This proposal should provide information, in molecular terms, about the mechanism(s) by which proteins are directed to various cellular locations. Since the process of protein localization appears to have been conserved in the evolution of cells of all species, E. coli can be employed as a model system. Then availability of sophisticated genetic methods, such as gene fusion, and the ease with which recombinant DNA technology can be applied make this problem particularly amenable to study in this organism. Studies will focus on proteins destined for the outer membrane; in particular the export of the major outer membrane protein LamB (the receptor for bacteriophage Lambda). Using lamB-lacZ (specifies the cytoplasmic enzyme Beta-galactosidase) gene fusions, four intragenic export signals, each less than or equal to 75 base pairs, have been identified. To determine precisely the molecular function of each of these signals a systematic mutational analysis is proposed. The effect of these mutations on expression will be determined using various lamB-lacZ fusion strains. The effect on localization will be determined by recombining the various mutations into an otherwise wild-type lamB gene and quantitating LamB export. To identify components of the cellular export machinery, a number of mutant strains in which the export process is altered will be isolated and characterized. These mutants provide experimental tools for the identification of important gene products. The long-term goal is to examine the effects of the mutations described above biochemically using an in vitro protein translocation system. A thorough understanding of the mechanism(s) of protein localization could provide insights into certain genetic disorders (I-cell disease), suggest new sites of action for antibiotics or antiproliferative agents. Moreover, it may provide a general means to simplify commercial production of medically important proteins.
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