title: Using native gel electrophoresis and phosphofluoroimaging to analyze GFP-tagged proteasomes.
abstract: Native polyacrylamide gel electrophoresis (PAGE) is an invaluable technique in biochemistry to characterize native protein complexes with high molecular mass. Thus, native PAGE is suited to resolve proteasomes, giant proteases responsible for the degradation of polyubiquitylated proteins. Proteasomes contain multiple subunits and exist in different configurations. All configurations have a common 20S core particle (CP). The CP encloses the proteolytic chamber and is composed of four stacked rings with C2 symmetry. The entrance to the CP is gated by central pores within the outer rings, which also provide the binding sites for the 19S regulatory complex (RP). Adjacent regulatory proteins, such as Blm10/PA200, are bound to specific proteasome species of low abundance and contribute to the heterogeneity of proteasome complexes. To get insight into the complexity of proteasome configurations in yeast, we developed a native PAGE system by which GFP-labelled variants of proteasomal subunits are visualized by phosphofluoroimaging. Following native PAGE, proteasome species can be subjected to in-gel activity assays, subsequent SDS-PAGE, and Western blotting.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada. cordula.enenkel@utoronto.ca
authors.forename: Cordula
authors.initials: C
authors.lastname: Enenkel
mesh_terms: D015153:Blotting, Western / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D025821:Polyubiquitin / Q000032:analysis / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry*
chemical_list: C500597:Blm10 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025821:Polyubiquitin; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex