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首页> 外文期刊>Molecular and Cellular Biology >Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae.
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Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae.

机译：Bul1，一种新蛋白，可与酿酒酵母中的Rsp5泛素连接酶结合。

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We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt- phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.
机译：我们表征了酿酒酵母的温度敏感突变体，其中微型染色体在高温下不稳定，并克隆了编码碱性和亲水性蛋白质（110 kDa）的新基因。该基因的破坏导致了与原始突变相同的温度敏感性生长。通过使用双杂交系统，我们进一步分离了编码一个公顷（与E6-AP C端同源）域的RSP5（反向Spt表型），作为一个编码泛素连接酶的基因。因此，我们将基因命名为BUL1（用于与泛素连接酶结合的蛋白质）。 BUL1似乎参与了泛素化途径，因为高剂量的UBI1编码泛素，部分抑制了bul1破坏剂和rsp5突变体的温度敏感性。 RSP5和BUL1在多拷贝质粒上的共表达对野生型细胞的有丝分裂生长有毒。脉冲追踪实验表明，野生型细胞中的Bul1保持稳定，而rsp5细胞中的Bul1的条带几乎未检测到。由于两种菌株中蛋白质的稳态水平与通过免疫印迹分析确定的相同，因此在没有完整Rsp5的情况下，免疫沉淀过程中Bul1可能很容易降解。此外，Bul1和Rsp5似乎都与通过蔗糖梯度离心分离的大复合物相关，并且Rsp5与Bul1共免疫沉淀。我们讨论在蛋白质泛素化中Bul1与Rsp5一起起作用的可能性。

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