CFTR DEGRADATION IN THE ENDOPLASMIC RETICULUM

这个项目将检验这样一个假设，即某些形式的CFTR 在细胞内的隔间里降解。最初的实验将是 在酵母中进行，利用一系列突变体 内质网中不同阶段分子的加工 (有或不有囊泡形成)、高尔基体或更远。野生型和 突变(deltaF508和其他)cftr原生形式，包含一个 标记序列，以便于免疫学鉴定，或融合到β- 为了便于报告和以后的基因分析，半乳糖苷酶将 遵循使用SEC、VPS、ERD和KAR2的处理序列 变种人。将利用β-半乳糖苷酶融合来促进 筛选不能处理或降解CFTR的突变体。基因 其中有缺陷的突变体将从酵母中克隆出来，而人类 寻找同源物。基因产物的性质将被确定并 想出了抑制它们的方法。这些实验将确定 在蛋白质加工的哪个阶段CFTR没有得到适当的处理，以及 被降解，并将分离出的蛋白质(S)负责。如果，如 根据程和同事的工作预测，囊肿症的一个主要原因 纤维化是突变形式的有缺陷的加工和降解 Cftr，因此永远不会在质膜上实现正确的位置， 这些结果可能允许我们设计干预措施，以允许 一些突变蛋白的加工和逃逸到膜上，在那里它 可能会激活它并恢复功能。

This project will test the hypothesis that some forms of CFTR are degraded in intracellular compartments. The initial experiments will be conducted in yeast, capitalizing on a series of mutants which halt processing of molecules at various stages in the endoplasmic reticulum (with or without vesicle formation), Golgi, or beyond. Wild type and mutant (deltaF508 and others) CFTR either in native form, containing a FLAG sequence for ease of immunologic identification, or fused to beta- galactosidase for ease of reporting and later genetic analysis will be followed along the processing sequence using sec, vps, erd, and kar2 mutants. The beta-galactosidase fusions will be utilized to facilitate screening for mutants which fail to process or degrade CFTR. The genes which are defective in the mutants will be cloned from yeast, and human homologs sought. The nature of the gene products will be determined and means of inhibiting them devised. These experiments will determine at which stage in protein processing CFTR fails to be properly processed and is degraded and will isolate the protein(s) responsible. If, as predicted by the work of Cheng and coworkers, a major cause of cystic fibrosis is the defective processing and degradation of mutant forms of CFTR, so that proper location at the plasma membrane is never achieved, these results might permit us to design interventions to permit processing and escape of some mutant protein to the membrane, where it may be possible to activate it and restore function.