Rustaeus S, Stillemark P, Lindberg K, Gordon D, Olofsson S O
Department of Medical Biochemistry and the Wallenberg Laboratory, University of Göteborg, Sweden and the Division of Metabolic Diseases, Bristol-Myers Squibb Co., Princeton, New Jersey 08543, USA.
J Biol Chem. 1998 Feb 27;273(9):5196-203. doi: 10.1074/jbc.273.9.5196.
In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded. Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium. These results indicate that the membrane-associated apoB-100 can be converted to VLDL. A new method was developed by which the major amount (88%) of microsomal apoB-100 but not integral membrane proteins could be extracted. The major effect of this method was to increase the recovery of apoB-100 that banded in the LDL and HDL density regions, suggesting that the membrane-associated form of apoB-100 is partially lipidated. We also investigated the role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable MTP inhibitor (BMS-192951). This compound strongly inhibited the assembly and secretion of apoB-100 VLDL when present during the translation of the protein. To investigate the importance of MTP during the later stages in the assembly process, the cells were preincubated with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and pulse-labeled (+BFA) and chased (+BFA) for 30 min to obtain full-length apoB-100 associated with the microsomal membrane. Inhibition of MTP after the 30-min chase blocked assembly of VLDL. This indicates that MTP is important for the conversion of full-length apoB-100 into VLDL. Results from experiments in which a second chase (-BFA) was introduced before the inactivation of MTP indicated that only early events in this conversion of full-length apoB-100 into VLDL were blocked by the MTP inhibitor. Together these results indicate that there is a MTP-dependent "window" in the VLDL assembly process that occurs after the completion of apoB-100 but before the major amount of lipids is added to the VLDL particle. Thus the assembly of apoB-100 VLDL from membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase, during which the major amount of lipid is added.
在那些脂蛋白组装过程已被布雷菲德菌素A（BFA）灭活的细胞中，与膜相关的载脂蛋白B-100消失了，既没有形成脂蛋白也没有被分泌，这表明它被降解了。在没有BFA的情况下对细胞进行追踪，从而使组装过程重新激活，结果培养基中极低密度脂蛋白（VLDL）部分的与膜相关的载脂蛋白B-100实现了定量恢复。这些结果表明，与膜相关的载脂蛋白B-100可以转化为VLDL。开发了一种新方法，通过该方法可以提取大部分（88%）的微粒体载脂蛋白B-100，但不能提取整合膜蛋白。该方法的主要作用是提高在低密度脂蛋白（LDL）和高密度脂蛋白（HDL）密度区域中条带化的载脂蛋白B-100的回收率，这表明与膜相关形式的载脂蛋白B-100部分被脂化。我们还使用可光活化的微粒体甘油三酯转移蛋白（MTP）抑制剂（BMS-192951）研究了微粒体甘油三酯转移蛋白（MTP）在载脂蛋白B-100 VLDL组装中的作用。当该化合物在蛋白质翻译期间存在时，它强烈抑制载脂蛋白B-100 VLDL的组装和分泌。为了研究MTP在组装过程后期阶段的重要性，将细胞用BFA预孵育（以可逆地抑制载脂蛋白B-100 VLDL的组装），进行脉冲标记（+BFA）并追踪（+BFA）30分钟，以获得与微粒体膜相关的全长载脂蛋白B-100。在30分钟的追踪后抑制MTP会阻断VLDL的组装。这表明MTP对于全长载脂蛋白B-100转化为VLDL很重要。在MTP失活之前引入第二次追踪（-BFA）的实验结果表明，MTP抑制剂仅阻断了全长载脂蛋白B-100转化为VLDL过程中的早期事件。这些结果共同表明，在VLDL组装过程中存在一个MTP依赖性的“窗口”，该窗口发生在载脂蛋白B-100完成后但在大量脂质添加到VLDL颗粒之前。因此，从与膜相关的载脂蛋白B-100组装载脂蛋白B-100 VLDL涉及一个早期的MTP依赖性阶段和一个后期的MTP非依赖性阶段，在后期阶段添加大量脂质。
