ARF-GAP–mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

Abstract

In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)–GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.

Interaction of SNAREs with ArfGAPs Precedes Recruitment of Sec18p/NSF

“…Previously, we reported that the ArfGAPs Glo3p and Gcs1p induce a conformational change in v-SNAREs involved in ER-Golgi and post-Golgi trafficking (Rein et al, 2002;Robinson et al, 2006). In this article, we extended these results to t-SNAREs (target-SNAREs on the target membrane) and show that Gcs1p accelerates v-t-SNARE complex formation in vitro.…”

“…Sec17p and Sec18p were expressed from pQE9 (QIAGEN, Valencia, CA), Sec18E350Qp was expressed from pQE30 (QIAGEN). The vectors for expression of Gcs1p, Glo3p, and Age2p and Arf1⌬N17p-Q71L have been described previously (Poon et al, 1996(Poon et al, , 1999(Poon et al, , 2001Rein et al, 2002).…”

“…His 6 -tagged SNARE proteins and His 6 -Sec17p were expressed in E. coli BL21* and purified using Ni 2ϩ -nitrilotriacetic acid (NTA)-agarose (QIAGEN) according to the manufacturer's instructions. Arf1⌬N17p was purified as described previously (Rein et al, 2002). His 6 -Sec18p and His 6 -Sec18E350Qp were purified according to Whiteheart et al, (1994) with the following modifications: Ni 2ϩ -NTAagarose with bound proteins was washed in batch twice with 10 column volumes of wash buffer 20 mM HEPES-KOH, pH 6.8, 400 mM KOAc, 0.5 mM ATP, 1 mM MgCl 2 , 1 mM 2-mercaptoethanol, and 10% glycerol, and 2 ϫ 10 column volumes of wash buffer20, 2 ϫ 10 column volumes of wash buffer50 (wash buffer containing 20 and 50 mM imidazole, respectively).…”

“…Other factors that intimately interact with SNAREs are the ArfGAPs Gcs1p and Glo3p, which have been shown to catalytically induce a conformational change in SNAREs involved in endoplasmic reticulum (ER)-Golgi trafficking (Rein et al, 2002). SNAREs in this altered conformation were able to recruit Arf1p.…”

“…This recruitment step required neither other factors nor the activation of the GTPase by a GEF. The interaction between Arf1p and SNAREs might provide a mechanism by which each vesicle carries at least one SNARE protein and would therefore be capable of fusing with the target membrane (Rein et al, 2002). However, membrane-anchored cargo proteins may also provide a platform for Arf1p binding.…”