Sequential restriction of SNARE-mediated fusion by the COPII inner coat and SNARE chaperones.

SNARE-mediated fusion requires assembly of four SNARE domains (R, Qa, Qb, and Qc) distributed across two membranes, with an SM (Sec1/Munc-18 family) chaperone catalyzing this assembly. We investigated topological requirements for the four SNAREs that mediate ER-Golgi fusion using in vitro assays. In the presence of the cognate SM Sly1, we find that the R SNARE must be in trans to the Qa and Qb SNAREs. The Qc SNARE functions on either membrane but is most active in cis to Qa and Qb. These results prompted us to look upstream, at COPII-SNARE interactions. The assembled COPII coat is known to block fusion. We discovered that of five COPII core subunits, the Sar1 GTPase and Sec23/Sec24 subunits were necessary and sufficient to prevent fusion for prolonged periods, and that Sar1 was dispensable for inhibition over short periods. When Sec24- interactions with R and Qc SNAREs were disrupted, inhibition was relieved, indicating that SNARE sequestration COPII on the nascent carrier prevents fusion. These observations help to explain how appropriate fusion events are facilitated while inappropriate events are deterred.