Coordinating different protein translocation machineries during assembly of a membrane protein

所有的细胞都被脂质膜包围着。然而，为了让重要的代谢物和其他物质通过，生物膜中含有蛋白质。位于脂质膜上的蛋白质是不寻常的，因为它们主要是疏水的（憎水），而不像其他蛋白质是亲水的（亲水）。这给细胞带来了一个问题，因为疏水膜蛋白必须从制造它们的含水细胞质插入到膜中。如果允许脂膜蛋白在细胞质中积聚，它们就会聚集，造成细胞压力并最终导致细胞死亡。因此，膜蛋白通常是共翻译合成的。这意味着蛋白质的疏水片段从未暴露在细胞质中，而是在由核糖体合成时有效地插入膜中。一种叫做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，有时与另一种叫做YidC的蛋白质相互作用。一旦进入脂质双分子层，膜蛋白就会折叠成最终的活性构象。我们最近报道了一个非常重要的例外，同时研究了来自色链霉菌的Rieske蛋白的膜插入机制。里斯克蛋白几乎存在于所有生物体中，它非常重要，因为它有助于在呼吸或光合作用过程中转移电子。Rieske蛋白的电子转移特性是由于铁硫簇的存在，这是一种铁离子，由无机硫保持在原位，最终以非共价方式与蛋白质中的半胱氨酸残基配位。正常情况下，细菌中的Rieske蛋白是在细胞质中合成的，它们结合铁硫簇，形成完全折叠的构象，并通过一个叫做Tat的大通道穿过脂质双分子层，这个通道大到足以让折叠的蛋白质通过。Rieske蛋白通过其n端一个短的、适度疏水的信号序列引导到Tat通道，最终将Rieske蛋白固定在膜上，其大的折叠结构域包含面向相反一侧的铁硫簇。我们注意到，来自色链霉菌及其密切相关的细菌的Rieske蛋白比预期的要大得多，更疏水，并预计这可能会改变膜插入的途径。事实上，我们能够证明蛋白质的前半部分需要Sec来实现其膜整合，但剩余的疏水序列和折叠的含铁硫簇结构域需要Tat途径。这是出乎意料的，因为Sec机制通常不会在膜蛋白的共翻译插入过程中释放核糖体，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，蛋白质折叠。这个项目的目的是在我们之前的发现的基础上，了解Sec和Tat机器是如何协调组装单个膜蛋白的。为此，我们将筛选Sec通路不能再释放Rieske蛋白的突变体。我们将使用类似的方法来选择不再允许蛋白质被Tat途径识别的突变体。虽然我们最初的研究发现Rieske蛋白是一种双重靶向膜蛋白，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物。我们还将分析其中两种新的膜蛋白，并确认它们靶向膜的途径。我们预计，我们的结果将坚定地确立双重靶向是在许多细菌和古细菌中发现的共同特征。

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.