Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum.
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.
Neeli
Indira
I
Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
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.
Lagakos
William S
WS
Department of Nutritional Sciences, Rutgers University, New Brunswick, New Jersey 08901.
Binas
Bert
B
Department of Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843.
Gheyi
Tarun
T
Department of Chemistry, University of Memphis, Memphis, Tennessee 38152.
Storch
Judith
J
Department of Nutritional Sciences, Rutgers University, New Brunswick, New Jersey 08901. Electronic address: storch@aesop.rutgers.edu.
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.
D000180:Acrylic Resins / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002914:Chylomicrons / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D050556:Fatty Acid-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000180:Acrylic Resins; D002914:Chylomicrons; D050556:Fatty Acid-Binding Proteins; D011994:Recombinant Proteins