Background: Nascent VLDL exits the ER in a specialized large vesicle, the VTV. Results: CideB interacts with COPII proteins, and CideB ablation abrogates VTV biogenesis. Conclusion: CideB forms an intricate COPII coat and regulates the formation of the VTV. Significance: New physiological role of CideB provides new insight into the mechanism that controls intracellular VLDL trafficking and secretion.

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.

The VLDL transport vesicle (VTV) mediates the transport of nascent VLDL particles from the ER to the Golgi and plays a key role in VLDL-secretion from the liver. The functionality of VTV is controlled by specific proteins; however, full characterization and proteomic profiling of VTV remain to be carried out. Here, we report the first proteomic profile of VTVs. VTVs were purified to their homogeneity and characterized biochemically and morphologically. Thin section transmission electron microscopy suggests that the size of VTV ranges between 100 nm to 120 nm and each vesicle contains only one VLDL particle. Immunoblotting data indicate VTV concentrate apoB100, apoB48 and apoAIV but exclude apoAI. Proteomic analysis based on 2D-gel coupled with MALDI-TOF identified a number of vesicle-related proteins, however, many important VTV proteins could only be identified using LC-MS/MS methodology. Our data strongly indicate that VTVs greatly differ in their proteome with their counterparts of intestinal origin, the PCTVs. For example, VTV contains Sec22b, SVIP, ApoC-I, reticulon 3, cideB, LPCAT3 etc. which are not present in PCTV. The VTV proteome reported here will provide a basic tool to study the mechanisms underlying VLDL biogenesis, maturation, intracellular trafficking and secretion from the liver.

Post-Golgi trafficking of mature VLDL (very-low-density lipoprotein) is crucial in maintaining normal TAG (triacylglycerol) homoeostasis of hepatocytes; however, the mechanism that regulates the exit of mature VLDL from the TGN (trans-Golgi network) is not known. We developed an in vitro TGN-budding assay that allowed us to examine the formation of secretory vesicles from the TGN in primary rat hepatocytes. We isolated TAG-rich PG-VTVs (post-TGN VLDL transport vesicles) using a continuous sucrose density gradient. PG-VTVs were distributed in low-density fractions, whereas protein transport vesicles were present in relatively higher-density fractions of the same sucrose gradient. EM revealed large intact PG-VTVs ranging 300–350 nm in size. The biogenesis of PG-VTVs from the TGN required cytosol, ATP, GTP hydrolysis and incubation at 37 °C. PG-VTVs concentrated the VLDL proteins: apolipoproteins apoB100, apoAIV, apoAI and apoE, but did not contain either albumin or transferrin. Proteinase K treatment did not degrade VLDL core proteins, suggesting that PG-VTVs were sealed. PG-VTVs were able to fuse with and deliver VLDL to the PM (plasma membrane) in a vectorial manner. We conclude that we have identified a new TGN-derived vesicle, the PG-VTV, which specifically transports mature VLDL from the TGN to the PM.

The transport of nascent very low density lipoprotein (VLDL) particles from the endoplasmic reticulum (ER) to the Golgi determines their secretion by the liver and is mediated by a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Our previous studies have shown that the formation of ER-derived VTV requires proteins in addition to coat complex II proteins. The VTV proteome revealed that a 9-kDa protein, small valosin-containing protein-interacting protein (SVIP), is uniquely present in these specialized vesicles. Our biochemical and morphological data indicate that the VTV contains SVIP. Using confocal microscopy and co-immunoprecipitation assays, we show that SVIP co-localizes with apolipoprotein B-100 (apoB100) and specifically interacts with VLDL apoB100 and coat complex II proteins. Treatment of ER membranes with myristic acid in the presence of cytosol increases SVIP recruitment to the ER in a concentration-dependent manner. Furthermore, we show that myristic acid treatment of hepatocytes increases both VTV budding and VLDL secretion. To determine the role of SVIP in VTV formation, we either blocked the SVIP protein using specific antibodies or silenced SVIP by siRNA in hepatocytes. Our results show that both blocking and silencing of SVIP lead to significant reduction in VTV formation. Additionally, we show that silencing of SVIP reduces VLDL secretion, suggesting a physiological role of SVIP in intracellular VLDL trafficking and secretion. We conclude that SVIP acts as a novel regulator of VTV formation by interacting with its cargo and coat proteins and has significant implications in VLDL secretion by hepatocytes.Aberrant secretion of very low density lipoproteins (VLDLs) from the liver contributes to the development of dyslipidemia, which constitutes a major risk factor for various metabolic disorders. A number of environmental and genetic factors have been identified that affect VLDL secretion. Insulin resistance is one of the most studied and a common factor that increases VLDL secretion from the liver as evident by both in vitro and in vivo studies (1-3). In contrast, hepatic infection with hepatitis C virus results in reduced VLDL secretion (4 -6). Although the significance of VLDL secretion and the factors affecting this process have been very well studied by numerous groups, the molecular mechanisms that control intracellular VLDL movement along the secretory pathway are poorly understood (7-9). We have adopted a proteomic, biochemical, and molecular approach to dissect the molecular machinery that controls intracellular VLDL trafficking and its eventual secretion (8, 10 -12).Several studies have shown that VLDLs are synthesized in the endoplasmic reticulum (ER) 3 and exported to the Golgi for their maturation (13,14). The transport of nascent VLDLs from the ER to the Golgi determines the rate of their eventual secretion, and this step is mediated by a dedicated vesicular system that utilizes a unique vesicle, the VLDL transport vesicle (VTV), which buds off the hepatic ER (7-9...