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J. Biol. Chem., Vol. 282, Issue 25, 17974-17984, June 22, 2007

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Liver Fatty Acid-binding Protein Initiates Budding of Pre-chylomicron Transport Vesicles from Intestinal Endoplasmic Reticulum^*

Indira Neeli, Shadab A. Siddiqi, Shahzad Siddiqi, James Mahan^¶, William S. Lagakos^||, Bert Binas^**, Tarun Gheyi¹, Judith Storch^||2, and Charles M. Mansbach, II^¶3

From the Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163, the Division of Gastroenterology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, the ^||Department of Nutritional Sciences, Rutgers University, New Brunswick, New Jersey 08901, the ^**Department of Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843, the Department of Chemistry, University of Memphis, Memphis, Tennessee 38152, and ^¶Veterans Affairs Medical Center, Memphis, Tennessee 38104

The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.

Received for publication, November 20, 2006 , and in revised form, April 19, 2007.

^* This work was supported by NIDDK Grants DK38760, DK74565 (to C. M. M.), and DK38389 (to J. S.) from the National Institutes of Health and by the Office of Research and Development Medical Research Service, Department of Veteran Affairs research funds (to C. M. M.). 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.

¹ Present address: SGX Pharmaceuticals, San Diego, CA 92121.

² To whom correspondence may be addressed: Dept. of Nutritional Science, Rutgers University, Cook College, 96 Lipman Dr., New Brunswick, NJ 08901-8525. Tel.: 732-932-1689; Fax: 732-932-3769; E-mail: storch{at}aesop.rutgers.edu. ³ To whom correspondence may be addressed: Division of Gastroenterology, the University of Tennessee Health Science Center, 920 Madison Ave., Ste. 240, Memphis, TN 38163. Tel.: 901-448-5813; Fax: 901-448-7091; E-mail: cmansbach{at}utmem.edu.

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