Schmidtke G, Schmidt M, Kloetzel P M
Zentrum für Experimentelle Medizin (ZEM), Institut für Biochemie, Charité, Humboldt Universität zu Berlin, Germany.
J Mol Biol. 1997 Apr 25;268(1):95-106. doi: 10.1006/jmbi.1997.0947.
The maturation of the eukaryotic 20 S proteasome complex occurs via 13 S and 16 S precursor complexes in a multistep assembly pathway. These precursor complexes contain alpha-subunits as well as unprocessed beta-subunit proproteins. We have purified and characterized the different proteasome assembly intermediates and analysed their ability to support beta-subunit proprotein processing in vitro. Our data show that 13 S and 16 S proteasome precursor complexes differ not only in size but also in their protein content and behaviour during hydrophobic chromatography. By establishing conditions which allowed us to analyse beta-prosubunit maturation in vitro we demonstrate that the processing of the homologous proproteins of the beta-subunits LMP2 and delta essentially takes place in 16 S precursor complexes. No proprotein processing activity was observed in 13 S precursor complexes. Furthermore, proprotein processing in vitro can be inhibited with a proteasome specific inhibitor, but with different efficiency for LMP2 and delta. A peptide, which represents the sequence of the proprotein processing site HGTT, exhibited no inhibitory effect on the processing of either subunit. These data provide further evidence that proprotein processing occurs via an autocatalytic mechanism. Our experiments also demonstrate that the chaperone protein hsc73 is associated with 16 S but not with 13 S precursor complexes. In support of the specificity of this interaction incubation with ATP leads to the dissociation of hsc73 from 16 S complexes and to the formation of high molecular weight aggregates. Prosubunit processing in isolated 16 S complexes does not, however, result in the formation of proteolytically active 20 S proteasomes which may be due to the fact that not all beta-subunits can be efficiently processed in vitro. In contrast to previous assumptions subunit processing and formation of proteolytic activity do not coincide and final 20 S complex assembly seems to represent in part a separate event which requires additional factors or proteins which are not present or active in the purified 16 S precursor complexes.
真核生物20 S蛋白酶体复合物的成熟是通过13 S和16 S前体复合物在多步骤组装途径中发生的。这些前体复合物包含α亚基以及未加工的β亚基前体蛋白。我们已经纯化并表征了不同的蛋白酶体组装中间体，并分析了它们在体外支持β亚基前体蛋白加工的能力。我们的数据表明，13 S和16 S蛋白酶体前体复合物不仅在大小上不同，而且在其蛋白质含量和疏水色谱过程中的行为也不同。通过建立允许我们在体外分析β前体亚基成熟的条件，我们证明β亚基LMP2和δ的同源前体蛋白的加工基本上发生在16 S前体复合物中。在13 S前体复合物中未观察到前体蛋白加工活性。此外，蛋白酶体特异性抑制剂可抑制体外前体蛋白加工，但对LMP2和δ的抑制效率不同。代表前体蛋白加工位点HGTT序列的肽对任一亚基的加工均无抑制作用。这些数据进一步证明前体蛋白加工是通过自催化机制发生的。我们的实验还表明，伴侣蛋白hsc73与16 S前体复合物相关，但与13 S前体复合物无关。为支持这种相互作用的特异性，与ATP孵育会导致hsc73从16 S复合物中解离并形成高分子量聚集体。然而，在分离的16 S复合物中前体亚基加工并不会导致形成具有蛋白水解活性的20 S蛋白酶体，这可能是因为并非所有β亚基都能在体外有效加工。与先前的假设相反，亚基加工和蛋白水解活性的形成并不一致，最终20 S复合物的组装似乎部分代表了一个独立的事件，这需要额外的因子或蛋白质，而这些因子或蛋白质在纯化的16 S前体复合物中不存在或不活跃。
