The identification of the SNARE complex required for the fusion of VLDL-transport vesicle with hepatic cis-Golgi.
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 approximately 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.
Siddiqi
Shaila
S
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Boulevard, Orlando, FL 32827, USA.
Mani
Arul M
AM
Siddiqi
Shadab A
SA
D000818:Animals; D001693:Biological Transport, Active; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D066298:In Vitro Techniques; D008079:Lipoproteins, VLDL / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology; D050765:Qa-SNARE Proteins / Q000378:metabolism; D050766:Qb-SNARE Proteins / Q000378:metabolism; D050683:R-SNARE Proteins / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism
D008079:Lipoproteins, VLDL; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C098784:Sec22a protein, rat
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