Dohmen R Jürgen, London Markus K, Glanemann Christoph, Ramos Paula C
Institute for Genetics, University of Cologne, Germany.
Methods Mol Biol. 2005;301:243-54. doi: 10.1385/1-59259-895-1:243.
The 20S proteasome is a complex multisubunit protease that is present in all phylae of life. Eukaryotic 26S proteasomes, which are composed of 20S proteasomes and 19S activator complexes, mediate the degradation of ubiquitylated proteins. Biogenesis of proteasomes involves a coordinated expression of proteasome genes as well as numerous assembly and maturation steps. Activation of proteolytic sites occurs via autocatalytic processing of the N-terminal propeptides of beta subunits. This process is coupled to the dimerization of half-proteasome precursor complexes and, in eukaryotes, requires the presence of the Ump1 maturation factor to occur efficiently. After activation of proteolytic sites the encased Ump1 is degraded rapidly. Here we describe methods that track assembly and maturation of proteasomes in bacteria and eukaryotic cells. Assembly intermediates and mature forms of the proteasome present in cells at steady state are analyzed by gel filtration and immunoblotting after sodium dodecyl sulfate (SDS)- and native polyacrylamide gel electrophoresis (PAGE). The kinetics of proteasome assembly is followed by pulse chase detection of beta subunit maturation or of Ump1 degradation.
20S蛋白酶体是一种复杂的多亚基蛋白酶，存在于所有生命谱系中。真核生物的26S蛋白酶体由20S蛋白酶体和19S激活复合物组成，介导泛素化蛋白的降解。蛋白酶体的生物合成涉及蛋白酶体基因的协调表达以及众多组装和成熟步骤。蛋白水解位点的激活通过β亚基N端前肽的自催化加工发生。该过程与半蛋白酶体前体复合物的二聚化相关，在真核生物中，需要Ump1成熟因子的存在才能有效发生。蛋白水解位点激活后，包裹的Ump1会迅速降解。在这里，我们描述了追踪细菌和真核细胞中蛋白酶体组装和成熟的方法。通过十二烷基硫酸钠（SDS）和天然聚丙烯酰胺凝胶电泳（PAGE）后的凝胶过滤和免疫印迹分析处于稳态的细胞中存在的蛋白酶体组装中间体和成熟形式。蛋白酶体组装的动力学通过脉冲追踪检测β亚基成熟或Ump1降解来跟踪。
