Fate of Cleaved Signal Peptides of the Endoplasmic Reticulum

内质网切割信号肽的命运

• 批准号: 9306875
• 负责人: Eve Perara
• 金额: $ 1.8万
• 依托单位: San Francisco State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-06-01 至 1995-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9306875&HistoricalAwards=false
• 关键词: Fate; Cleaved; Signal; Peptides; Endoplasmic

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.

蛋白质跨特定细胞内膜的转运机制是细胞生物学的中心问题。 该项目重点研究指导分泌蛋白跨内质网 (ER) 膜转移的疏水信号序列，这是细胞分泌蛋白的第一步。 信号序列的重要性已得到充分证实，但一旦通过内质网膜蛋白信号肽酶的作用从前体蛋白中去除，它们的命运尚不清楚。 进行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，以努力确定非洲爪蟾卵母细胞中所提出的信号肽水解酶活性。 将观察工程化和未修饰的切割信号肽的细胞内命运，并表征它们的降解特性。 这些研究的结果将用于开发非洲爪蟾卵母细胞中信号肽水解酶的工作检测方法。 这种测定对于鉴定和纯化特异性代谢切割信号序列的蛋白质至关重要。 这些蛋白质可能不仅对于从细胞中清除信号序列很重要，而且对于跨内质网膜易位的实际机制也很重要。 该技术将用于追踪非洲爪蟾卵母细胞中切割信号肽的命运，可能为剖析活细胞中蛋白质跨内质网膜转移的离散步骤提供强大的工具。 %%% 在真核细胞中合成蛋白质时，蛋白质穿过内质网膜的转移是将蛋白质运输到各种目的地（包括溶酶体和细胞外空间（分泌））的关键的第一步。 我们对这一基本过程的理解的进步不仅对于一般理解细胞功能很重要，而且对于生物技术也很重要。 该项目解决了切割信号序列的命运问题，即穿过内质网膜转运所需的新合成蛋白质片段，但它不是“最终”蛋白质产物的一部分，因此在转移完成后很快就被酶促地从蛋白质的其余部分中切割下来。 尽管分泌过程其他方面的研究活动水平很高，但这是一个很少受到关注的细胞生物学领域。 这项工作很可能会产生不可预见的新的、潜在的重要见解。