PROTEIN TRANSLOCATION IN BACTERIA AND HIGHER EUKARYOTES

我们的长期目标是了解分子机制， 哪些分泌蛋白是靶向的，并跨膜转运， 在真核生物和原核生物中都有合适的细胞质膜。我们 一般的策略是集中在单一的蛋白质，麦芽糖， 结合蛋白（MBP）。MBP的机制 从E.大肠杆菌的特性很好。详细比较 这种蛋白质在同源细菌系统中以及在 异源真核系统将揭示相似性和差异 在两个系统中MBP易位的机制。而且，当 在真核系统中表达，MBP将作为新的探针， 蛋白质易位的机制。 我们提出了三个具体的目标来探讨MBP的机制 原核生物和真核生物中的易位： 1.表征MBP跨哺乳动物ER膜的易位。 MBP可以通过一种潜在的新的 翻译后机制我们将进一步描述 MBP在哺乳动物无细胞系统中的转运， 分离和重组以确定分子内相互作用 MBP与细胞质和细胞膜中的辅助蛋白之间的相互作用。 2.确定特定信号阻断易位的步骤 序列突变体我们将描述真核细胞的信号易位 MBP的序列突变体以比较细菌中的信号序列功能 和真核生物已知选定的突变体不完全易位 穿过细菌内膜，代表了一系列 易位中间体 3.鉴定与MBP相互作用的细胞溶质和膜蛋白， 在其穿过内质网和细菌内部转运的不同阶段， 膜。野生型和突变型MBP的化学交联将是 用来解剖出分子成分， MBP靶向和易位的步骤。分子和生化 方法将用于表征交联分子。

Our long term objectives are to understand the molecular mechanisms by which secretory proteins are targeted to, and translocated across, the appropriate cytoplasmic membrane in both eukaryotes and prokaryotes. Our general strategy is to concentrate on a single protein, the maltose- binding protein (MBP) from Escherichia coli. The mechanism(s) by which MBP is exported from E. coli is well-characterized. Detailed comparisons of the translocation of this protein in homologous bacterial systems and in heterologous eukaryotic systems will reveal similarities and differences in the mechanisms for MBP translocation in the two systems. Moreover, when expressed in eukaryotic systems, MBP will serve as a probe for novel mechanisms of protein translocation. We propose three specific aims to probe the mechanisms of MBP translocation in prokaryotes and eukaryotes: 1. Characterize the translocation of MBP across the mammalian ER membrane. MBP can be translocated across mammalian microsomes by a potentially novel post-translational mechanism. We will further characterize the translocation of MBP in mammalian cell-free systems using biochemical fractionation and reconstitution to define intramolecular interactions between MBP and accessory proteins in the cytosol and the membrane. 2. Determine steps at which translocation is blocked by specific signal sequence mutants. We will characterize eukaryotic translocation of signal sequence mutants of MBP to compare signal sequence function in bacteria and eukaryotes. Selected mutants are known to be incompletely translocated across the bacterial inner membrane and represent a series of translocation intermediates. 3. Identify cytosolic and membrane proteins which interact with MBP at different stages in its translocation across the ER and bacterial inner membranes. Chemical cross-linking of wild-type and mutant MBP will be employed to dissect out molecular components which facilitate different steps in MBP targeting and translocation. Molecular and biochemical approaches will be used to characterize cross-linking molecules.