Assays for proteasome assembly and maturation.
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.
Dohmen
R Jürgen
RJ
Institute for Genetics, University of Cologne, Germany.
London
Markus K
MK
Glanemann
Christoph
C
Ramos
Paula C
PC
D000251:Adenosine Triphosphatases / Q000032:analysis*; D000818:Animals; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000032:analysis*; D002460:Cell Line; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000032:analysis*; D006801:Humans; D018832:Molecular Chaperones / Q000032:analysis*; D046988:Proteasome Endopeptidase Complex / Q000032:analysis*; D025801:Ubiquitin / Q000032:analysis
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D025801:Ubiquitin; C110856:proteasome maturation protein; D010450:Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C119011:26S proteasome non-ATPase regulatory subunit 13; D000251:Adenosine Triphosphatases