title: ATP binding by proteasomal ATPases regulates cellular assembly and substrate-induced functions of the 26 S proteasome.
abstract: We examined the role of ATP binding by six different ATPase subunits (Rpt1-6) in the cellular assembly and molecular functions of mammalian 26 S proteasome. Four Rpt subunits (Rpt1-4) with ATP binding mutations were incompetent for cellular assembly into 26 S proteasome. In contrast, analogous mutants of Rpt5 and Rpt6 were incorporated normally into 26 S proteasomes in both intact cells and an in vitro assembly assay. Surprisingly, purified 26 S proteasomes containing either mutant Rpt5 or Rpt6 had normal basal ATPase activity and substrate gate opening for hydrolysis of short peptides. However, these mutant 26 S proteasomes were severely defective for ATP-dependent in vitro degradation of ubiquitylated and non-ubiquitylated proteins and did not display substrate-stimulated ATPase and peptidase activities characteristic of normal proteasomes. These results reveal differential roles of ATP binding by various Rpt subunits in proteasome assembly and function. They also indicate that substrate-stimulated ATPase activity and gating depend on the concerted action of a full complement of Rpt subunits competent for ATP binding and that this regulation is essential for normal proteolysis. Thus, protein substrates appear to promote their own degradation by stimulating proteasome functions involved in proteolysis.
authors.affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
authors.forename: Young-Chan
authors.initials: YC
authors.lastname: Kim
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Li
authors.forename: David
authors.initials: D
authors.lastname: Thompson
authors.forename: George N
authors.initials: GN
authors.lastname: DeMartino
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002846:Chromatography, Affinity; D057809:HEK293 Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000302:isolation & purification / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D059748:Proteolysis; D013379:Substrate Specificity; D054875:Ubiquitination
chemical_list: D046912:Multiprotein Complexes; D050505:Mutant Proteins; C085834:PA700 proteasome activator; D010455:Peptides; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases