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.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.