title: Structural and biochemical properties of an extreme 'salt-loving' proteasome activating nucleotidase from the archaeon Haloferax volcanii.
abstract: In eukaryotes, the 26S proteasome degrades ubiquitinylated proteins in an ATP-dependent manner. Archaea mediate a form of post-translational modification of proteins termed sampylation that resembles ubiquitinylation. Sampylation was identified in Haloferax volcanii, a moderate halophilic archaeon that synthesizes homologs of 26S proteasome subunits including 20S core particles and regulatory particle triple-A ATPases (Rpt)-like proteasome-associated nucleotidases (PAN-A/1 and PAN-B/2). To determine whether sampylated proteins associate with the Rpt subunit homologs, PAN-A/1 was purified to homogeneity from Hfx. volcanii and analyzed for its subunit stoichiometry, nucleotide-hydrolyzing activity and binding to sampylated protein targets. PAN-A/1 was found to be associated as a dodecamer (630 kDa) with a configuration in TEM suggesting a complex of two stacked hexameric rings. PAN-A/1 had high affinity for ATP (K m of ~0.44 mM) and hydrolyzed this nucleotide with a specific activity of 0.33 ± 0.1 μmol Pi/h per mg protein and maximum at 42 °C. PAN-A1 was stabilized by 2 M salt with a decrease in activity at lower concentrations of salt that correlated with dissociation of the dodecamer into trimers to monomers. Binding of PAN-A/1 to a sampylated protein was demonstrated by modification of a far Western blotting technique (derived from the standard Western blot method to detect protein-protein interaction in vitro) for halophilic proteins. Overall, our results support a model in which sampylated proteins associate with the PAN-A/1 AAA+ ATPase in proteasome-mediated proteolysis and/or protein remodeling and provide a method for assay of halophilic protein-protein interactions.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA, lprunetti@ufl.edu.
authors.forename: Laurence
authors.initials: L
authors.lastname: Prunetti
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Reuter
authors.forename: Nathaniel L
authors.initials: NL
authors.lastname: Hepowit
authors.forename: Yifei
authors.initials: Y
authors.lastname: Wu
authors.forename: Luisa
authors.initials: L
authors.lastname: Barrueto
authors.forename: Hugo V
authors.initials: HV
authors.lastname: Miranda
authors.forename: Karen
authors.initials: K
authors.lastname: Kelly
authors.forename: Julie A
authors.initials: JA
authors.lastname: Maupin-Furlow
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D019622:Haloferax volcanii / Q000201:enzymology* / Q000502:physiology; D009708:Nucleotidases / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D055049:Salt Tolerance
chemical_list: D019843:Archaeal Proteins; D009708:Nucleotidases; C030287:nucleotidase; D046988:Proteasome Endopeptidase Complex