Shahzad Siddiqi

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.

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.

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.

Sec24C is required for docking the prechylomicron transport vesicle with the Golgi

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.

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

Background: ATP is required to generate the pre-chylomicron transport vesicle from intestinal ER. Results: FABP1, part of a four protein complex, is split from the complex by phosphorylation of Sar1b enabling ER binding. Conclusion: Phosphorylation of Sar1b enables FABP1 to bind to the ER. Significance: Control of Sar1b phosphorylation controls chylomicron exit from the ER. 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.

Dietary and biliary phosphatidylcholine activates PKCζ in rat intestine

Chylomicron output by the intestine is proportional to intestinal phosphatidylcholine (PC) delivery. Using five different variations of PC delivery to the intestine, we found that lyso-phosphatidylcholine (lyso-PC), the absorbed form of PC, concentrations in the cytosol (0 to 0.45 nM) were proportional to the input rate. The activity of protein kinase C (PKC)ζ, which controls prechylomicron output rate by the endoplasmic reticulum (ER), correlated with the lyso-PC concentration suggesting that it may be a PKCζ activator. Using recombinant PKCζ, the Km for lyso-PC activation was 1.49 nM and the Vmax 1.12 nM, more than the maximal lyso-PC concentration in cytosol, 0.45 nM. Among the phospholipids and their lyso derivatives, lyso-PC was the most potent activator of PKCζ and the only one whose cytosolic concentration suggested that it could be a physiological activator because other phospholipid concentrations were negligible. PKCζ was on the surface of the dietary fatty acid transport vesicle, the caveolin-1-containing endocytic vesicle. Once activated, PKCζ, eluted off the vesicle. A conformational change in PKCζ on activation was suggested by limited proteolysis. We conclude that PKCζ on activation changes its conformation resulting in elution from its vesicle. The downstream effect of dietary PC is to activate PKCζ, resulting in greater chylomicron output by the ER.

Control of chylomicron export from the intestine

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.

181 VESICLE-ASSOCIATED MEMBRANE PROTEIN 7 IS CRUCIAL FOR LIPID ABSORPTION.

The rate-limiting step in lipid absorption is the exit of triacylglycerols (TAG) from the endoplasmic reticulum (ER) in a specialized transport vesicle, the prechylomicron transport vesicle (PCTV). SNARE proteins direct vesicles to target membranes. Newly synthesized proteins in vesicles constantly move to the cis Golgi. We questioned if intermittent, meal-derived PCTV contained a unique SNARE. Vesicle-associated membrane protein 7 (VAMP7), a SNARE protein previously found only in the post Golgi compartment, was identified by 2D gels in PCTV. We sought its presence in its parent, intestinal ER, and questioned its functionality in lipid transport. Methods We used our novel antirat VAMP7 antibody that identifies a 25 kDa protein on a 2D gel immunoblot (IB) to identify VAMP7. We isolated ER both by sucrose density and iodixanol gradients. Immunohistochemistry (IH) resolved by deconvolution was used to colocalize marker proteins with VAMP7 and immunoelectron microscopy (IEM) was used to colocalize VAMP7 to ER marker proteins on PCTV. Results Our intestinal, liver and kidney ER preparations were not contaminated by endosomes or Golgi as judged by the lack of rab11, syntaxin8, and GOS28 in the area of the gradient occupied by the ER proteins calreticulin and sec22. Intestinal ER but not liver or kidney ER contained VAMP7 by IB. ER-derived PCTV contained VAMP7 and the ER proteins Sar1 and rBet1 by IEM. IH showed VAMP7 to be colocalized with the ER protein PDI. To test the functionality of VAMP7 in TAG transport to the Golgi, we incubated intestinal ER with anti-VAMP7 antibody and observed its effect on delivery of ER-14 C-TAG and protein to the Golgi. Only 10% of the 14 C-TAG was transported to the Golgi on anti-VAMP7 antibody treatment as compared to preincubation of the ER with IgG. Newly synthesized 3 H-protein transport was unaffected by anti-VAMP7 antibody as was ER to Golgi TAG transport if the Golgi was preincubated with the antibody. In coimmunoprecipitation studies, VAMP7 was associated with apolipoprotein B48 (apoB48). VAMP7 does not bind to apoB48 in intestinal ER treated with proteinase K, which left a 170 kDa apoB48 fragment. Conclusions We have localized VAMP7 to intestinal ER by IH and IEM and by cell organelle separation techniques and shown that intestine but not liver or kidney ER contains VAMP7. We have also shown that VAMP7 in intestinal ER is functional in the movement of TAG in PCTV from ER to the Golgi. We speculate that VAMP7 binds to a cytosolic exposed portion of apoB48, which enables selection of the prechylomicron for inclusion in PCTV.