Protein Export in Escherichia coli

University Of North Dakota Main Campus, Grand Forks ND

Investigators

Abstract

This research investigates a problem faced by all living organisms: how are proteins transported across membranes? These studies will use the model organism, Escherichia coli, which shares many aspects of the protein export process with higher organisms. In particular, the process of bacterial protein export is similar to that of eukaryotic endoplasmic reticulum targeting. The wealth of genetic and biochemical tools available make the use of E. coli ideal for these studies. This project combines bacterial genetics and biochemical assays to create novel approaches to study the protein export pathway. Most proteins are translocated across the cytoplasmic membrane of E. coli by the multisubunit Sec translocase. The essential components of the Sec system are SecA, SecE and SecY, while SecD, SecF and SecG enhance the efficiency of translocation. Export of a secretory protein requires sequential, productive, and seemingly complex interactions between the Sec proteins. The long-term goals of Dr. Flower's laboratory are to understand the functions of the Sec proteins and to elucidate the requirements for productive interactions amongst them. Undergraduate and graduate students will participate in the conduct of this research. Three specific goals will be pursued in this project. 1) The inter- and intra-molecular interactions between proteins of the translocation complex will be examined by genetic analyses, including synthetic lethality suppression and multiple mutation experiments. Novel mutations are expected from these experiments that will shed light on the interactions between SecE and SecY. 2) The mechanism of action of mutant forms of SecE and SecY, including new mutants isolated from goal 1, will be investigated using in vitro translocation and ATPase assays. The combination of new genetic screens and the biochemical assays provides a unique approach to understanding the functions of SecE and SecY. 3) The role of membrane phospholipids in protein export will be explored by examination of the effect of mutations in phospholipid biosynthesis pathways on export and by determination of phospholipid ratios in strains with specific export defects. Results obtained previously indicate that SecG function is related to membrane phospholipid content; this hypothesis will now be tested. These experiments will further our understanding of the formation and function of a complex multisubunit nanoscale biological machine, and will contribute to the training of the next generation of scientists and scientifically literate citizens.