LIPOPROTEIN--MEMBRANE INSERTION/MODIFICATION/PROCESSING

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The overall objective of this project is to understand the molecular details of the biosynthesis and export of prolipoproteins. This process involves prior modification with glycerol and fatty acids before processing of the lipid-modified prolipoprotein by specific lipoprotein signal peptidase. The understanding of the mechanism of protein secretion is important not only because many enzymes, hormones, toxins, antibodies are secreted but also because of the recent advent of recombinant DNA technology of making medically important proteins. Many lipoproteins are also important in human metabolism, e.g. low- density and high-density lipoproteins. The overall process of prolipoprotein translocation will be studied in terms of its component steps: insertion, modification, and processing. The requirements and the specificity of each step will be examined by a combination of genetic manipulation and biochemical studies in an in vitro translocation system with Escherichia coli inverted cytoplasmic membrane vesicles. The structural requirements for the insertion of prolipoprotein will employ mutants with altered signal sequence and mature region, and the lipid specificity for the insertion will be examined with liposomes formed with synthetic lipids. The roles of ATP hydrolysis, cytoplasmic soluble factors, and membrane proteins in modification and in processing, and the importance of precursor competency for translocation will be assessed with purified components for each distinct step. Chemical crosslinking with bifunctional reagents, followed by immunoprecipitation and partial reconstitution with membrane vesicles and liposomes, will be used to identify the components involved. Finally, non- lipoprotein precursors with minimal structural change from corresponding prolipoproteins will be compared to test the hypothesis that spontaneous insertion into membrane lipids is the first step of protein translocation.