A biochemical analysis of the constraints of tail-anchored protein biogenesis
A biochemical analysis of the constraints of tail-anchored protein biogenesis
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DOI：
10.1042/bj20101737
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发表时间：
2011-06-15
影响因子：
4.1
通讯作者：
High, Stephen
中科院分区：
文献类型：
--
作者：
Leznicki, Pawel;Warwicker, Jim;High, Stephen
TA（尾锚定）蛋白利用不同的生物合成途径，包括TRC 40（40 kDa的跨膜结构域识别复合物）介导的、伴侣依赖的和/或无辅助的途径到达ER（内质网）膜。我们已经解决了参与这些途径的胞质组分的灵活性，并探索了它们的膜插入的热力学约束，通过利用重组形式的Sec 61 β和Cytb 5（细胞色素B（5））携带共价修饰在其TA区域。在这两种情况下，有效的膜插入依赖于胞质因子能够容纳一个令人惊讶的范围内的共价修饰的TA区。对于Sec 61 β，我们发现SGTA（富含谷氨酰胺的小三角肽重复序列的蛋白α）和TRC 40都可以用单个聚乙二醇化的TA区域结合该底物。然而，通过引入两个PEG [聚（乙二醇）]部分，可以防止TRC 40结合，导致随后的膜整合的阻断。尽管TRC 40可以结合在不同位置处单聚乙二醇化的Sec 61 β多肽，但膜插入对PEG附着的精确位置更敏感。建模和实验表明，这种后TRC 40效应是由于将不同的PEG化TA区域插入脂质双层中的能量成本增加而导致的。因此，我们提出，通过TRC 40传递的TA蛋白的膜整合强烈依赖于基本的热力学，并推测它们的插入是通过磷脂介导的过程。
TA (tail-anchored) proteins utilize distinct biosynthetic pathways, including TRC40 (transmembrane domain recognition complex of 40 kDa)-mediated, chaperone-dependent and/or unassisted routes to the ER (endoplasmic reticulum) membrane. We have addressed the flexibility of cytosolic components participating in these pathways, and explored the thermodynamic constraints of their membrane insertion, by exploiting recombinant forms of Sec61 beta and Cytb5 (cytochrome b(5)) bearing covalent modifications within their TA region. In both cases, efficient membrane insertion relied on cytosolic factors capable of accommodating a surprising range of covalent modifications to the TA region. For Sec61 beta, we found that both SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) and TRC40 can bind this substrate with a singly PEGylated TA region. However, by introducing two PEG [poly(ethylene glycol)] moieties, TRC40 binding can be prevented, resulting in a block of subsequent membrane integration. Although TRC40 can bind Sec61 beta polypeptides singly PEGylated at different locations, membrane insertion is more sensitive to the precise location of PEG attachment. Modelling and experimentation indicate that this post-TRC40 effect results from an increased energetic cost of inserting different PEGylated TA regions into the lipid bilayer. We therefore propose that the membrane integration of TA proteins delivered via TRC40 is strongly dependent upon underlying thermodynamics, and speculate that their insertion is via a phospholipid-mediated process.