Charles M. Mansbach

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.

Overexpression of Apolipoprotein A-IV Enhances Lipid Secretion in IPEC-1 Cells by Increasing Chylomicron Size

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a “pig-like” human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a “chicken-like” apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

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.

Phospholipases: Old Enzymes With New Meaning

Phospholipases are enzymes that hydrolyze specific portions of phospholipid molecules. Their role in the digestion of exogenous phospholipids and as the active principle in snake and bee venoms has long been appreciated. Interest has increased in phospholipases recently because of new data implicating them in the inflammatory response. The ability of phospholipases to hydrolyze bacterial phospholipids has also received considerable attention. These new data have brought pertinence to studies of the physicochemical nature of potential substrates that greatly influence enzyme activity. Interest in the regulation of enzyme activity, both by physiological and pharmacological means, has increased as the importance of the phospholipases in response to various stimuli has become better appreciated. Finally, considerable interest has focused on the role of the phospholipases in response to hormones in a variety of cell systems. Data pertinent to all of these areas of interest will be discussed in this review with a view toward stimulating those with an interest in gastrointestinal physiology to apply them to their own areas of research in the gastrointestinal tract or liver.

Intestinal caveolin-1 is important for dietary fatty acid absorption.

How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound (3)H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the (3)H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the (3)H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with (3)H-oleate or (3)H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed (3)H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.