The Identification of a Novel Endoplasmic Reticulum to Golgi SNARE Complex Used by the Prechylomicron Transport Vesicle

doi:10.1074/jbc.M601401200

The Identification of a Novel Endoplasmic Reticulum to Golgi SNARE Complex Used by the Prechylomicron Transport Vesicle^*

^‡Division of Gastroenterology, University of Tennessee Health Science Center, Memphis, Tennessee 38163 and ^§Veterans Affairs Medical Center, Memphis, Tennessee 38104, the ^¶Department of Medicine, Veterans Affairs Healthcare, and ^∥Yale University School of Medicine, New Haven, Connecticut 06516

1 To whom correspondence should be addressed: Division of Gastroenterology, the University of Tennessee Health Science Center, 920 Madison Ave., Suite 240, Memphis, TN 38163. Tel.: 901-448-5813; Fax: 901-448-7091; E-mail: cmansbach{at}utmem.edu.

Abstract

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 [³H]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.

Footnotes

↵2 The abbreviations used are: TAG, triacylglycerol(s); ER, endoplasmic reticulum; PCTV, prechylomicron transport vesicle(s); SNARE, N-ethylmaleimide-sensitive factor attachment protein receptor; ATPγS, adenosine 5′-O-(thiotriphosphate); PBS, phosphate-buffered saline.
↵* This study was supported by NIDDK, National Institutes of Health (NIH), Grants DK38760 (to C. M. M.) and DK54201 (to F. S. G.) and NIH Medical Student Research Program Grant T35DK07405. The Stout Neuroscience Laboratory was supported by NIH Grant RR1052 and National Science Foundation Grant DB1960633, a Veterans Administration Senior Career Development Award (to F. S. G.), and Office of Research and Development (R & D) Medical Research Service, Department of Veteran Affairs, research funds (to C. M. M. and F. S. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received February 14, 2006.
- Revision received May 23, 2006.
The American Society for Biochemistry and Molecular Biology, Inc.