Coordinating different protein translocation machineries during assembly of a membrane protein

在膜蛋白组装过程中协调不同的蛋白质易位机制

• 批准号: BB/L000768/1
• 负责人: Tracy Palmer
• 金额: $ 51.77万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL000768%2F1
• 关键词: Coordinating; different; protein; translocation; machineries

All cells are surrounded by lipid membranes. However, in order to allow the passage of important metabolites and other substances biological membranes contain proteins. Proteins located in the lipid membrane are unusual because they are mainly hydrophobic (water-hating) unlike other proteins which are hydrophilic (water-loving). This presents a problem for the cell because the hydrophobic membrane proteins must be inserted into the membrane from the aqueous cytoplasm where they are made. If greasy membrane proteins are allowed to accumulate in the cytoplasm they aggregate, causing cell stress and ultimately cell death. Therefore membrane proteins are usually made co-translationally. This means that the hydrophobic segments of proteins are never exposed to the cytoplasm but are effectively threaded into the membrane as they are synthesised by the ribosome. A transmembrane channel in a specialised membrane protein complex called Sec receives the unfolded hydrophobic segments and releases them sideways into the membrane where they interact with lipids and sometimes with another protein called YidC. Once in the lipid bilayer the membrane protein will fold into its final, active conformation.We recently reported a very important exception whilst examining the mechanism of membrane insertion of the Rieske protein from Streptomyces coelicolor. Rieske proteins are found in almost all organisms and are very important because they help to transfer electrons during respiration or photosynthesis. The electron transfer properties of the Rieske protein are due to the presence of an iron sulphur cluster, which is an iron ion, held in place by inorganic sulphur, and ultimately co-ordinated in a non-covalent manner to cysteine residues in the protein. Normally Rieske proteins in bacteria are synthesised in the cytoplasm, they bind their iron-sulphur cluster, attain their fully folded conformation and are moved across the lipid bilayer through a large channel called Tat which is big enough for folded proteins to pass through. The Rieske protein is guided to the Tat channel by a short, moderately hydrophobic signal sequence at its N-terminus which ultimately anchors the Rieske protein into the membrane, with its large folded domain containing the iron sulphur cluster facing the opposite side. We noted that the Rieske protein from Streptomyces coelicolor and closely related bacteria was much larger and more hydrophobic than expected and anticipated that this might alter the route of membrane insertion. Indeed we were able to show that the first half of the protein required Sec for its membrane integration, but that the remaining hydrophobic sequence and the folded iron sulphur cluster-containing domain required the Tat pathway. This was unexpected because the Sec machinery does not normally release the ribosome during co-translational insertion of membrane proteins, but must do so in this case to allow the remainder of the protein to be synthesised at the cytoplasmic side of the membrane, the iron sulphur cluster to be bound and the protein to fold. This project aims to build on our previous discovery to understand just how the Sec and Tat machineries coordinate to assemble a single membrane protein. To this end we will screen for mutants in which the Sec pathway can no longer release the Rieske protein. We will use similar approaches to select mutants that can no longer allow the protein to be recognised by the Tat pathway. Although our initial studies identified the Rieske protein as a dual targeted membrane protein, we have now analysed the genome sequences of many more bacteria and archaea and have identified new candidates for dual targeting. We will also analyse two of these novel membrane proteins and confirm the route/s by which they are targeted to the membrane. We anticipate that our results will firmly establish that dual targeting is a common feature found in many bacteria and archaea.

所有细胞都被脂质膜包围。然而，为了允许重要的代谢物和其他物质通过，生物膜含有蛋白质。位于脂质膜中的蛋白质是不寻常的，因为它们主要是疏水性的（憎水），不像其他蛋白质是亲水性的（亲水）。这给细胞带来了一个问题，因为疏水膜蛋白必须从它们产生的含水细胞质插入膜中。如果允许脂膜蛋白在细胞质中积累，它们就会聚集，导致细胞应激并最终导致细胞死亡。因此，膜蛋白通常是协同合成的。这意味着蛋白质的疏水片段从不暴露于细胞质，而是在核糖体合成时有效地穿入膜中。一种名为Sec的特殊膜蛋白复合物中的跨膜通道接收未折叠的疏水片段，并将它们侧向释放到膜中，在那里它们与脂质相互作用，有时与另一种名为YidC的蛋白质相互作用。一旦在脂质双分子层的膜蛋白将折叠成其最终的，活性conformation.We最近报道了一个非常重要的例外，同时检查膜插入的机制从天蓝色链霉菌的Rieske蛋白。Rieske蛋白几乎存在于所有生物体中，非常重要，因为它们有助于在呼吸或光合作用期间转移电子。Rieske蛋白的电子转移性质是由于存在铁硫簇，其是铁离子，通过无机硫保持在适当位置，并最终以非共价方式与蛋白质中的半胱氨酸残基配位。通常，细菌中的Rieske蛋白质在细胞质中合成，它们结合铁硫簇，获得完全折叠的构象，并通过称为达特的大通道穿过脂质双层，该通道足够大以供折叠的蛋白质通过。Rieske蛋白通过其N-末端的短的、适度疏水的信号序列被引导至达特通道，该信号序列最终将Rieske蛋白锚定到膜中，其含有铁硫簇的大折叠结构域朝向相反侧。我们注意到天蓝色链霉菌和密切相关的细菌的Rieske蛋白比预期的要大得多，疏水性更强，并预计这可能会改变膜插入的途径。事实上，我们能够表明，蛋白质的前半部分需要Sec进行膜整合，但其余的疏水序列和折叠的含铁硫簇结构域需要达特途径。这是出乎意料的，因为Sec机制在膜蛋白的共翻译插入期间通常不释放核糖体，但在这种情况下必须这样做以允许蛋白质的剩余部分在膜的细胞质侧合成，铁硫簇被结合并且蛋白质折叠。这个项目的目的是建立在我们以前的发现，以了解SEC和达特机制如何协调组装一个单一的膜蛋白。为此，我们将筛选Sec途径不再释放Rieske蛋白的突变体。我们将使用类似的方法来选择突变体，这些突变体不再允许蛋白质被达特途径识别。尽管我们最初的研究将Rieske蛋白确定为双重靶向膜蛋白，但我们现在已经分析了更多细菌和古细菌的基因组序列，并确定了双重靶向的新候选蛋白。我们还将分析这些新的膜蛋白中的两种，并确认它们靶向膜的途径。我们预计，我们的研究结果将坚定地确立双重靶向是许多细菌和古细菌中的共同特征。

项目成果

Spotlight onTracy Palmer.

聚焦特雷西·帕尔默。

• DOI: 10.1093/femsle/fnw271
• 发表时间: 2016
• 期刊: FEMS microbiology letters
• 影响因子: 2.1
• 作者: Palmer T

Ferric Citrate Regulator FecR Is Translocated across the Bacterial Inner Membrane via a Unique Twin-Arginine Transport-Dependent Mechanism.

柠檬酸铁调节剂 FecR 通过独特的双精氨酸运输依赖机制跨细菌内膜转运。

• DOI: 10.1128/jb.00541-19
• 发表时间: 2020
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Passmore IJ