Thompson David, Hakala Kevin, DeMartino George N
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
J Biol Chem. 2009 Sep 11;284(37):24891-903. doi: 10.1074/jbc.M109.023218. Epub 2009 Jul 9.
We have identified, purified, and characterized three subcomplexes of PA700, the 19 S regulatory complex of the 26 S proteasome. These subcomplexes (denoted PS-1, PS-2, and PS-3) collectively account for all subunits present in purified PA700 but contain no overlapping components or significant levels of non-PA700 proteins. Each subcomplex contained two of the six AAA subunits (Rpt1-6) that form the binding interface of PA700 with the 20 S proteasome, the protease component of the 26 S proteasome. Unlike intact PA700, no individual PA700 subcomplex displayed ATPase activity or proteasome activating activity. However, both activities were manifested by ATP-dependent in vitro reconstitution of PA700 from the subcomplexes. We exploited functional reconstitution to define and distinguish roles of different PA700 subunits in PA700 function by selective alteration of subunits within individual subcomplexes prior to reconstitution. Carboxypeptidase treatment of either PS-2 or PS-3, subcomplexes containing specific Rpt subunits previously shown to have important roles in 26 S proteasome assembly and activation, inhibited these processes but did not affect PA700 reconstitution or ATPase activity. Thus, the intact C termini of both subunits are required for 26 S proteasome assembly and activation but not for PA700 reconstitution. Surprisingly, carboxypeptidase treatment of PS-1 also inhibited 26 S proteasome assembly and activation upon reconstitution with untreated PS-2 and PS-3. These results suggest a previously unidentified role for other PA700 subunits in 26 S proteasome assembly and activation. Our results reveal relative structural and functional relationships among the AAA subunits of PA700 and new insights about mechanisms of 26 S proteasome assembly and activation.
我们已经鉴定、纯化并表征了PA700（26S蛋白酶体的19S调节复合物）的三个亚复合物。这些亚复合物（分别表示为PS - 1、PS - 2和PS - 3）共同构成了纯化的PA700中存在的所有亚基，但不包含重叠的成分或显著水平的非PA700蛋白。每个亚复合物都包含六个AAA亚基（Rpt1 - 6）中的两个，这些亚基形成了PA700与20S蛋白酶体（26S蛋白酶体的蛋白酶成分）的结合界面。与完整的PA700不同，单个PA700亚复合物均未表现出ATP酶活性或蛋白酶体激活活性。然而，通过亚复合物进行的ATP依赖性体外重组可表现出这两种活性。我们利用功能重组，通过在重组前选择性改变单个亚复合物中的亚基，来定义和区分不同PA700亚基在PA700功能中的作用。用羧肽酶处理PS - 2或PS - 3（这两个亚复合物含有先前已证明在26S蛋白酶体组装和激活中起重要作用的特定Rpt亚基），会抑制这些过程，但不影响PA700的重组或ATP酶活性。因此，这两个亚基完整的C末端是26S蛋白酶体组装和激活所必需的，但不是PA700重组所必需的。令人惊讶的是，用羧肽酶处理PS - 1后，在用未处理的PS - 2和PS - 3进行重组时，也会抑制26S蛋白酶体的组装和激活。这些结果表明，PA700的其他亚基在26S蛋白酶体组装和激活中具有先前未被识别的作用。我们的结果揭示了PA700的AAA亚基之间相对的结构和功能关系，以及关于26S蛋白酶体组装和激活机制的新见解。
