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/1). 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 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 (Km 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 2M 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.

Conformational switching in the coiled-coil domains of a proteasomal ATPase regulates substrate processing

“…These prior studies indicate that CC domains are allosterically linked to the ATPase domains, yet no studies have shown how the CCs function mechanistically to regulate protein degradation by the proteasome. Much like the 19S ATPases, some studies suggest that the CC domains from PAN are also involved in substrate binding, although it is thought that PAN can achieve this without using additional substrate receptors that are found in the 19S 30 , 31 , 33 – 36 .…”

The Role of Stress Proteins in Haloarchaea and Their Adaptive Response to Environmental Shifts

“…There is little information about the proteasome concerning halophilic archaea; but the proteasome system in Hfx. volcanii has been widely studied in the presence of some stressors (high temperature, low salt, and the addition of l -canavanine), making it the best studied organism in haloarchaea [ 126 , 127 ]. This research concludes that psmA and panA are required to withstand low salt stress and l -canavanine, while psmA helps to overcome the hypersensitive responses of high temperature stress.…”

Prokaryotic Ubiquitin-Like Protein Modification

“…Thus, the PANs may form weak associations with the SAMPs alone. Far Western experiments demonstrate that the full-length PAN-A/1 binds the sampylation target MoaE only when covalently linked to SAMP1 (101). Thus, at least PAN-A/1 appears to have a higher affinity for SAMP1 when covalently bound to a target protein compared with SAMP1 alone, suggesting sampylation triggers association of protein targets with the proteasomal ATPase.…”