title: Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase.
abstract: The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms used by the proteasomal heterohexameric AAA+ unfoldase to drive substrate degradation remain poorly understood. To perform systematic mutational analyses of individual ATPase subunits, we heterologously expressed the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases have distinct roles in degradation, corresponding to their positions in the spiral staircases adopted by the AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from alternate subunits. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Robyn
authors.initials: R
authors.lastname: Beckwith
authors.forename: Eric
authors.initials: E
authors.lastname: Estrin
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Worden
authors.forename: Andreas
authors.initials: A
authors.lastname: Martin
mesh_terms: D049071:Endopeptidase Clp / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D049071:Endopeptidase Clp; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease