Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
FEBS J. 2011 Dec;278(24):4833-44. doi: 10.1111/j.1742-4658.2011.08383.x. Epub 2011 Oct 28.
Polyubiquitin receptors execute the targeting of polyubiquitylated proteins to the 26S proteasome. In vitro studies indicate that disturbance of the physiological balance among different receptor proteins impairs the proteasomal degradation of polyubiquitylated proteins. To study the physiological consequences of shifting the in vivo equilibrium between the p54/Rpn10 proteasomal and the Dsk2/dUbqln extraproteasomal polyubiquitin receptors, transgenic Drosophila lines were constructed in which the overexpression or RNA interference-mediated silencing of these receptors can be induced. Flies overexpressing Flag-p54 were viable and fertile, without any detectable morphological abnormalities, although detectable accumulation of polyubiquitylated proteins demonstrated a certain level of proteolytic disturbance. Flag-p54 was assembled into the 26S proteasome and could fully complement the lethal phenotype of a p54 null mutant Drosophila line. The overexpression of Dsk2 caused severe morphological abnormalities in the late pupal stages, leading to pharate adult lethality, accompanied by a huge accumulation of highly polyubiquitylated proteins. The lethal phenotype of Dsk2 overexpression could be rescued in a double transgenic line coexpressing Flag-Dsk2 and Flag-p54. Although the double transgenic line was viable and fertile, it did not restore the proteolytic defects; the accumulation of the highly polyubiquitylated proteins was even more severe in the double transgenic line. Significant differences were found in the Dsk2-26S proteasome interaction in Drosophila melanogaster as compared with Saccharomyces cerevisiae. In yeast, Dsk2 can interact only with ΔRpn10 proteasomes and not with the wild-type one. In Drosophila, Dsk2 does not interact with Δp54 proteasomes, but the interaction can be fully restored by complementing the Δp54 deletion with Flag-p54.
多聚泛素受体执行多聚泛素化蛋白靶向 26S 蛋白酶体的过程。体外研究表明，不同受体蛋白之间生理平衡的干扰会损害多聚泛素化蛋白的蛋白酶体降解。为了研究体内 p54/Rpn10 蛋白酶体与 Dsk2/dUbqln 外蛋白酶体多聚泛素受体之间平衡转移的生理后果，构建了这些受体的过表达或 RNA 干扰介导的沉默可以诱导的转基因果蝇系。过表达 Flag-p54 的果蝇存活和繁殖能力正常，没有任何可检测到的形态异常，尽管可检测到的多聚泛素化蛋白积累表明存在一定程度的蛋白水解干扰。Flag-p54 组装到 26S 蛋白酶体中，并能完全弥补 p54 缺失突变型果蝇系的致死表型。Dsk2 的过表达导致晚期蛹期严重的形态异常，导致拟成虫致死，同时高度多聚泛素化蛋白大量积累。在共表达 Flag-Dsk2 和 Flag-p54 的双转基因系中，Dsk2 过表达的致死表型可以得到挽救。尽管双转基因系存活和繁殖能力正常，但它并没有恢复蛋白水解缺陷；高度多聚泛素化蛋白的积累在双转基因系中更为严重。与酿酒酵母相比，在黑腹果蝇中发现 Dsk2 与 26S 蛋白酶体的相互作用存在显著差异。在酵母中，Dsk2 只能与 ΔRpn10 蛋白酶体相互作用，而不能与野生型相互作用。在果蝇中，Dsk2 与 Δp54 蛋白酶体不相互作用，但通过用 Flag-p54 补充 Δp54 缺失可以完全恢复相互作用。
