Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, 32611-0700, USA,
Extremophiles. 2014 Mar;18(2):283-93. doi: 10.1007/s00792-013-0615-8. Epub 2013 Dec 17.
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.
在真核生物中，26S 蛋白酶体以 ATP 依赖性方式降解泛素化蛋白。古菌介导了一种称为 sampylation 的蛋白质翻译后修饰形式，类似于泛素化。Sampylation 在中度嗜盐古菌 Haloferax volcanii 中被鉴定出来，该菌合成 26S 蛋白酶体亚基的同源物，包括 20S 核心颗粒和调节颗粒三 A 型 ATP 酶（Rpt）样蛋白酶体相关核苷酸酶（PAN-A/1 和 PAN-B/2）。为了确定 sampylated 蛋白是否与 Rpt 亚基同源物结合，从 Hfx. volcanii 中纯化到均一的 PAN-A/1 并分析其亚基比例、核苷酸水解活性和与 sampylated 蛋白靶标的结合。发现 PAN-A/1 以十二聚体（630 kDa）的形式与 TEM 中的配置相关，提示两个堆叠的六聚体环的复合物。PAN-A/1 对 ATP 具有高亲和力（K m 约为 0.44 mM），并以 0.33±0.1 μmol Pi/h/mg 蛋白的比活性水解该核苷酸，在 42°C 时达到最大值。PAN-A1 被 2 M 盐稳定，在较低浓度的盐下活性降低，这与十二聚体分解为三聚体和单体相关。通过修改远 Western 印迹技术（源自标准 Western 印迹方法以检测体外蛋白质-蛋白质相互作用）来证明 PAN-A/1 与 sampylated 蛋白的结合，该技术适用于嗜盐蛋白。总体而言，我们的结果支持一种模型，即 sampylated 蛋白与 PAN-A/1 AAA+ATP 酶在蛋白酶体介导的蛋白水解和/或蛋白重塑中结合，并提供了一种用于检测嗜盐蛋白-蛋白相互作用的方法。
