least some coatomer complexes from vesicles appears to happen late in the life cycle of a transport vesicle as the ER tethering complex can recognize coatomer (Andag et al 2001;Vanrheenen et al 2001), and Sec18/NSF can displace Arf1 from SNAREs in vitro (Schindler and Spang 2007).…”