Biomedical subjects
Publications and source records attributed to S D Emr.
The amino-terminal signal sequence is required for initiation of transmembrane protein transfer of the Escherichia coli lambda receptor protein. Mutations leading to insertion of charged amino acids into or deletion of amino acids from the hydrophobic segment of this sequence prevent export of this outer membrane protein.
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.
We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this problem particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid proteins produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided evidence that the molecular mechanism of cellular protein localization is strinkingly similar in both bacteria and animal cells.
Two mutant strains of Escherichia coli have been isolated in which the cellular location of an outer membrane protein, the phage lambda receptor (the lamB gene product), is altered. These mutations were initially selected in a strain containing a lamB-lacZ fusion. In the parent strain the protein coded for by the hybrid gene is located, at least in part, in the outer membrane. In the mutants it is located in the cytoplasm. The mutations responsible for the alteration of cellular location lie very early in the lamB gene, in a region corresponding to the NH2-terminus of the lambda receptor protein. One of these mutations is a small deletion internal to the lamB gene. When this mutation is present in an otherwise wild-type lamB gene, the protein produced is of lower molecular weight than normal receptor. The other mutation behaves as a point mutation; when it is present in an otherwise normal lamB gene, reversion can be demonstrated. The molecular weight of this mutant protein, which is located in the cytoplasm, is larger than that of the wild-type gene product by approximately 2000. It is suggested that these two mutations are in the portion of the lamB gene coding for a signal sequence and thereby block export of the protein.