INTRODUCTION Protein transport between membrane compartments along the secretory pathway is primarily mediated by vesicles that bud from a donor compartment followed by targeting to, docking onto and fusion with an acceptor membrane (Pryer et al., 1992; Rothman, 1994; Rothman and Wieland, 1996; Schekman and Orci, 1996). NSF and SNAPs have been implicated in diverse transport events in the vesicle docking/fusion stage and it has been proposed that they function as general fusion proteins (Whiteheart and Kubalek, 1995). Sec18p (Eakle et al., 1988) is the yeast counterpart of NSF (Wilson et al., 1989). Three mammalian forms of SNAPs have been identified and are referred to as α-, β-, and γ-SNAP, respectively (Clary et al., 1990; Whiteheart et al., 1993). β-SNAP is brain specific and highly homologous to α-SNAP, while α- and γ-SNAPs are widely distributed and exhibit 25% amino acid identity. Sec17p is the yeast SNAP and can be functionally substituted for mammalian α-SNAP (Clary et al., 1990; Griff et al., 1992). To account for the specificity of vesicle transport, the SNAP receptor (SNARE) hypothesis proposes that vesicles for a defined transport event have unique integral membrane proteins termed v-SNAREs (vesicle SNAREs) that will interact specifically with the cognate SNAREs on the target membrane (t-SNAREs) (Ferro-Novick and Jahn, 1994; Pfeffer, 1996; Rothman, 1994; Rothman and Warren, 1994; Rothman and Wieland, 1996; Scheller, 1995; Söllner et al., 1993). Synaptobrevins (or VAMPs) function as v-SNAREs associated with the synaptic vesicles while syntaxin 1 and SNAP-25 function as t-SNAREs on the presynaptic membrane (Scheller, 1995; Söllner et al., 1993; Südhof, 1995). The correct pairing between v- and t-SNAREs then triggers the recruitment of SNAPs and subsequently NSF onto the paired SNAREs, leading to the formation of the v-SNAREs-t-SNAREs-SNAPs- NSF fusion complex (or 20S SNARE complex). This complex is intimately associated with vesicle fusion to the target membrane through a process that involves ATP hydrolysis by NSF and the disassembly of the 20S complex. The pairing between the v- and t-SNAREs and the formation of the SNARE complex can be regulated by other proteins such as members of the Rab/Sec4p/Ypt1p family of small GTPases (Lian et al., 1994; Novick and Brennwald, 1993; Nuoffer and Balch, 1994; Simons and Zerial, 1993; Sögaard et al., 1994), and/or other types of proteins that interact with either v-SNAREs (such as synaptophysin) (Calakos and Scheller, 1994; Edelmann et al., 1995) or t-SNAREs (such as Sec1p-like proteins) (Scheller, 2625 Journal of Cell Science 111, 2625-2633 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS9749 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. Key words: SNAP, Vesicular transport, Docking/fusion, ER, Golgi SUMMARY 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 Frank Peter, Siew Heng Wong, V. Nathan Subramaniam, Bor Luen Tang and Wanjin Hong* Membrane Biology Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore *Author for correspondence Accepted 19 June; published on WWW 13 August 1998