Targeting of Proteins for Export in Escherichia coli

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DESCRIPTION (provided by applicant): The Sec-dependent protein export pathway of Escherichia coli is responsible for translocation of secretory proteins across the inner membrane to final destinations in the outer membrane or periplasmic space. SecA, SecE, and SecY are the essential subunits of translocase, and homologs of SecE and SecY have been identified in other eubacteria, archae, yeast and mammals. Further, the mechanisms of the translocation process seem to be conserved across species. Therefore, information gained from continued studies of the E. coli translocation pathway will be directly applicable to a greater understanding of targeting of proteins to the endoplasmic reticulum. Secretory proteins are synthesized in precursor form with an amino terminal signal sequence that is cleaved upon traversal of the inner membrane. Despite the central role of the signal sequence, it has been shown that it is neither necessary nor sufficient to direct export of a secretory protein. Previous results in this lab and others demonstrate that secretory proteins contain information in addition to the signal sequence that contributes to recognition by the Sec proteins. This hypothesis is based on the following data: deletion of the signal sequence significantly reduces export of a secretory protein, but does not completely abolish export; addition of a signal sequence to a non-secretory protein is not necessarily sufficient to target that protein for export; and mutant secretory proteins that completely lack a signal sequence are exported quite efficiently in a Prl suppressor strain. We propose to (1) examine the role of folding in protein export through a genetic screen that will identify slow-folding cytoplasmic proteins that are exported in a Prl suppressor strain, and (2) select for cytoplasmic localization of a secreted protein by removal of targeting information. The proposed studies will contribute to the general understanding of the normal export process and allow us to define the characteristics of a secretory protein that target it to the export pathway.