The identification of the SNARE complex required for the fusion of VLDL-transport vesicle with hepatic cis-Golgi

Abstract

VLDLs (very-low-density lipoproteins) are synthesized in the liver and play an important role in the pathogenesis of atherosclerosis. Following their biogenesis in hepatic ER (endoplasmic reticulum), nascent VLDLs are exported to the Golgi which is a physiologically regulatable event. We have previously shown that a unique ER-derived vesicle, the VTV (VLDL-transport vesicle), mediates the targeted delivery of VLDL to the Golgi lumen. Because VTVs are different from other ER-derived transport vesicles in their morphology and biochemical composition, we speculated that a distinct set of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins would form a SNARE complex which would eventually facilitate the docking/fusion of VTVs with Golgi. Our results show that Sec22b is concentrated in VTVs as compared with the ER. Electron microscopic results show that Sec22b co-localizes with p58 and Sar1 on the VTV surface. Pre-treatment of VTV with antibodies against Sec22b inhibited VTV–Golgi fusion, indicating its role as a v-SNARE (vesicle SNARE). To isolate the SNARE complex, we developed an in vitro docking assay in which VTVs were allowed to dock with the Golgi, but fusion was prevented to stabilize the SNARE complex. After the docking reaction, VTV–Golgi complexes were collected, solubilized in 2% Triton X-100 and the SNARE complex was co-immunoprecipitated using anti-Sec22b or GOS28 antibodies. A ~ 110 kDa complex was identified in non-boiled samples that was dissociated upon boiling. The components of the complex were identified as Sec22b, syntaxin 5, rBet1 and GOS28. Antibodies against each SNARE component significantly inhibited VTV–Golgi fusion. We conclude that the SNARE complex required for VTV–Golgi fusion is composed of Sec22b, syntaxin 5, rBet1 and GOS28.

Phosphorylation of Sar1b Protein Releases Liver Fatty Acid-binding Protein from Multiprotein Complex in Intestinal Cytosol Enabling It to Bind to Endoplasmic Reticulum (ER) and Bud the Pre-chylomicron Transport Vesicle

“…Furthermore, the cargo-selective function of the COPII proteins, especially Sec24 (34,35), for protein vesicles appears to play a secondary role for PCTV. At least three of the COPII proteins, Sar1b, Sec23, and Sec24C, interact with the quintessential protein of the developing chylomicron, apolipoprotein B48 (36), but this binding is not required for incorporation of the chylomicron cargo into PCTV (12). These data are in contrast to the required engagement of Sec24 with cargo proteins in the protein vesicle system (34).…”

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

“…Numerous studies have shown that Gos28 is evenly distributed across the Golgi, including the cis-, medial-, and trans-cisternae (31)(32)(33). Consistent with these findings, Gos28 has been implicated in a variety of transport steps, including anterograde transport between the endoplasmic reticulum (ER) and the Golgi, intraGolgi transport in both directions, and retrograde transport from the early/recycling endosome back to the trans-Golgi network (28,29,(32)(33)(34)(35)(36). Finally, some of these studies have defined Gos28 as a v-SNARE, whereas some have identified it as a t-SNARE.…”

“…To date, Gos28 has been isolated in three distinct SNARE complexes as follows: one with Ykt6, syntaxin 5, and Bet1 (36); a second with Ykt6, syntaxin 5, and Gos15 (23,35,79,80); and a third with Sec22b, syntaxin 5, and Bet1 (34). This complexity is compounded by the fact that each of Gos28's interacting partners may also be involved in additional SNARE complexes that do not involve Gos28.…”

Recent advances in physiological lipoprotein metabolism

“…The VLDL transport vesicle fuses with liver cis-Golgi to deliver VLDL to the Golgi lumen. The components of the SNARE complex that play a role in docking and fusion of the VLDL transport vesicles have been identified as Sec22b (vesicle SNARE), syntaxin 5, rBet1 and Gos28 (target membrane SNARE) [120]. The ER to Golgi movement of VLDL is prerequisite for their ultimate secretion from hepatocytes ( Figure 3).…”