Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18p/NSF.
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 alpha-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.
Schindler
Christina
C
Biozentrum, University of Basel, CH-4056 Basel, Switzerland.
Spang
Anne
A
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050600:SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; C089147:GCS1 protein, S cerevisiae; D020690:GTPase-Activating Proteins; C514182:Glo3 protein, S cerevisiae; D050505:Mutant Proteins; C075137:SEC17 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; D020823:ADP-Ribosylation Factor 1