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Shadab A Siddiqi - One of the best experts on this subject based on the ideXlab platform.
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reticulon 3 regulates very low density lipoprotein secretion by controlling very low density lipoprotein Transport Vesicle biogenesis
Canadian Journal of Physiology and Pharmacology, 2018Co-Authors: Shaila Siddiqi, Olga Zhelyabovska, Shadab A SiddiqiAbstract:Secretion of very low density lipoprotein (VLDL) by the liver is an important physiological process; however, the rate of VLDL secretion is determined by its Transport from the endoplasmic reticulum (ER) to the Golgi. This Transport event is facilitated by a specialized ER-derived Vesicle, the VLDL Transport Vesicle (VTV). We have reported earlier a detailed VTV proteome, which revealed that reticulon 3 (RTN3) is uniquely present in the VTV. Our immunoblotting and electron microscopic data demonstrate that RTN3 is enriched in the VTV; however, other ER-derived Vesicles do not contain RTN3. Co-immunoprecipitation data coupled with confocal microscopic analyses strongly suggest that RTN3 interacts with VLDL core protein, apoB100, at the ER level. Our data show that either blocking of RTN3 using specific antibodies or RTN3 knockdown resulted in significant reduction in VTV biogenesis from hepatic ER membranes. Additionally, VLDL secretion from hepatocytes was significantly decreased when RTN3 was silenced by...
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silencing of small valosin containing protein interacting protein svip reduces very low density lipoprotein vldl secretion from rat hepatocytes by disrupting its endoplasmic reticulum er to golgi trafficking
Journal of Biological Chemistry, 2016Co-Authors: Samata Tiwari, Shaila Siddiqi, Olga Zhelyabovska, Shadab A SiddiqiAbstract:Abstract The Transport of nascent very low density lipoprotein (VLDL) particles from the endoplasmic reticulum (ER) to the Golgi determines their secretion by the liver and is mediated by a specialized ER-derived Vesicle, the VLDL Transport Vesicle (VTV). Our previous studies have shown that the formation of ER-derived VTV requires proteins in addition to coat complex II proteins. The VTV proteome revealed that a 9-kDa protein, small valosin-containing protein-interacting protein (SVIP), is uniquely present in these specialized Vesicles. Our biochemical and morphological data indicate that the VTV contains SVIP. Using confocal microscopy and co-immunoprecipitation assays, we show that SVIP co-localizes with apolipoprotein B-100 (apoB100) and specifically interacts with VLDL apoB100 and coat complex II proteins. Treatment of ER membranes with myristic acid in the presence of cytosol increases SVIP recruitment to the ER in a concentration-dependent manner. Furthermore, we show that myristic acid treatment of hepatocytes increases both VTV budding and VLDL secretion. To determine the role of SVIP in VTV formation, we either blocked the SVIP protein using specific antibodies or silenced SVIP by siRNA in hepatocytes. Our results show that both blocking and silencing of SVIP lead to significant reduction in VTV formation. Additionally, we show that silencing of SVIP reduces VLDL secretion, suggesting a physiological role of SVIP in intracellular VLDL trafficking and secretion. We conclude that SVIP acts as a novel regulator of VTV formation by interacting with its cargo and coat proteins and has significant implications in VLDL secretion by hepatocytes.
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cideb protein is required for the biogenesis of very low density lipoprotein vldl Transport Vesicle
Journal of Biological Chemistry, 2013Co-Authors: Samata Tiwari, Shaila Siddiqi, Shadab A SiddiqiAbstract:Abstract Nascent very low density lipoprotein (VLDL) exits the endoplasmic reticulum (ER) in a specialized ER-derived Vesicle, the VLDL Transport Vesicle (VTV). Similar to protein Transport Vesicles (PTVs), VTVs require coat complex II (COPII) proteins for their biogenesis from the ER membranes. Because the size of the VTV is large, we hypothesized that protein(s) in addition to COPII components might be required for VTV biogenesis. Our proteomic analysis, supported by Western blotting data, shows that a 26-kDa protein, CideB, is present in the VTV but not in other ER-derived Vesicles such as PTV and pre-chylomicron Transport Vesicle. Western blotting and immunoelectron microscopy analyses suggest that CideB is concentrated in the VTV. Our co-immunoprecipitation data revealed that CideB specifically interacts with VLDL structural protein, apolipoprotein B100 (apoB100), but not with albumin, a PTV cargo protein. Confocal microscopic data indicate that CideB co-localizes with apoB100 in the ER. Additionally, CideB interacts with COPII components, Sar1 and Sec24. To investigate the role of CideB in VTV biogenesis, we performed an in vitro ER budding assay. We show that the blocking of CideB inhibits VTV budding, indicating a direct requirement of CideB in VTV formation. To confirm our findings, we knocked down CideB in primary hepatocytes and isolated ER and cytosol to examine whether they support VTV budding. Our data suggest that CideB knockdown significantly reduces VTV biogenesis. These findings suggest that CideB forms an intricate COPII coat and regulates the VTV biogenesis.
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intracellular trafficking and secretion of vldl
Arteriosclerosis Thrombosis and Vascular Biology, 2012Co-Authors: Samata Tiwari, Shadab A SiddiqiAbstract:Steady increase in the incidence of atherosclerosis is becoming a major concern not only in the United States but also in other countries. One of the major risk factors for the development of atherosclerosis is high concentrations of plasma low-density lipoprotein, which are metabolic products of very low-density lipoprotein (VLDL). VLDLs are synthesized and secreted by the liver. In this review, we discuss various stages through which VLDL particles go from their biogenesis to secretion in the circulatory system. Once VLDLs are synthesized in the lumen of the endoplasmic reticulum, they are Transported to the Golgi. The Transport of nascent VLDLs from the endoplasmic reticulum to Golgi is a complex multistep process, which is mediated by a specialized Transport Vesicle, the VLDL Transport Vesicle. The VLDL Transport Vesicle delivers VLDLs to the cis -Golgi lumen where nascent VLDLs undergo a number of essential modifications. The mature VLDL particles are then Transported to the plasma membrane and secreted in the circulatory system. Understanding of molecular mechanisms and identification of factors regulating the complex intracellular VLDL trafficking will provide insight into the pathophysiology of various metabolic disorders associated with abnormal VLDL secretion and identify potential new therapeutic targets.
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Proteomic Analysis of the Very Low Density Lipoprotein (VLDL) Transport Vesicles
Journal of Proteomics, 2012Co-Authors: Abdul Rahim, Shaila Siddiqi, Erika Nafi-valencia, Riyaz Basha, Chukwuemeka C. Runyon, Shadab A SiddiqiAbstract:The VLDL Transport Vesicle (VTV) mediates the Transport of nascent VLDL particles from the ER to the Golgi and plays a key role in VLDL-secretion from the liver. The functionality of VTV is controlled by specific proteins; however, full characterization and proteomic profiling of VTV remain to be carried out. Here, we report the first proteomic profile of VTVs. VTVs were purified to their homogeneity and characterized biochemically and morphologically. Thin section transmission electron microscopy suggests that the size of VTV ranges between 100 nm to 120 nm and each Vesicle contains only one VLDL particle. Immunoblotting data indicate VTV concentrate apoB100, apoB48 and apoAIV but exclude apoAI. Proteomic analysis based on 2D-gel coupled with MALDI-TOF identified a number of Vesicle-related proteins, however, many important VTV proteins could only be identified using LC-MS/MS methodology. Our data strongly indicate that VTVs greatly differ in their proteome with their counterparts of intestinal origin, the PCTVs. For example, VTV contains Sec22b, SVIP, ApoC-I, reticulon 3, cideB, LPCAT3 etc. which are not present in PCTV. The VTV proteome reported here will provide a basic tool to study the mechanisms underlying VLDL biogenesis, maturation, intracellular trafficking and secretion from the liver.
Randy Schekman - One of the best experts on this subject based on the ideXlab platform.
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the structure of the copii Transport Vesicle coat assembled on membranes
eLife, 2013Co-Authors: Giulia Zanetti, Randy Schekman, Simone Prinz, Sebastian Daum, Annette Meister, Kirsten Bacia, John A G BriggsAbstract:Proteins often need to move between different compartments within cells. To do this they are packaged into Transport pods called Vesicles. Many trafficked proteins are synthesized in an organelle called the endoplasmic reticulum, or ER; these proteins are Transported away from the ER in ‘COPII’ Vesicles, which are formed when the COPII proteins assemble on the ER membrane and force it to bulge outward. The bulge pinches off from the ER membrane, forming the Vesicle, which can then move to, and fuse with, a different compartment in the cell. The COPII proteins assemble in a particular order to form the Vesicle—Sar1 inserts into the membrane of the ER; Sec23 and Sec24 form an inner coat and capture the proteins that the Vesicle will Transport; and Sec13 and Sec31 form an outer coat. Although the structures of the coat proteins are known, how they bind to each other—and to the ER membrane—to form Vesicles of many shapes and sizes is less well understood. Now, Zanetti et al. show how the inner and outer coat proteins can interact flexibly to accommodate a variety of cargoes. Zanetti et al. mixed purified Sar1 and COPII coat proteins with artificial membranes in vitro. As in cells, the proteins assembled a coat on the membranes, creating bulges and Vesicles of different shapes. These coats were imaged using an electron microscope, and the images were analysed using computational image-analysis methods. In this way, Zanetti et al. produced a detailed 3D view of the assembled coat. It was found that the inner and outer proteins each arranged to form lattice structures—like fishing nets—which showed flexibility and variability in the way the individual proteins interact, as well as imperfections in the arrangement. Both coats may help to reshape the membrane, and the inner-coat and outer-coat lattices were also found to move with respect to each other. These flexible properties could allow the coat to assemble on membranes with different shapes and curvatures, forming COPII Vesicles with distinct sizes and shapes that can carry a range of cargoes.
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sec24b selectively sorts vangl2 to regulate planar cell polarity during neural tube closure
Nature Cell Biology, 2010Co-Authors: Janna Merte, Randy Schekman, Devon Jensen, Kevin M Wright, Sarah Sarsfield, Yanshu Wang, David D GintyAbstract:Mouse mutants for Sec24b, a component of COPII-coated ER-to-Golgi Vesicles, have defects in convergent extension, neural tube closure and other phenotypes related to planar cell polarity (PCP). The PCP component Vangl2 is sorted by Sec24b, and Vangl2 mutants defective in convergent extension do not exit the ER. Craniorachischisis is a rare but severe birth defect that results in a completely open neural tube. Mouse mutants in planar cell polarity (PCP) signalling components have deficits in the morphological movements of convergent extension that result in craniorachischisis. Using a forward genetic screen in mice, we identified Sec24b, a cargo-sorting member of the core complex of the endoplasmic reticulum (ER)-to-Golgi Transport Vesicle COPII, as critical for neural tube closure. Sec24bY613 mutant mice exhibit craniorachischisis, deficiencies in convergent extension and other PCP-related phenotypes. Vangl2, a key component of the PCP-signalling pathway critical for convergent extension, is selectively sorted into COPII Vesicles by Sec24b. Moreover, Sec24bY613 genetically interacts with a loss-of-function Vangl2 allele (Vangl2LP), causing a marked increase in the prevalence of spina bifida. Interestingly, the Vangl2 looptail point mutants Vangl2D255E and Vangl2S464N, known to cause defects in convergent extension, fail to sort into COPII Vesicles and are trapped in the ER. Thus, during COPII Vesicle formation, Sec24b shows cargo specificity for a core PCP component, Vangl2, of which proper ER-to-Golgi Transport is essential for the establishment of PCP, convergent extension and closure of the neural tube.
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bi directional protein Transport between the er and golgi
Annual Review of Cell and Developmental Biology, 2004Co-Authors: Elizabeth A Miller, Jonathan Goldberg, L Orci, Randy SchekmanAbstract:▪ Abstract The endoplasmic reticulum (ER) and the Golgi comprise the first two steps in protein secretion. Vesicular carriers mediate a continuous flux of proteins and lipids between these compartments, reflecting the Transport of newly synthesized proteins out of the ER and the retrieval of escaped ER residents and Vesicle machinery. Anterograde and retrograde Transport is mediated by distinct sets of cytosolic coat proteins, the COPII and COPI coats, respectively, which act on the membrane to capture cargo proteins into nascent Vesicles. We review the mechanisms that govern coat recruitment to the membrane, cargo capture into a Transport Vesicle, and accurate delivery to the target organelle.
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Sec16p potentiates the action of COPII proteins to bud Transport Vesicles
Journal of Cell Biology, 2002Co-Authors: Frantisek Supek, David T. Madden, Susan Hamamoto, Lelio Orci, Randy SchekmanAbstract:SEC16 encodes a 240-kD hydrophilic protein that is required for Transport Vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes, hence it is not one of the cytosolic proteins required to reconstitute Transport Vesicle budding in a cell-free reaction. However, Sec16p is removed from the membrane by salt washes, and using such membranes we have reconstituted a Vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII Vesicle budding is promoted by GTP or a nonhydrolyzable analogue, guanylimide diphosphate (GMP-PNP), Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated Vesicle budding were explored with synthetic liposomes composed of a mixture of lipids, including acidic phospholipids (major–minor mix), or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major–minor mix liposomes and facilitates the recruitment of COPII proteins and Vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major–minor mix are required to recruit pure Sec16p to liposomes, PC/PE liposomes bind Sar1p-GTP, which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP–dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.
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sec24p and iss1p function interchangeably in Transport Vesicle formation from the endoplasmic reticulum in saccharomyces cerevisiae
Molecular Biology of the Cell, 2000Co-Authors: Tatsuo Kurihara, Randy Schekman, Susan Hamamoto, Chris A Kaiser, Ruth E Gimeno, Tohru YoshihisaAbstract:The Sec23p/Sec24p complex functions as a component of the COPII coat in Vesicle Transport from the endoplasmic reticulum. Here we characterize Saccharomyces cerevisiae SEC24, which encodes a protei...
Suzanne R. Pfeffer - One of the best experts on this subject based on the ideXlab platform.
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Transport Vesicle tethering at the trans golgi network coiled coil proteins in action
Frontiers in Cell and Developmental Biology, 2016Co-Authors: Pak Yan Cheung, Suzanne R. PfefferAbstract:The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound Transport Vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of Transport Vesicles at the trans Golgi network (TGN). How do long tethering proteins actually catch Vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and Vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress toward understanding these questions and remaining, unresolved mysteries will be discussed.
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an update on Transport Vesicle tethering
Molecular Membrane Biology, 2010Co-Authors: Frank C Brown, Suzanne R. PfefferAbstract:Membrane trafficking involves the collection of cargo into nascent Transport Vesicles that bud off from a donor compartment, translocate along cytoskeletal tracks, and then dock and fuse with their target membranes. Docking and fusion involve initial interaction at a distance (tethering), followed by a closer interaction that leads to pairing of Vesicle SNARE proteins (v-SNAREs) with target membrane SNAREs (t-SNAREs), thereby catalyzing Vesicle fusion. When tethering cannot take place, Transport Vesicles accumulate in the cytoplasm. Tethering is generally carried out by two broad classes of molecules: extended, coiled-coil proteins such as the so-called Golgin proteins, or multi-subunit complexes such as the Exocyst, COG or Dsl complexes. This review will focus on the most recent advances in terms of our understanding of the mechanism by which tethers carry out their roles, and new structural insights into tethering complex transactions.
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a functional role for the gcc185 golgin in mannose 6 phosphate receptor recycling
Molecular Biology of the Cell, 2006Co-Authors: Jonathan V Reddy, Alondra Schweizer Burguete, Khambhampaty Sridevi, Ian G Ganley, Ryan M Nottingham, Suzanne R. PfefferAbstract:Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during Transport Vesicle formation. Rab9-bearing Vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these Vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing Transport Vesicles at the TGN.
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Transport-Vesicle targeting: tethers before SNAREs
Nature Cell Biology, 1999Co-Authors: Suzanne R. PfefferAbstract:Protein secretion and the Transport of proteins between membrane-bound compartments are mediated by small, membrane-bound Vesicles. Here I review what is known about the process by which Vesicles are targeted to the correct destination. A growing family of proteins, whose precise modes of action are far from established, is involved in targeting. Despite the wide diversity in the identities of the players, some common themes are emerging that may explain how Vesicles can identify their targets and release their cargo at the correct time and place in eukaryotic cells.
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Transport Vesicle docking snares and associates
Annual Review of Cell and Developmental Biology, 1996Co-Authors: Suzanne R. PfefferAbstract:Proteins that function in Transport Vesicle docking are being identified at a rapid rate. So-called v- and t-SNAREs form the core of a Vesicle docking complex. Additional accessory proteins are required to protect SNAREs from promiscuous binding and to deprotect SNAREs under conditions in which Transport Vesicle docking should occur. Because access to SNAREs must be regulated, other proteins must also contain specificity determinants to accomplish delivery of Transport Vesicles to their distinct and specific membrane targets.
Eckart D Gundelfinger - One of the best experts on this subject based on the ideXlab platform.
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Molecular organization and assembly of the presynaptic active zone of neurotransmitter release
Results and Problems in Cell Differentiation, 2006Co-Authors: Anna Fejtova, Eckart D GundelfingerAbstract:At chemical synapses, neurotransmitter is released at a restricted region of the presynaptic plasma membrane, called the active zone. At the active zone, a matrix of proteins is assembled, which is termed the presynaptic grid or cytomatrix at the active zone (CAZ). Components of the CAZ are thought to localize and organize the synaptic Vesicle cycle, a series of membrane trafficking events underlying regulated neurotransmitter exocytosis. This review is focused on a set of specific proteins involved in the structural and functional organization of the CAZ. These include the multi-domain Rab3-effector proteins RIM1alpha and RIM2alpha; Bassoon and Piccolo, two multi-domain CAZ scaffolding proteins of enormous size; as well as members of the CAST/ERC family of CAZ-specific structural proteins. Studies on ribbon synapses of retinal photoreceptor cells have fostered understanding the molecular design of the CAZ. In addition, the analysis of the delivery pathways for Bassoon and Piccolo to presynaptic sites during development has produced new insights into assembly mechanisms of brain synapses during development. Based on these studies, the active zone Transport Vesicle hypothesis was formulated, which postulates that active zones, at least in part, are pre-assembled in neuronal cell bodies and Transported as so-called Piccolo-Bassoon Transport Vesicles (PTVs) to sites of synaptogenesis. Several PTVs can fuse on demand with the presynaptic membrane to rapidly form an active zone.
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Unitary assembly of presynaptic active zones from Piccolo-Bassoon Transport Vesicles
Neuron, 2003Co-Authors: Mika Shapira, N. E. Ziv, Tal Bresler, Viviana I. Torres, R. Grace Zhai, Thomas Dresbach, Eckart D Gundelfinger, Craig C. GarnerAbstract:Recent studies indicate that active zones (AZs) - sites of neurotransmitter release - may be assembled from preassembled AZ precursor Vesicles inserted into the presynaptic plasma membrane. Here we report that one putative AZ precursor Vesicle of CNS synapses - the Piccolo-Bassoon Transport Vesicle (PTV) - carries a comprehensive set of AZ proteins genetically and functionally coupled to synaptic Vesicle exocytosis. Time-lapse imaging reveals that PTVs are highly mobile, consistent with a role in intracellular Transport. Quantitative analysis reveals that the Bassoon, Piccolo, and RIM content of individual PTVs is, on average, half of that of individual presynaptic boutons and shows that the synaptic content of these molecules can be quantitatively accounted for by incorporation of integer numbers (typically two to three) of PTVs into presynaptic membranes. These findings suggest that AZs are assembled from unitary amounts of AZ material carried on PTVs.
Charles M Mansbach - One of the best experts on this subject based on the ideXlab platform.
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Control of chylomicron export from the intestine
American Journal of Physiology-gastrointestinal and Liver Physiology, 2016Co-Authors: Charles M Mansbach, Shahzad SiddiqiAbstract:The control of chylomicron output by the intestine is a complex process whose outlines have only recently come into focus. In this review we will cover aspects of chylomicron formation and prechylomicron Vesicle generation that elucidate potential control points. Substrate (dietary fatty acids and monoacylglycerols) availability is directly related to the output rate of chylomicrons. These substrates must be converted to triacylglycerol before packaging in prechylomicrons by a series of endoplasmic reticulum (ER)-localized acylating enzymes that rapidly convert fatty acids and monoacylglycerols to triacylglycerol. The packaging of the prechylomicron with triacylglycerol is controlled by the microsomal triglyceride Transport protein, another potential limiting step. The prechylomicrons, once loaded with triacylglycerol, are ready to be incorporated into the prechylomicron Transport Vesicle that Transports the prechylomicron from the ER to the Golgi. Control of this exit step from the ER, the rate-limiting step in the transcellular movement of the triacylglycerol, is a multistep process involving the activation of PKCζ, the phosphorylation of Sar1b, releasing the liver fatty acid binding protein from a heteroquatromeric complex, which enables it to bind to the ER and organize the prechylomicron Transport Vesicle budding complex. We propose that control of PKCζ activation is the major physiological regulator of chylomicron output.
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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
Journal of Biological Chemistry, 2012Co-Authors: Shahzad Siddiqi, Charles M MansbachAbstract:Native cytosol requires ATP to initiate the budding of the pre-chylomicron Transport Vesicle from intestinal endoplasmic reticulum (ER). When FABP1 alone is used, no ATP is needed. Here, we test the hypothesis that in native cytosol FABP1 is present in a multiprotein complex that prevents FABP1 binding to the ER unless the complex is phosphorylated. We found on chromatography of native intestinal cytosol over a Sephacryl S-100 HR column that FABP1 (14 kDa) eluted in a volume suggesting a 75-kDa protein complex that contained four proteins on an anti-FABP1 antibody pulldown. The FABP1-containing column fractions were chromatographed over an anti-FABP1 antibody adsorption column. Proteins co-eluted from the column were identified as FABP1, Sar1b, Sec13, and small VCP/p97-interactive protein by immunoblot, LC-MS/MS, and MALDI-TOF. The four proteins of the complex had a total mass of 77 kDa and migrated on native PAGE at 75 kDa. When the complex was incubated with intestinal ER, there was no increase in FABP1-ER binding. However, when the complex member Sar1b was phosphorylated by PKCζ and ATP, the complex completely disassembled into its component proteins that migrated at their monomer molecular weight on native PAGE. FABP1, freed from the complex, was now able to bind to intestinal ER and generate the pre-chylomicron Transport Vesicle (PCTV). No increase in ER binding or PCTV generation was observed in the absence of PKCζ or ATP. We conclude that phosphorylation of Sar1b disrupts the FABP1-containing four-membered 75-kDa protein complex in cytosol enabling it to bind to the ER and generate PCTV.
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a novel multiprotein complex is required to generate the prechylomicron Transport Vesicle from intestinal er
Journal of Lipid Research, 2010Co-Authors: Shahzad Siddiqi, Umair Saleem, Nada A Abumrad, Nicholas O Davidson, Judith Storch, Shadab A Siddiqi, Charles M MansbachAbstract:Dietary lipid absorption is dependent on chylo- micron production whose rate-limiting step across the intes- tinal absorptive cell is the exit of chylomicrons from the endoplasmic reticulum (ER) in its ER-to-Golgi Transport Vesicle, the prechylomicron Transport Vesicle (PCTV). This study addresses the composition of the budding complex for PCTV. Immunoprecipitation (IP) studies from rat intes- tinal ER solubilized in Triton X-100 suggested that Vesicle- associated membrane protein 7 (VAMP7), apolipoprotein B48 (apoB48), liver fatty acid-binding protein (L-FABP), CD36, and the COPII proteins were associated on incuba- tion of the ER with cytosol and ATP. This association was confi rmed by chromatography of the solubilized ER over Sephacryl S400-HR in which these constituents cochromato- graphed with an apparent kDa of 630. No multiprotein com- plex was detected when the ER was chromatographed in the absence of PCTV budding activity (resting ER or PKC de- pletion of ER and cytosol). Treatment of the ER with anti- apoB48 or anti-VAMP7 antibodies or using gene disrupted L-FABP or CD36 mice all signifi cantly inhibited PCTV gen- eration. A smaller complex (no COPII proteins) was formed when only rL-FABP was used to bud PCTV. The data sup- port the conclusion that the PCTV budding complex in in- testinal ER is composed of VAMP7, apoB48, CD36, and L-FABP, plus the COPII proteins. —Siddiqi, S., U. Saleem, N. Abumrad, N. Davidson, J. Storch, S. A. Siddiqi, and C. M. Mansbach II. A novel multiprotein complex is required to generate the prechylomicron Transport Vesicle from intesti- nal ER. J. Lipid Res. 2010. 51: 1918-1928.
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Sec24C is required for docking the prechylomicron Transport Vesicle with the Golgi.
Journal of Lipid Research, 2009Co-Authors: Shahzad Siddiqi, Shadab A Siddiqi, Charles M MansbachAbstract:The rate-limiting step in the transit of dietary fat across the intestinal absorptive cell is its exit from the endoplasmic reticulum (ER) in a specialized ER-to-Golgi Transport Vesicle, the prechylomicron Transport Vesicle (PCTV). PCTV bud off from the ER membranes and have unique features; they are the largest ER-derived Vesicles (average diameter 250 nm), do not require GTP and COPII proteins for their formation, and utilize VAMP7 as a v-N-ethylmaleimide sensitive factor attachment protein receptor (SNARE). However, PCTV require COPII proteins for their fusion with the Golgi, suggesting a role for them in Golgi target recognition. In support of this, PCTV contained each of the five COPII proteins when docked with the Golgi. When PCTV were fused with the Golgi, the COPII proteins were present in greatly diminished amounts, indicating they had cycled back to the cytosol. Immuno-depletion of Sec31 from the cytosol did not affect PCTV-Golgi docking, but depletion of Sec23 resulted in a 25% decrease. Immuno-depletion of Sec24C caused a nearly complete cessation of PCTV docking activity, but on the addition of recombinant Sec24C, docking activity was restored. We conclude that the COPII proteins are present at docking of PCTV with the Golgi and that Sec24C is required for this event. Sec23 plays a less important role.
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PKCζ-mediated phosphorylation controls budding of the pre-chylomicron Transport Vesicle
Journal of Cell Science, 2008Co-Authors: Shadab A Siddiqi, Charles M MansbachAbstract:Dietary triacylglycerols are absorbed by enterocytes and packaged in the endoplasmic reticulum (ER) in the intestinal specific lipoprotein, the chylomicron, for export into mesenteric lymph. Chylomicrons exit the ER in an ER-to-Golgi Transport Vesicle, the pre-chylomicron Transport Vesicle (PCTV), which is the rate-limiting step in the transit of chylomicrons across the cell. Here, we focus on potential mechanisms of control of the PCTV-budding step from the intestinal ER. We incubated intestinal ER with intestinal cytosol and ATP to cause PCTV budding. The budding reaction was inhibited by 60 nM of the PKC inhibitor Go 6983, suggesting the importance of PKCζ in the generation of PCTV. Immunodepletion of PKCζ from the cytosol and the use of washed ER greatly inhibited the generation of PCTVs, but was restored following the addition of recombinant PKCζ. Intestinal ER incubated with intestinal cytosol and [γ- 32 P]ATP under conditions supporting the generation of PCTVs showed the phosphorylation of a 9-kDa band following autoradiography. The phosphorylation of this protein correlated with the generation of PCTVs but not the formation of protein Vesicles and was inhibited by depletion of PKCζ. Phosphorylation of the 9-kDa protein was restored following the addition of recombinant PKCζ. The association of the 9-kDa protein with proteins that are important for PCTV budding was phosphorylation dependent. We conclude that PKCζ activity is required for PCTV budding from intestinal ER, and is associated with phosphorylation of a 9-kDa protein that might regulate PCTV budding. Summary
