Wu X, Zhou M, Huang L S, Wetterau J, Ginsberg H N
Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
J Biol Chem. 1996 Apr 26;271(17):10277-81. doi: 10.1074/jbc.271.17.10277.
Microsomal triglyceride (TG) transfer protein (MTP) is an endoplasmic reticulum lumenal protein consisting of a 97-kDa subunit and protein disulfide isomerase. It is believed that MTP delivers TG to nascent apoB molecules during the assembly of lipoprotein particles in the secretory pathway. Although in vitro studies have established the mechanism of TG transfer between donor and acceptor membranes, the mechanism of action of MTP in vivo remains unknown. The present studies were undertaken to examine whether or not the transfer of TG to nascent apoB in the endoplasmic reticulum involves the physical interaction between MTP and apoB. HepG2 cells were labeled with [3H]leucine, lysed in a nondenaturing homogenizing buffer, and immunoprecipitated with anti-MTP antiserum. We found that labeled apoB and protein disulfide isomerase were co-immunoprecipitated by this procedure. In addition, we were able to detect the 97-kDa subunit of MTP in these immunoprecipitates by immunoblot. The association of MTP and apoB, as assessed in pulse-labeled cells by co-immunoprecipitation, was transient; apoB was prominent on fluorgraphy at 10 min of chase but minimal thereafter. Oleic acid treatment, which protects apoB from rapid intracellular degradation by increasing TG availability, increased both the degree and the duration of association between MTP and apoB dramatically. Inhibition of TG synthesis by Triacsin D, on the other hand, significantly decreased the MTP-apoB binding. N-Acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor, which directly protects apoB from rapid intracellular degradation but does not affect TG synthesis, increased the interaction between MTP and apoB only slightly, although it did prolong it. Our results suggest that direct interaction between MTP and apoB occurs during the assembly of apoB-containing lipoproteins in HepG2 cells.
微粒体甘油三酯（TG）转运蛋白（MTP）是一种内质网腔蛋白，由一个97 kDa的亚基和蛋白二硫键异构酶组成。据信，MTP在分泌途径中脂蛋白颗粒组装过程中将TG传递给新生的载脂蛋白B（apoB）分子。尽管体外研究已经确立了供体膜和受体膜之间TG转移的机制，但MTP在体内的作用机制仍然未知。本研究旨在探讨内质网中TG向新生apoB的转移是否涉及MTP与apoB之间的物理相互作用。用[3H]亮氨酸标记HepG2细胞，在非变性匀浆缓冲液中裂解，并用抗MTP抗血清进行免疫沉淀。我们发现，通过该程序，标记的apoB和蛋白二硫键异构酶被共免疫沉淀。此外，我们能够通过免疫印迹在这些免疫沉淀物中检测到MTP的97 kDa亚基。通过共免疫沉淀在脉冲标记细胞中评估的MTP与apoB的结合是短暂的；在追踪10分钟时，apoB在荧光图上很明显，但此后最少。油酸处理通过增加TG的可用性来保护apoB免于快速的细胞内降解，显著增加了MTP与apoB之间结合的程度和持续时间。另一方面，用Triacsin D抑制TG合成显著降低了MTP-apoB的结合。N-乙酰-亮氨酰-亮氨酰-正亮氨酸，一种半胱氨酸蛋白酶抑制剂，它直接保护apoB免于快速的细胞内降解，但不影响TG合成，尽管确实延长了时间，但仅略微增加了MTP与apoB之间的相互作用。我们的结果表明，在HepG2细胞中含apoB脂蛋白组装过程中发生了MTP与apoB之间的直接相互作用。
