The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle.
Dietary long chain fatty acids are absorbed in the intestine, esterified to triacylglycerol, and packaged in the unique lipoprotein of the intestine, the chylomicron. The rate-limiting step in the transit of chylomicrons through the enterocyte is the exit of chylomicrons from the endoplasmic reticulum in prechylomicron transport vesicles (PCTV) that transport chylomicrons to the cis-Golgi. Because chylomicrons are 250 nm in average diameter and lipid absorption is intermittent, we postulated that a unique SNARE pairing would be utilized to fuse PCTV with their target membrane, cis-Golgi. PCTV loaded with [(3)H]triacylglycerol were incubated with cis-Golgi and were separated from the Golgi by a sucrose step gradient. PCTV-chylomicrons acquire apolipoprotein-AI (apoAI) only after fusion with the Golgi. PCTV became isodense with Golgi upon incubation and were considered fused when their cargo chylomicrons acquired apoAI but docked when they did not. PCTV, docked with cis-Golgi, were solubilized in 2% Triton X-100, and proteins were immunoprecipitated using VAMP7 or rBet1 antibodies. In both cases, a 112-kDa complex was identified in nonboiled samples that dissociated upon boiling. The constituents of the complex were VAMP7, syntaxin 5, vti1a, and rBet1. Antibodies to each SNARE component significantly inhibited fusion of PCTV with cis-Golgi. Membrin, Sec22b, and Ykt6 were not found in the 112-kDa complex. We conclude that the PCTV-cis-Golgi SNARE complex is composed of VAMP7, syntaxin 5, Bet1, and vti1a.
Siddiqi
Shadab A
SA
Division of Gastroenterology, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
Siddiqi
Shahzad
S
Division of Gastroenterology, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
Mahan
James
J
Veterans Affairs Medical Center, Memphis, Tennessee 38104.
Peggs
Kiffany
K
Division of Gastroenterology, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
Gorelick
Fred S
FS
Department of Medicine, Veterans Affairs Healthcare, New Haven, Connecticut 06516; Yale University School of Medicine, New Haven, Connecticut 06516.
Mansbach
Charles M
CM
Division of Gastroenterology, University of Tennessee Health Science Center, Memphis, Tennessee 38163; Veterans Affairs Medical Center, Memphis, Tennessee 38104. Electronic address: cmansbach@utmem.edu.
D000818:Animals; D002914:Chylomicrons / Q000737:chemistry*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000737:chemistry; D050766:Qb-SNARE Proteins / Q000737:chemistry; D050767:Qc-SNARE Proteins / Q000737:chemistry; D050683:R-SNARE Proteins / Q000737:chemistry; D051381:Rats; D050600:SNARE Proteins / Q000737:chemistry*; D014280:Triglycerides / Q000737:chemistry
C098783:BET1 protein, rat; D002914:Chylomicrons; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D014280:Triglycerides; C496926:Vamp7 protein, rat; C491417:Vti1a protein, rat