The rate-limiting step in lipid absorption is the exit of pre-chylomicrons from the endoplasmic reticulum, which occurs by budding the pre-chylomicron transport vesicle from the surface of the endoplasmic reticulum membrane. This sealed vesicle transports chylomicrons anterograde to the cis Golgi where it fuses with the Golgi, delivers its chylomicron cargo to the Golgi lumen, and within the Golgi, the chylomicron acquires apolipoprotein Al on its surface. We propose to identify the site on apolipoprotein B48 to which Sar1 b and other cargo selective proteins bind as this will confirm if they all bind to the same apolipoprotein B48 peptide or if different binding sites are required for different proteins. We will also identify the proteins required for budding the pre-chylomicron transport vesicle, enable it to be vectorially transported to the Golgi, and to fuse with it, using both immunological and other approaches to determine protein-protein interactions. The GTP binding pocket in Sar1b is mutated in Chylomicron Retention Disease/Anderson's Disease leading to the question of promiscuous cargo selection for inclusion in the pre-chylomicron transport vesicle and production of a vesicle, which does not fuse, with the cis Golgi. We will determine why the COPII proteins, which are associated with the pre-chylomicron transport vesicle, are not uncoated prior to the docking of the vesicle with the cis Golgi unlike vesicles that transport proteins from the ER to the cis Golgi. This may be due to inhibition of the GTPase activating function of Sec23 by Sec23 Interactive Protein or by differential phosphorylation. Both possibilities will be tested for using recombinant Sec23 Interactive Protein and antibody directed towards it, and by using g32P-GTP loaded Sar1 b. In the absence of COPII proteins on the surface of the vesicles, no fusion with the Golgi occurs suggesting their functionality in SNARE pairing, a possibility that will be tested using specific antibodies. We will also mutate Sar1b to mimic Chylomicron Retention Disease/Anderson's Disease to test its function in a fusion assay of the vesicle with the cis Golgi. Experiments will be performed to test if Sar1 b activity is rate limiting for the generation of fusion competent pre-chylomicron transport vesicles and to test if one function of Sar1 b is to restrict access of budding competent proteins to the cargo selection protein, apolipoprotein B48. In the absence of the COPII proteins, vesicle budding increases 6 to 10 fold. We will compare the ability of Sar1a and Sar1b to bud with pre-chylomicron and protein vesicles to produce vesicles that are fusion competent with the cis Golgi to determine the specificity of each Sar1 for both vesicle types.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Gastrointestinal Cell and Molecular Biology Study Section (GCMB)
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Grey, Michael J
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University of Tennessee Health Science Center
Internal Medicine/Medicine
Schools of Medicine
United States
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Siddiqi, Shahzad; Sheth, Atur; Patel, Feenalie et al. (2013) Intestinal caveolin-1 is important for dietary fatty acid absorption. Biochim Biophys Acta 1831:1311-21
Siddiqi, Shahzad; Mansbach 2nd, Charles M (2012) Phosphorylation of Sar1b protein releases liver fatty acid-binding protein from multiprotein complex in intestinal cytosol enabling it to bind to endoplasmic reticulum (ER) and bud the pre-chylomicron transport vesicle. J Biol Chem 287:10178-88
Siddiqi, Shahzad; Saleem, Umair; Abumrad, Nada A et al. (2010) A novel multiprotein complex is required to generate the prechylomicron transport vesicle from intestinal ER. J Lipid Res 51:1918-28
Siddiqi, Shahzad; Siddiqi, Shadab A; Mansbach 2nd, Charles M (2010) Sec24C is required for docking the prechylomicron transport vesicle with the Golgi. J Lipid Res 51:1093-100
Mansbach, Charles M; Siddiqi, Shadab A (2010) The biogenesis of chylomicrons. Annu Rev Physiol 72:315-33
Siddiqi, Shadab A; Mansbach 2nd, Charles M (2008) PKC zeta-mediated phosphorylation controls budding of the pre-chylomicron transport vesicle. J Cell Sci 121:2327-38
Mansbach 2nd, Charles M; Gorelick, Fred (2007) Development and physiological regulation of intestinal lipid absorption. II. Dietary lipid absorption, complex lipid synthesis, and the intracellular packaging and secretion of chylomicrons. Am J Physiol Gastrointest Liver Physiol 293:G645-50
Neeli, Indira; Siddiqi, Shadab A; Siddiqi, Shahzad et al. (2007) Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum. J Biol Chem 282:17974-84