Genetic studies on mechanisms of protein localization in Escherichia coli K-12.

In the last few years, several laboratories have demonstrated that many proteins (both from eukaryotic and prokaryotic organisms) that are destined to be localized in noncytoplasmic locations initially are synthesized as a precursor with a 15-30 amino acid extension at the NH2-terminal end of the molecule. This extra peptide has been termed the signal sequence, and it has been proposed that this signal plays a role in the localization of the extracytoplasmic protein. We are studying the process by which proteins are exported to the envelope region of Escherichia coli. Our work deals primarily with the outer membrane proteins, lambda receptor, the product of the lamB gene, and the major outer membrane (porin) proteins 1a and 1b, products of the ompF and ompC genes. Using techniques of gene fusion, we have demonstrated that information specifying the cellular location of the lambda receptor is contained within the lamB gene. Furthermore, we have shown that this information is capable of directing even a normally cytoplasmic protein, beta-galactosidase, to the outer membrane. Some of this information is contained within the signal sequence. Mutations that alter this sequence prevent export of the lambda receptor protein. Again using techniques of gene fusion, we have shown that the signal sequence alone is not sufficient to cause export of beta-galactosidase from the cytoplasm. Other information within the lamB gene is required. Selection procedures have been developed to isolate mutations that exhibit a general alteration in the export process. Genetic analysis of these mutations has provided evidence for the involvement of the ribosome in the process of protein localization. The structural genes for the porin proteins, 1a and 1b, are regulated at the transcriptional level by the ompB locus. This has permitted us to extend our studies on outer membrane protein localization to protein 1. With this genetic system, it should be possible to determine if E coli employs more than a single mechanism for the export of proteins to the outer membrane.