Regulation of Intestinal Lipid Transport

描述（由申请人提供）：肠道的主要功能是吸收膳食脂肪，并通过肠道特异性中性脂质转运载体和乳糜微粒将其输出。我们实验室一直致力于确定这一过程中的限速步骤。我们已经确定脂质从内质网（ER）的出口是限制步骤。进一步的研究表明，我们称之为乳糜微粒前转运囊泡（PCTV）的囊泡是乳糜微粒从内质网输出并特异性进入肠道高尔基体的机制。肝脏和肾脏高尔基体不能接受pctv -乳糜微粒。最近的工作表明，参与从内质网出芽的PCTV的蛋白质不是涂层蛋白（COPII），而是一组待发现的蛋白质，这是本应用的目的之一。相比之下，我们发现COPII蛋白位于将新合成的蛋白质运输到高尔基体的囊泡表面，并且它们是蛋白质囊泡出芽所必需的。令人惊讶的是，当其中一种COPII蛋白Sar1被免疫耗尽时，PCTV的输出增加，而蛋白囊泡的形成被完全阻断，这表明两种囊泡途径之间可能存在对共同资源的竞争。我们提出这种资源是磷脂，特别是磷脂酰胆碱，并设计了实验来验证这一假设，作为应用的另一个目的。最后，我们发现尽管PCTV出芽不需要COPII蛋白，但它们位于PC-IV表面，具有与高尔基体融合的重要结合蛋白的推测功能。我们已经确定了几种出芽所需的蛋白质，其中一些尚未在内质网到高尔基囊泡运输中描述。这些蛋白质如何发挥作用以及是什么赋予肠道高尔基体特异性是本研究的另一个目的。

DESCRIPTION (provided by applicant): 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.