MECHANICS AND FUNCTIONS OF THE N-END RULE PATHWAY

泛素是一种76个残基的蛋白质，存在于细胞中，可以是自由存在的，也可以与许多其他蛋白质结合。通过依赖泛素/蛋白酶体的途径选择性降解蛋白质，在许多生物学过程中发挥作用，包括细胞生长和分化、信号转导和对应激的反应。一种依赖泛素/蛋白酶体的蛋白分解系统是N-端规则通路，由实验室于1986年发现。N-端规则将蛋白质在体内的半衰期与其N-端残基的同一性联系起来。从哺乳动物到细菌，所有被研究的生物中都存在N端规则途径。如进度报告所述，目前处于第11个年头的本赠款(“N-End规则路径的机制和功能”，DK39520)支持的研究对N-End规则的功能和机制产生了重要的见解。本次更新申请中描述的研究的目的是促进对泛素系统的N-端规则通路和相关方面的理解。具体目标是：1)N-端规则通路对多肽输入的调节：生化和遗传学研究；2)基于融合的酿酒酵母N-端规则通路生理底物的筛选；3)N-端规则通路调节子的分离和分析；4)筛选其生存需要N-端r的突变体；5)使用抑制消减杂交来鉴定N-端规则通路的新功能；6)分析酿酒酵母Ntalp N-端酰胺酶的线粒体功能；7)鉴定生理N-末端规则底物的生物化学遗传学方法；8)N-末端规则途径的“双头”抑制物；9)“双泛素”技术及其在共翻译蛋白分解问题中的应用；10)UBRI编码的NRecogin和Ubr2p的生化和遗传学剖析，Ubrlp是最近在酿酒酵母中发现的Ubrlp的同源物。

Ubiquitin is a 76-residue protein that exists in cells either free or conjugated to many other proteins. Selective degradation of proteins by the ubiquitin/proteasome-dependent pathways plays a role in a multitude of biological processes, including cell growth and differentiation, signal transduction and responses to stress. One ubiquitin/proteasome-dependent proteolytic system is the N-end rule pathway, identified by the laboratory in 1986. The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is present in all organisms examined, from mammals to bacteria. Studies supported by the present grant ("Mechanics and Functions of the N-end Rule Pathway", DK39520), currently in its 11th year, have yielded significant insights into the N-end rule's functions and mechanisms, as described in the Progress Report. The objective of the research described in this renewal application is to advance the understanding of the N-end rule pathway and related aspects of the ubiquitin system. Specific aims are: 1) Regulation of peptide import by the N-end rule pathway: biochemical and genetic studies; 2) A fusion-based screen for physiological substrates of the N-end rule pathway in S. cerevisiae; 3) Isolation and analysis of regulators of the N-end rule pathway; 4) Screens for mutants whose viability requires the presence of the N-end r; 5) The use of suppression subtractive hybridization to identify new functions of the N-end rule pathway; 6) Analysis of a mitochondrial function of the Ntalp N-terminal amidase in S. cerevisiae; 7) A biochemico-genetic approach to identification of physiological N-end rule substrates; 8) A "double-headed" inhibitor of the N-end rule pathway; 9) The "two-ubiquitin" technique and its application to the problem of cotranslational proteolysis; and 10) Biochemical and genetic dissection of the UBRI-encoded Nrecognin and Ubr2p, a recently identified homolog of Ubrlp in S. cerevisiae.