Shadab A. Siddiqi

COPII proteins are required for Golgi fusion but not for endoplasmic reticulum budding of the pre-chylomicron transport vesicle

The budding of vesicles from endoplasmic reticulum (ER) that contains nascent proteins is regulated by COPII proteins. The mechanisms that regulate lipid-carrying pre-chylomicron transport vesicles (PCTVs) budding from the ER are unknown. To study the dependence of PCTV-ER budding on COPII proteins we examined protein and PCTV budding by using ER prepared from rat small intestinal mucosal cells prelabeled with 3H-oleate or 14C-oleate and 3H-leucine. Budded 3H-oleate-containing PCTVs were separated by sucrose density centrifugation and were revealed by electron microscopy as 142-500 nm vesicles. Our results showed the following: (1) Proteinase K treatment did not degrade the PCTV cargo protein, apolipoprotein B-48, unless Triton X-100 was added. (2) PCTV budding was dependent on cytosol and ATP. (3) The COPII proteins Sar1, Sec24 and Sec13/31 and the membrane proteins syntaxin 5 and rBet1 were associated with PCTVs. (4) Isolated PCTVs were able to fuse with intestinal Golgi. (5) Antibodies to Sar1 completely inhibited protein vesicle budding but increased the generation of PCTV; these changes were reversed by the addition of recombinant Sar1. (6) PCTVs formed in the absence of Sar1 did not contain the COPII proteins Sar1, Sec24 or Sec31 and did not fuse with the Golgi complex. Together, these findings suggest that COPII proteins may not be required for the exit of membrane-bound chylomicrons from the ER but that they or other proteins may be necessary for PCTV fusion with the Golgi.

The Biogenesis of Chylomicrons

The absorption of dietary fat is of increasing concern given the rise of obesity not only in the United States but throughout the developed world. This review explores what happens to dietary fat within the enterocyte. Absorbed fatty acids and monoacylglycerols are required to be bound to intracellular proteins and/or to be rapidly converted to triacylglycerols to prevent cellular membrane disruption. The triacylglycerol produced at the level of the endoplasmic reticulum (ER) is either incorporated into prechylomicrons within the ER lumen or shunted to triacylglycerol storage pools. The prechylomicrons exit the ER in a specialized transport vesicle in the rate-limiting step in the intracellular transit of triacylglycerol across the enterocyte. The prechylomicrons are further processed in the Golgi and are transported to the basolateral membrane via a separate vesicular system for exocytosis into the intestinal lamina propria. Fatty acids and monoacylglycerols entering the enterocyte via the basolateral membrane are also incorporated into triacylglycerol, but the basolaterally entering lipid is much more likely to enter the triacylglycerol storage pool than the lipid entering via the apical membrane.

Insulin enhances post-translational processing of nascent SREBP-1c by promoting its phosphorylation and association with COPII vesicles

The regulation of lipid homeostasis by insulin is mediated in part by the enhanced transcription of the gene encoding SREBP-1c (sterol regulatory element-binding protein-1c). Nascent SREBP-1c is synthesized and embedded in the endoplasmic reticulum (ER) and must be transported to the Golgi in coatomer protein II (COPII) vesicles where two sequential cleavages generate the transcriptionally active NH2-terminal fragment, nSREBP-1c. There is limited indirect evidence to suggest that insulin may also regulate the posttranslational processing of the nascent SREBP-1c protein. Therefore, we designed experiments to directly assess the action of insulin on the post-translational processing of epitope-tagged full-length SREBP-1c and SREBP-2 proteins expressed in cultured hepatocytes. We demonstrate that insulin treatment led to enhanced post-translational processing of SREBP-1c, which was associated with phosphorylation of ER-bound nascent SREBP-1c protein that increased affinity of the SREBP-1c cleavage-activating protein (SCAP)-SREBP-1c complex for the Sec23/24 proteins of the COPII vesicles. Furthermore, chemical and molecular inhibitors of the phosphoinositide 3-kinase pathway and its downstream kinase protein kinase B (PKB)/Akt prevented both insulin-mediated phosphorylation of nascent SREBP-1c protein and its posttranslational processing. Insulin had no effect on the proteolysis of nascent SREBP-2 under identical conditions. We also show that in vitro incubation of an active PKB/Akt enzyme with recombinant full-length SREBP-1c led to its phosphorylation. Thus, insulin selectively stimulates the processing of SREBP-1c in rat hepatocytes by enhancing the association between the SCAP-SREBP-1c complex and COPII proteins and subsequent ER to Golgi transport and proteolytic cleavage. This effect of insulin is tightly linked to phosphoinositide 3-kinase and PKB/Akt-dependent serine phosphorylation of the precursor SREBP-1c protein.

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.

Intracellular Trafficking and Secretion of VLDL

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.

A novel multiprotein complex is required to generate the prechylomicron transport vesicle from intestinal ER.

Dietary lipid absorption is dependent on chylomicron production whose rate-limiting step across the intestinal 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 intestinal 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 incubation of the ER with cytosol and ATP. This association was confirmed by chromatography of the solubilized ER over Sephacryl S400-HR in which these constituents cochromatographed with an apparent kDa of 630. No multiprotein complex was detected when the ER was chromatographed in the absence of PCTV budding activity (resting ER or PKCζ depletion 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 significantly inhibited PCTV generation. A smaller complex (no COPII proteins) was formed when only rL-FABP was used to bud PCTV. The data support the conclusion that the PCTV budding complex in intestinal ER is composed of VAMP7, apoB48, CD36, and L-FABP, plus the COPII proteins.

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

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.

Vesicle-associated membrane protein 7 is expressed in intestinal ER

Intestinal dietary triacylglycerol absorption is a multi-step process. Triacylglycerol exit from the endoplasmic reticulum (ER) is the rate-limiting step in the progress of the lipid from its apical absorption to its basolateral membrane export. Triacylglycerol is transported from the ER to the cis Golgi in a specialized vesicle, the pre-chylomicron transport vesicle (PCTV). The vesicle-associated membrane protein 7 (VAMP7) was found to be more concentrated on PCTVs compared with ER membranes. VAMP7 has been previously identified associated with post-Golgi sites in eukaryotes. To examine the potential role of VAMP7 in PCTV trafficking, antibodies were generated that identified a 25 kDa band consistent with VAMP7 but did not crossreact with VAMP1,2. VAMP7 was concentrated on intestinal ER by immunofluorescence microscopy. Immunoelectron microscopy showed that the ER proteins Sar1 and rBet1 were present on PCTVs and colocalized with VAMP7. Iodixanol gradient centrifugation showed VAMP7 to be isodense with ER and endosomes. Although VAMP7 localized to intestinal ER, it was not present in the ER of liver and kidney. Anti-VAMP7 antibodies reduced the transfer of triacylglycerol, but not newly synthesized proteins, from the ER to the Golgi by 85%. We conclude that VAMP7 is enriched in intestinal ER and that it plays a functional role in the delivery of triacylglycerol from the ER to the Golgi.

VLDL exits from the endoplasmic reticulum in a specialized vesicle, the VLDL transport vesicle, in rat primary hepatocytes

The movement of VLDL [very-LDL (low-density lipoprotein)] from the ER (endoplasmic reticulum) to the Golgi is required for its eventual secretion from hepatocytes and represents a potential target in controlling elevated concentrations of its metabolite LDL, the major determinant of atherosclerosis. To study this process, an in vitro ER-budding assay was developed to examine the generation of the VTV (VLDL transport vesicle) and PTV (protein transport vesicles) using ER isolated from [(14)C]TAG (triacylglycerol) and [(3)H]protein-labelled primary rat hepatocytes. VTVs do not contain albumin, as determined by immunoblots. VTVs were distributed in light-density fractions, whereas PTVs were mainly in the mid-portion of the sucrose gradient. Electron microscopy revealed that VTVs were larger ( approximately 100-120 nm) in size than PTVs ( approximately 55-70 nm). ER from 0.4 mM OA (oleic acid)-treated hepatocytes budded VTVs of a lighter density as compared with VTVs budded from ER of 0.1 mM or 0.004 mM OA-treated hepatocytes. The generation of VTVs from rat hepatic ER required cytosol, ATP, Sar1 (a GTPase) and incubation at 37 degrees C. Proteinase K treatment did not degrade the VTV cargo protein, apoB100 (apolipoprotein 100), indicating that VTVs were sealed. Immunoblots showed that VTV concentrated apoB100, Sar1 and rSec22b, and excluded albumin and calnexin. VTVs were shown to fuse with cis-Golgi and delivered their cargo to the Golgi lumen, as determined by in vitro fusion, and acquired endoglycosidase H resistance. These results suggest that a new ER-derived transport vesicle (VTV) has been identified and characterized which transports nascent VLDL from the hepatic ER to the Golgi.

The identification of the SNARE complex required for the fusion of VLDL-transport vesicle with hepatic cis-Golgi

VLDLs (very-low-density lipoproteins) are synthesized in the liver and play an important role in the pathogenesis of atherosclerosis. Following their biogenesis in hepatic ER (endoplasmic reticulum), nascent VLDLs are exported to the Golgi which is a physiologically regulatable event. We have previously shown that a unique ER-derived vesicle, the VTV (VLDL-transport vesicle), mediates the targeted delivery of VLDL to the Golgi lumen. Because VTVs are different from other ER-derived transport vesicles in their morphology and biochemical composition, we speculated that a distinct set of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins would form a SNARE complex which would eventually facilitate the docking/fusion of VTVs with Golgi. Our results show that Sec22b is concentrated in VTVs as compared with the ER. Electron microscopic results show that Sec22b co-localizes with p58 and Sar1 on the VTV surface. Pre-treatment of VTV with antibodies against Sec22b inhibited VTV-Golgi fusion, indicating its role as a v-SNARE (vesicle SNARE). To isolate the SNARE complex, we developed an in vitro docking assay in which VTVs were allowed to dock with the Golgi, but fusion was prevented to stabilize the SNARE complex. After the docking reaction, VTV-Golgi complexes were collected, solubilized in 2% Triton X-100 and the SNARE complex was co-immunoprecipitated using anti-Sec22b or GOS28 antibodies. A approximately 110 kDa complex was identified in non-boiled samples that was dissociated upon boiling. The components of the complex were identified as Sec22b, syntaxin 5, rBet1 and GOS28. Antibodies against each SNARE component significantly inhibited VTV-Golgi fusion. We conclude that the SNARE complex required for VTV-Golgi fusion is composed of Sec22b, syntaxin 5, rBet1 and GOS28.