LIPOPROTEIN - MEMBRANE INSERTION/MODIFICATION/PROCESSING

本项目的总体目标是了解 生物合成和输出的分子细节 前脂蛋白 该过程涉及预先修改， 甘油和脂肪酸，然后加工脂质改性的 前脂蛋白通过特异性脂蛋白信号肽酶。 的 了解蛋白质分泌的机制很重要 不仅是因为许多酶、激素、毒素、抗体 也是因为最近出现的重组DNA 制造重要医学蛋白质的技术。 许多 脂蛋白在人体代谢中也很重要，例如低- 密度和高密度脂蛋白。 将研究前脂蛋白易位的整个过程 就其组成步骤而言：插入、修改和 处理. 每个步骤的要求和具体性将 通过基因操作和 体外易位系统中的生化研究， 大肠杆菌倒置胞质膜囊泡。 的 插入前脂蛋白的结构要求将 使用具有改变的信号序列和成熟区的突变体，和 插入的脂质特异性将用 由合成脂质形成的脂质体。 ATP的作用 水解，细胞质可溶性因子和膜蛋白， 改性和在加工过程中，以及前体的重要性 易位能力将用纯化的 每一步都有不同的组成部分。 化学交联， 双功能试剂，然后免疫沉淀， 用膜囊泡和脂质体部分重构， 用于识别所涉及的组件。 最后，非- 脂蛋白前体，其结构变化极小， 将比较相应的前脂蛋白，以测试 假设自发插入膜脂质是 蛋白质转运的第一步

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.