丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

Assays for Proteasome Assembly and Maturation

互联网

822
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 β 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 β subunit maturation or of Ump1 degradation.
提问
扫一扫
丁香实验小程序二维码
实验小助手
丁香实验公众号二维码
扫码领资料
反馈
TOP
打开小程序