Genetic approaches to structure-function analysis of the E. coli Tat translocon components

一些细菌蛋白质在细胞外部起作用，例如细菌病原体产生的毒素。由于所有的蛋白质都是在细菌内部制造的，因此胞外蛋白质必须穿过通常不可渗透的细胞膜移出细胞。这一任务是由位于细胞膜上的蛋白质转运蛋白完成的。一种类型的转运蛋白移动未折叠的蛋白质，将它们穿过细胞膜，就像穿过针眼的线一样。与此相反，第二种类型的转运蛋白，我们称之为达特系统，将折叠的蛋白质跨膜移动。在细菌E.在大肠杆菌中，达特转运蛋白是由TatA、TatB和TatC 3种蛋白质组成的大蛋白复合体。我们希望了解这些蛋白质在转运蛋白中的功能。为了做到这一点，我们希望分离TatA，TatB和TatC蛋白中阻止转运蛋白发挥作用的突变体。这些突变体将为我们提供有关达特蛋白分子重要部分的信息。我们已经在TatA、B和C蛋白中进行了突变，引入了一种称为半胱氨酸的氨基酸。半胱氨酸是一种特殊的氨基酸，因为如果它在某些条件下接近第二个半胱氨酸残基，它可以形成共价键。这种共价键锁定蛋白质并阻止它们移动。因此，我们可以检查TatA、B和C蛋白的部分，这些蛋白需要移动以使达特系统活跃。最后，我们认为TatA的一部分可能需要穿过细胞膜才能转运蛋白质。我们想通过在TatA蛋白上放置特殊的标签来测试这一点，并观察当达特系统运行时，标签是否出现或可以标记在膜的另一侧。

Some bacterial proteins operate on the outside of the cell, for example the toxins produced by bacterial pathogens. Since all proteins are made inside the bacterium the extracellular proteins must be moved out of the cell across the normally impermeable cell membrane. This task is carried out by machines termed protein transporters that are located in the cell membrane. One type of transporter moves unfolded proteins, threading them across the membrane like string through the eye of a needle. In contrast, a second type of transporter, which we term the Tat system, moves folded proteins across the membrane. In the bacterium E. coli, the Tat transporter is a large protein complex made up of 3 types of proteins, TatA, TatB and TatC. We wish to understand how each of these proteins functions in the transporter. To do that we want to isolate mutants in the TatA, TatB and TatC proteins that prevent the transporter from functioning. These mutants will give us information about important parts of the Tat protein molecules. We have made mutations throughout the TatA, B and C proteins that introduce an amino acid called cysteine. Cysteine is a special amino acid because if it comes close to a second cysteine residue under certain conditions it can form a covalent bond. This covalent bond locks the proteins and prevents them from moving. Therefore we can examine the parts of the TatA, B and C proteins that need to move in order to allow the Tat system to be active. Finally we believe that part of TatA may need to move across the membrane to allow proteins to be transported. We want to test this by putting special tags on the TatA protein and looking to see if the tag appears or can be labelled at the other side of the membrane when the Tat system is operating.