| Autor: |
Prunetti L; Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA, lprunetti@ufl.edu., Reuter CJ, Hepowit NL, Wu Y, Barrueto L, Miranda HV, Kelly K, Maupin-Furlow JA |
| Jazyk: |
angličtina |
| Zdroj: |
Extremophiles : life under extreme conditions [Extremophiles] 2014 Mar; Vol. 18 (2), pp. 283-93. Date of Electronic Publication: 2013 Dec 17. |
| DOI: |
10.1007/s00792-013-0615-8 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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