A major function of the intestine is to absorb dietary fat and to export it in the intestinally specific, neutral lipid transport vehicle, and the chylomicron. Our laboratory has been engaged in determining the rate limiting step in this process. We have identified the egress of lipid from the endoplasmic reticulum (ER) as the limiting step. Further studies have shown that a vesicle, which we have named the pre-chylomicron transport vesicle (PCTV), is the mechanism by which chylomicrons are exported from the ER and go specifically to the intestinal Golgi. Liver and kidney Golgi are not competent acceptors of PCTV-chylomicrons. Recent work has shown that the proteins involved in budding PCTV from the ER are not coatomer proteins (COPII) but rather a to be discovered group of proteins which is one of the Aims of the present application. By contrast, we found that COPII proteins were on the surface of vesicles that transported newly synthesized proteins to the Golgi and that they were required for protein vesicle budding. Surprisingly, when one of the COPII proteins, Sar1, was immuno-depleted, the output of PCTV increased whereas protein vesicle formation was completely blocked suggesting that there may be competition for a common resource between the two vesicle pathways. We propose that this resource is phospholipid, specifically phosphatidlylcholine, and have designed experiments to test this hypothesis as another Aim of the application. Finally, we have found that although COPII proteins were not required for PCTV budding, they are on the surface of PC-IV with a putative function of binding proteins important for fusion with the Golgi. We have identified several proteins that are required for budding and some of these have not been heretofore described in ER to Golgi vesicle transport. How these proteins function and what lends specificity to intestinal Golgi is another Aim of this proposal.
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