α-SNAP but not γ-SNAP is required for ER-Golgi transport after vesicle budding and the Rab1-requiring step but before the EGTA-sensitive step

Abstract

N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) have been implicated in diverse vesicular transport events; yet their exact role and site of action remain to be established. Using an established in vitro system, we show that antibodies against α-SNAP inhibit vesicle transport from the ER to the cis-Golgi and that recombinant α-SNAP enhances/stimulates the process. Cytosol immunodepleted of α-SNAP does not support normal transport unless supplemented with recombinant α-SNAP but not γ-SNAP. In marked contrast, cytosol immunodepleted of γ-SNAP supports ER-Golgi transport to the normal level. Neither antibodies against γ-SNAP nor recombinant γ-SNAP have any effect on ER-Golgi transport. These results clearly establish an essential role for α-SNAP but not γ-SNAP in ER-Golgi transport. When the transport assay is performed with cytosol immunodepleted of α-SNAP, followed by incubation with cytosol immunodepleted of a COPII subunit, normal transport is achieved. In marked contrast, no transport is detected when the assay is first performed with cytosol depleted of the COPII subunit followed by α-SNAP-depleted cytosol, suggesting that α-SNAP is required after a step that requires COPII (the budding step). In combination with cytosol immunodepleted of Rab1, it is seen that α-SNAP is required after a Rab1-requiring step. It has been shown previously that EGTA blocks ER-Golgi transport at a step after vesicle docking but before fusion and we show here that α-SNAP acts before the step that is blocked by EGTA. Our results suggest that α-SNAP may be involved in the pre-docking or docking but not the fusion process.