Johnson E S, Ma P C, Ota I M, Varshavsky A
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
J Biol Chem. 1995 Jul 21;270(29):17442-56. doi: 10.1074/jbc.270.29.17442.
Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
先前的研究表明，带有“不可去除”N端泛素（Ub）部分的融合蛋白在体内寿命较短，该融合蛋白的Ub起到降解信号的作用。通过分析干扰该途径的突变，在酿酒酵母中剖析了所涉及的蛋白水解系统，即所谓的UFD途径（泛素融合降解途径）。由此鉴定出的五个基因中的两个，UFD1和UFD5，在UFD途径的泛素化后步骤发挥作用。UFD3在控制细胞中Ub的浓度方面发挥作用：ufd3突变体中游离Ub的水平大大降低，并且通过过表达Ub可以恢复这些突变体中Ub融合蛋白的降解。UFD2和UFD4似乎影响与融合蛋白的Ub部分相连的多聚泛素链的形成和拓扑结构。UFD1、UFD2和UFD4分别编码以前未描述的40 kDa、110 kDa和170 kDa的蛋白质。Ufd4p最后约280个残基的序列与E6AP相似，E6AP是一种人类蛋白质，它与致癌乳头瘤病毒的E6蛋白和肿瘤抑制蛋白p53都结合，其泛素依赖性降解涉及E6AP。UFD5与先前鉴定的SON1相同，SON1是作为损害蛋白质向细胞核转运的sec63等位基因的基因外抑制子分离得到的。UFD5对于UFD途径和N端规则途径（后者系统降解带有某些N端残基的蛋白质）的活性都是必需的。我们还表明，融合蛋白的Ub部分中第29位或第48位的赖氨酸向精氨酸的转化极大地降低了Ub与β-半乳糖苷酶融合蛋白的泛素化和降解。相比之下，Ub与二氢叶酸还原酶融合蛋白的泛素化和降解受到UbR29的抑制，但不受UbR48部分的抑制。ufd4突变体无法在第29位赖氨酸处对融合蛋白的Ub部分进行泛素化，而ufd2突变体在第48位赖氨酸处的泛素化受损。这些以及相关的发现表明，底物连接的多聚泛素链中的Ub-Ub异肽键不仅涉及先前鉴定的赖氨酸48，还涉及Ub的赖氨酸29，并且结构不同的多聚泛素链在泛素依赖性蛋白质降解中具有不同的功能。
