Membrane lipoproteins with covalently linked lipids are ubiquitous components of biological membrane. In both gram-negative and gram-positive bacteria, an increasing number of membrane proteins have been found to contain glyceride-cysteine. Our long-term goal is to undertand the structures, functions and mode of assembly of membrane lipoproteins in bacteria. Recent studies have revealed that the export of lipoproteins in bacteria shares common step(s) in the initiation of the export process. Later in this process, the consensus tetrapeptide sequence of Leu-Ala-Gly-Cys which is present at the cleavage site of prolipoproteins, provides a unique recognition site for prolipoprotein modification and processing enzymes. Processing of prolipoprotein by prolipoprotein signal peptidase (SPase II) requires prior modification of prolipoprotein with glyceride and is specifically inhibited by globomycin. We propose to continue our biochemical and genetic studies of lipoprotein biogenesis in both gram-negative and gram-positive bacteria. Our goals will be (1) to identify prolipoprotein modification enzymes, to isolate mutants defective in the activities of these enzymes and to clone the structural genes encoding these enzymes; (2) to elucidate the mechanism of regulation of the expression of the x-ileS-lsp operon in E. coli; and (3) to clone 1sp gene (and other structural genes in this pathways) from B. subtilis or other bacteria in order to gain further insights into the structures of SPase II and genomic organization of 1sp (and ileS) in other bacteria. Techniques to be employed include microbial genetics, recombinant DNA technology, membrane biochemistry and bacterial physiological studies. We will continue to use Braun's lipoprotein in E. coli, and B. licheniformis penicillinase in B. subtilis as our model systems. We will also compare protein secretion in general with lipoprotein export to further define divergence of these two pathways from a common initiation of the export process.
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