The mechanism of SecA-dependent substrate recognition and delivery in Escherichia coli

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The ultimate goal of our research is to understand how proteins are transported across the cytoplasmic membrane (CM) in bacteria starting with synthesis of a protein in the cytoplasm and ending with its maturation on the other side of the CM.The CM plays a critical role in the definition of a cell and thus our definition of life. Cells are sometimes described as "bags of proteins", in which the CM is the "bag". It divides the contents of the cell (the cytoplasm) from the extracellular milieu, and it protects the cell from changes in the environment. Without the bag, there is no cell. However, the CM also poses a fundamental problem. A large number of proteins carry out (often essential) functions outside the cell. In the bacterium Escherichia coli, these proteins comprise ~30% of all proteins synthesized. Protein synthesis occurs exclusively in the cytoplasm, but proteins cannot cross the CM on their own. Thus, the cell has evolved a number of complex machineries to transport proteins across the CM. Of these, the Sec machinery is responsible for the vast majority of protein export. The central component of the Sec machinery is a universally conserved channel in the CM. In bacteria, most proteins are transported through this channel by a pump protein, SecA, only after they have been fully (or nearly fully) synthesized. Recognition of protein substrates by the Sec machinery is rapid, efficient, and very accurate, but despite decades of research, the process by which substrate proteins are recognized remains an unsolved problem. However, recent research by DH sheds new light on this old problem. This research suggests that SecA itself recognizes substrate proteins, and recognition occurs as the substrate protein is still being made-long before transport across the CM commences. The research described in this proposal aims to understand: (i) how substrate proteins are recognized by SecA, (ii) how substrate proteins are subsequently delivered to the channel in the CM, and (iii) how the timing of recognition and delivery affects the subsequent maturation of the substrate protein on the other side of the CM.Previous research in this area has been directly applied in the development of new tools for biotechnology. For example, during his PhD research, DH identified a set of signals that could target proteins to be transported across the CM by a second parallel pathway that is more efficient than the SecA-dependent pathway, and these signals have been widely used to secrete proteins that are otherwise refractory to protein transport. However, export by this parallel pathway has a number of significant limitations, including low protein expression levels, cellular toxicity, and in some cases, defective protein maturation. Greater insight into the mechanism of SecA-dependent transport could lead to improved methods for protein production.This research also has the potential to contribute to new approaches for combating bacterial infections. Some bacteria such as E. coli contain a second membrane outside the cell that serves as a barrier to many antimicrobial compounds, and the proper maturation of transported proteins is important for maintaining the integrity this barrier. Thus, the strong link between route by which proteins are delivered to the channel in the CM and the maturation of the transported protein could be exploited to increase the sensitivity of these bacteria to many otherwise useless antimicrobial compounds.Finally, many of the techniques developed in this grant can be adapted for use in other studies. For example, the high-throughput sequencing (HTS) methods developed to examine the structure of SecA-ribosome complex can be used to examine the interaction of other proteins with other large RNA or DNA molecules, and the use of HTS to identify novel pathways involved in OM biogenesis can be extended to research into other biological pathways.