MECHANICS AND FUNCTIONS OF THE N-END RULE

摘要 使用一种新的方法，最近发现 胞内氨基末端残基的识别 蛋白质调节蛋白质的代谢稳定性，并 显然，还有可能调节其稳定性(1)。 本提案中所述研究的目标是 探索这种洞察的含义(“N-End规则”途径 (1)。 具体目标摘要： 1)构建泛素与测试蛋白的融合 β-半乳糖苷酶(β-GAL)，特别是与小鼠的融合 二氢叶酸还原酶(DHFR)。利用这些泛素融合来 用N-端规则蛋白水解物解决靶向机制 路径。 2)确定用beta定义的N-End规则是否 用不相关的测试蛋白来定义GAL是相同的。 3)确定是否按照 酵母菌在大肠杆菌、青蛙中的定义是相同的。 卵母细胞和培养的哺乳动物细胞。 4)映射泛素部分在靶向内的位置， 多种泛素化蛋白质。 5)分离、纯化和分子遗传分析 从酵母中提取泛素的特定工艺。该酶用于生产 原核生物中N-端规则的直接测试(E.Coli)。 6)利用一种新的筛选方法分离酵母菌突变体 选择性蛋白质周转。 7)体外选择性蛋白质周转的机理研究 泛素-β半乳糖融合(1)和类似的明确定义的 底物。 8)调控的生化和分子遗传学分析 和氨基酸的翻译后共轭功能 蛋白质的氨基末端。 对选择性蛋白质周转的详细机制理解， 这仍然是一个遥远的目标，预计将被证明是有帮助的，如果不是必要的话 解决了许多目前悬而未决的生物问题， 从细胞周期进程的调控到分化、应激 反应、致癌和衰老。

Abstract Using a new approach, it has been recently found that the recognition of an amino-terminal residue in an intracellular protein mediates the metabolic stability of the protein and apparently also the potential for regulation of its stability (1). The objective of the research described in this proposal is to explore the implications of this insight (the "N-end rule" pathway (1)). Summary of Specific Aims: 1) Construction of ubiquitin fusions with test proteins other than beta-galactosidase (beta gal), in particular fusions with the mouse dihydrofolate reductase (DHFR). Use of these ubiquitin fusions to address the mechanism of targeting by the N-end rule proteolytic pathway. 2) Determination of whether the N-end rule as defined with beta gal is the same when defined with unrelated test proteins. 3) Determination of whether the N-end rule as defined in the yeast S. cerevisiae is the same when defined in E. coli, frog oocytes and cultured mammalian cells. 4) Mapping positions of ubiquitin moieties within a targeted, mulitply ubiquitinated protein. 5) Isolation, purification and molecular genetic analysis of the ubiquitin-specific processing from yeast. Use of this protease for direct testing of the N-end rule in prokaryotes (E. coli). 6) Use of a novel screening approach to isolate yeast mutants in selective protein turnover. 7) Mechanistic studies on selective protein turnover in vitro using both ubiquitin-beta gal fusions (1) and analogous well-defined substrates. 8) Biochemical and molecular genetic analyses of the regulation and function of posttranslational conjugation of amino acids to amino-termini of proteins. Detailed mechanistic understanding of selective protein turnover, still a distant goal, is expected to prove helpful if not essential for solving many of the currently outstanding biological problems, from regulation of cell cycle progression to differentiation, stress responses, carcinogenesis and aging.