Fate of Cleaved Signal Peptides of the Endoplasmic Reticulum

蛋白质跨特定细胞膜的转运机制是细胞生物学的核心问题。这个项目的重点是疏水信号序列，它指导分泌蛋白通过内质网（ER）膜的转移，这是细胞分泌蛋白质的第一步。信号序列的重要性已经得到证实，但是它们的命运，一旦被内质网膜蛋白信号肽酶的作用从前体蛋白中移除，是未知的。实验的目的是研究含有功能信号序列的不同多肽的蛋白水解，以确定非洲爪蟾卵母细胞中拟议的信号肽水解酶的活性。将观察工程和未修饰的裂解信号肽的细胞内命运，并对其降解特性进行表征。这些研究结果将用于开发非洲爪蟾卵母细胞信号肽水解酶的工作试验。这样的实验对于鉴定和纯化特异性代谢裂解信号序列的蛋白质是必不可少的。这些蛋白可能不仅对细胞信号序列的清除很重要，而且在跨内质网膜易位的实际机制中也很重要。该技术将用于跟踪非洲爪蟾卵母细胞中裂解信号肽的命运，可能为解剖活细胞中蛋白质跨内质网膜转移的离散步骤提供有力的工具。真核细胞中合成的蛋白质穿过内质网膜的转移是蛋白质运输到各种目的地（包括溶酶体和细胞外空间（分泌））的关键第一步。我们对这一基本过程的理解的进步不仅对细胞功能的一般理解很重要，而且对生物技术也很重要。这个项目解决了被切割的信号序列的命运问题，也就是说，新合成的蛋白质片段需要通过内质网膜运输，但它不是“完成”蛋白质产物的一部分，因此在转移完成后很快就被酶从蛋白质的其余部分中切割出来。这是细胞生物学的一个领域，尽管在分泌过程的其他方面进行了高水平的研究活动，但很少受到关注。很有可能从工作中产生不可预见的新见解和潜在的重要见解。

The mechanism of protein transport across specific intracellular membranes is a central issue in cell biology. This project focuses on the hydrophobic signal sequences which direct the transfer of secretory proteins across the endoplasmic reticulum (ER) membrane, the first step in the secretion of proteins from the cell. The importance of signal sequences has been well-established, but their fate, once removed from precursor proteins by the action of the ER membrane protein signal peptidase, is unknown. The objective of the experiments to be carried out is to investigate the proteolysis of different polypeptides containing functional signal sequences in an effort to define a proposed signal peptide hydrolase activity in Xenopus laevis oocytes. The intracellular fates of both engineered and unmodified cleaved signal peptides will be observed, and the properties of their degradation will be characterized. The results of these investigations will be used to develop a working assay for signal peptide hydrolases in Xenopus oocytes. Such an assay is essential to efforts to identify and purify the proteins which specifically metabolize cleaved signal sequences. These proteins may be important not only for clearance of signal sequences from cells, but also in the actual mechanism of translocation across the ER membrane. The technique which will be employed to follow the fate of cleaved signal peptides in Xenopus oocytes may provide a powerful tool for the dissection of discrete steps in the transfer of proteins across the ER membrane in living cells. %%% The transfer of proteins across the ER membrane as they are synthesized in eukaryotic cells is a critical first step in the trafficking of proteins to a variety of destinations, including lysosomes and the extracellular space (secretion). Advances in our understanding of this fundamental process are important not only for general understanding of how cells function, but also for biotechnology. This project addresses the question of the fate of the cleaved signal sequence, that is, the piece of the newly- synthesized protein which is required for transport across the ER membrane, but which is not part of the "finished" protein product and is therefore enzymatically cleaved from the rest of the protein very soon after the transfer is effected. This is an area of cell biology which has received very little attention, despite the high level of research activity on other aspects of the secretory process. There is a good likelihood that unforeseen new and potentially important insights will emerge from the work.