PROTEIN TRANSLOCATION IN BACTERIA AND HIGHER EUKARYOTES

我们的长期目标是通过以下方式了解分子机制 哪些分泌蛋白是靶向的，并在 在真核生物和原核生物中都有适当的细胞膜。我们的 一般的策略是专注于单一的蛋白质，麦芽糖- 大肠杆菌结合蛋白(MBP)。MBP的作用机制(S) 是从大肠杆菌输出的，具有很好的特性。详细比较了 这种蛋白在同源细菌系统中的移位和在 异源真核系统将揭示相似和不同 MBP在这两个系统中的移位机制。此外，当 在真核系统中表达的MBP将作为新的 蛋白质转位的机制。 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.研究MBP在哺乳动物内质网膜上的转运。 MBP可以通过一种潜在的新的方式在哺乳动物的微生物体之间转移 翻译后机制。我们将进一步描述 用生化方法研究MBP在哺乳动物无细胞系统中的移位 分级和重组以确定分子内相互作用 胞浆和细胞膜中的MBP和辅助蛋白之间。 2.确定特定信号阻止转位的步骤 序列突变体。我们将描述真核细胞的信号转运体 比较细菌信号序列功能的MBP序列突变体 和真核生物。已知选定的突变体是不完全易位的。 穿过细菌内膜，代表了一系列 易位中间体。 3.鉴定与MBP相互作用的细胞质和细胞膜蛋白 它在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 用来解剖出促进不同 MBP靶向和转位的步骤。分子和生化 将使用各种方法来表征交联剂分子。

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.