title: Selective chemical inactivation of AAA proteins reveals distinct functions of proteasomal ATPases.
abstract: BACKGROUND
  The 26S proteasome contains six highly related ATPases of the AAA family. We have developed a strategy that allows selective inhibition of individual proteasomal ATPases in the intact proteasome. Mutation of a threonine in the active site of Sug1/Rpt6 or Sug2/Rpt4 to a cysteine sensitizes these proteins to inactivation through alkylation by the sulfhydryl modifying agent NEM. Using this technique the individual contributions of Sug1 and Sug2 to proteasome function can be assessed.
  
  
  RESULTS
  We show that both Sug1 and Sug2 can be selectively alkylated by NEM in the context of the intact 26S complex and as predicted by structural modeling, this inactivates the ATPase function. Using this technique we demonstrate that both Sug 1 and 2 are required for full peptidase activity of the proteasome and that their functions are not redundant. Kinetic analysis suggests that Sug2 may have an important role in maintaining the interaction between the 19S regulatory complex and the 20S proteasome. In contrast, inhibition of Sug1 apparently decreases peptidase activity of the 26S proteasome by another mechanism.
  
  
  CONCLUSIONS
  These results describe a useful technique for the selective inactivation of AAA proteins. In addition, they also demonstrate that the functions of two related proteasomal AAA proteins are not redundant, suggesting differential roles of proteasomal AAA proteins in protein degradation.
authors.affiliation: Center for Biomedical Inventions, Department of Medicine, University of Texas-Southwestern Meidcal School, Dallas, 75390-8573, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Russell
authors.forename: F
authors.initials: F
authors.lastname: Gonzalez
authors.forename: L
authors.initials: L
authors.lastname: Joshua-Tor
authors.forename: S A
authors.initials: SA
authors.lastname: Johnston
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D000478:Alkylation; D020816:Amino Acid Motifs; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000032:analysis; D046988:Proteasome Endopeptidase Complex; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D015003:Yeasts / Q000201:enzymology
chemical_list: D005656:Fungal Proteins; D009097:Multienzyme Complexes; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C103015:RPT4 protein, S cerevisiae; C069670:RPT6 protein, S cerevisiae; D005033:Ethylmaleimide