Coordinating different protein translocation machineries during assembly of a membrane protein

所有的细胞都被脂膜包围。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。位于脂膜中的蛋白质是不寻常的，因为它们主要是疏水的(憎水)，而不像其他亲水的蛋白质(喜欢水)。这给细胞带来了一个问题，因为疏水性膜蛋白必须从制造它们的水生细胞质插入膜中。如果允许油膜蛋白在细胞质中积累，它们会聚集在一起，导致细胞应激，最终导致细胞死亡。因此，膜蛋白通常是共翻译的。这意味着蛋白质的疏水片段永远不会暴露在细胞质中，而是在核糖体合成时有效地进入细胞膜。一种称为SEC的特殊膜蛋白复合体中的跨膜通道接收未折叠的疏水片段，并将它们侧面释放到膜中，在那里它们与脂质相互作用，有时还与另一种称为YidC的蛋白质相互作用。一旦进入脂质双层，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，同时研究了天蓝色链霉菌Rieske蛋白的膜插入机制。Rieske蛋白几乎存在于所有生物体中，而且非常重要，因为它们有助于在呼吸作用或光合作用中传递电子。Rieske蛋白质的电子转移性质是由于铁硫簇的存在，这是一种铁离子，被无机硫固定在适当的位置，并最终以非共价方式与蛋白质中的半胱氨酸残基配位。正常情况下，细菌中的Rieske蛋白是在细胞质中合成的，它们与铁硫簇结合，达到完全折叠的构象，并通过一条名为TAT的大通道跨脂双层移动，该通道足够大，可以让折叠的蛋白质通过。Rieske蛋白通过其N端的一个短的、中等疏水的信号序列被引导到TAT通道，最终将Rieske蛋白锚定到膜上，其大的折叠结构域包含面向对面的铁硫簇。我们注意到来自天蓝色链霉菌和密切相关细菌的Rieske蛋白比预期的要大得多，疏水性也更强，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一半需要SEC来进行膜整合，但其余的疏水序列和折叠的含铁硫簇的结构域需要TAT途径。这是意想不到的，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，以允许剩余的蛋白质在膜的细胞质一侧合成，铁硫簇被结合，蛋白质折叠。这个项目旨在以我们之前的发现为基础，了解SEC和TAT机制是如何协调组装单个膜蛋白的。为此，我们将筛选SEC途径不能再释放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确定Rieske蛋白是一种双靶向膜蛋白，但我们现在已经分析了更多细菌和古菌的基因组序列，并确定了双靶向的新候选者。我们还将分析其中两种新的膜蛋白，并确定它们靶向膜的途径/S。我们预计，我们的结果将坚定地确立，双重靶向是许多细菌和古菌的共同特征。

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.