Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence.
Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence.
复制标题
脱辅基蛋白 B100 与易位子有长时间的相互作用，在此期间，其脂化和易位从依赖微粒体甘油三酯转移蛋白变为独立。
DOI：
10.1073/pnas.95.25.14733
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发表时间：
1998
影响因子：
11.1
通讯作者：
Fisher,EA
中科院分区：
文献类型：
--
作者：
Mitchell,DM;Zhou,M;Pariyarath,R;Wang,H;Aitchison,JD;Ginsberg,HN;Fisher,EA
当脂质合成在HepG 2细胞中受到限制时，载脂蛋白B100（apoB 100）不被分泌，而是通过泛素-蛋白酶体途径快速降解。为了进一步研究载脂蛋白B100的生物合成和分泌，在脂质合成、蛋白酶体活性和微粒体甘油三酯转移蛋白（MTP）脂质转移活性变化的条件下，研究了用于降解或脂蛋白组装的载脂蛋白B100的物理和功能状态。用蛋白酶体抑制剂预处理细胞（在整个实验过程中与细胞保持在一起），并放射性标记15 min。在追踪期间，标记的apoB 100仍与微粒体相关。此外，通过将sec 61 β与apoB 100交联，我们表明apoB 100保持接近易位子，同时可以检测到apoB 100-泛素缀合物。当向chase培养基中加入油酸（OA）刺激脂质合成和脂蛋白组装/分泌时，apoB 100被去泛素化，其与sec 61 β的相互作用被破坏，这意味着伴随着脂蛋白颗粒形成的易位完成。MTP参与apoB 100的转位和脂蛋白的组装。在OA的存在下，当MTP脂质转移活性在脉冲标记结束时被抑制时，apoB 100分泌被取消。相反，当标记的apoB 100在加入OA和抑制剂之前允许在细胞中积累60分钟时，apoB 100脂化和分泌不再受损。总的来说，这些数据意味着，在大多数与内质网的协会，apoB 100是接近或内的易位子，是可访问的泛素-蛋白酶体和脂蛋白组装途径。此外，MTP脂质转移活性似乎是必要的，只有早期易位和脂质化事件。
When lipid synthesis is limited in HepG2 cells, apoprotein B100 (apoB100) is not secreted but rapidly degraded by the ubiquitin-proteasome pathway. To investigate apoB100 biosynthesis and secretion further, the physical and functional states of apoB100 destined for either degradation or lipoprotein assembly were studied under conditions in which lipid synthesis, proteasomal activity, and microsomal triglyceride transfer protein (MTP) lipid-transfer activity were varied. Cells were pretreated with a proteasomal inhibitor (which remained with the cells throughout the experiment) and radiolabeled for 15 min. During the chase period, labeled apoB100 remained associated with the microsomes. Furthermore, by crosslinking sec61β to apoB100, we showed that apoB100 remained close to the translocon at the same time apoB100–ubiquitin conjugates could be detected. When lipid synthesis and lipoprotein assembly/secretion were stimulated by adding oleic acid (OA) to the chase medium, apoB100 was deubiquitinated, and its interaction with sec61β was disrupted, signifying completion of translocation concomitant with the formation of lipoprotein particles. MTP participates in apoB100 translocation and lipoprotein assembly. In the presence of OA, when MTP lipid-transfer activity was inhibited at the end of pulse labeling, apoB100 secretion was abolished. In contrast, when the labeled apoB100 was allowed to accumulate in the cell for 60 min before adding OA and the inhibitor, apoB100 lipidation and secretion were no longer impaired. Overall, the data imply that during most of its association with the endoplasmic reticulum, apoB100 is close to or within the translocon and is accessible to both the ubiquitin-proteasome and lipoprotein-assembly pathways. Furthermore, MTP lipid-transfer activity seems to be necessary only for early translocation and lipidation events.