Kiss Petra, Szabó Aron, Hunyadi-Gulyás Eva, Medzihradszky Katalin F, Lipinszki Zoltán, Pál Margit, Udvardy Andor
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary.
Biochem J. 2005 Oct 15;391(Pt 2):301-10. doi: 10.1042/BJ20050523.
In the presence of Zn2+, the Drosophila 26 S proteasome disassembles into RP (regulatory particle) and CP (catalytic particle), this process being accompanied by the dissociation of subunit Rpn10/p54, the ubiquitin receptor subunit of the proteasome. The dissociation of Rpn10/p54 induces extensive rearrangements within the lid subcomplex of the RP, while the structure of the ATPase ring of the base subcomplex seems to be maintained. As a consequence of the dissociation of the RP, the peptidase activity of the 26 S proteasome is lost. The Zn2+-induced structural and functional changes are fully reversible; removal of Zn2+ is followed by reassociation of subunit Rpn10/p54 to the RP, reassembly of the 26 S proteasome and resumption of the peptidase activity. After the Zn2+-induced dissociation, Rpn10/p54 interacts with a set of non-proteasomal proteins. Hsp82 (heat-shock protein 82) has been identified by MS as the main Rpn10/p54-interacting protein, suggesting its role in the reassembly of the 26 S proteasome after Zn2+ removal. The physiological relevance of another Rpn10/p54-interacting protein, the Smt3 SUMO (small ubiquitin-related modifier-1)-activating enzyme, detected by chemical cross-linking, has been confirmed by yeast two-hybrid analysis. Besides the Smt3 SUMO-activating enzyme, the Ubc9 SUMO-conjugating enzyme also exhibited in vivo interaction with the 5'-half of Rpn10/p54 in yeast cells. The mechanism of 26 S proteasome disassembly after ATP depletion is clearly different from that induced by Zn2+. Rpn10/p54 is permanently RP-bound during the ATP-dependent assembly-disassembly cycle, but during the Zn2+ cycle it reversibly shuttles between the RP-bound and free states.
在锌离子存在的情况下，果蝇26S蛋白酶体分解为调节颗粒（RP）和催化颗粒（CP），这一过程伴随着蛋白酶体泛素受体亚基Rpn10/p54的解离。Rpn10/p54的解离会诱导RP盖子亚复合体内部发生广泛的重排，而基部亚复合体的ATP酶环结构似乎得以维持。由于RP的解离，26S蛋白酶体的肽酶活性丧失。锌离子诱导的结构和功能变化是完全可逆的；去除锌离子后，亚基Rpn10/p54会重新与RP结合，26S蛋白酶体重组并恢复肽酶活性。锌离子诱导解离后，Rpn10/p54与一组非蛋白酶体蛋白相互作用。通过质谱鉴定出热休克蛋白82（Hsp82）是与Rpn10/p54相互作用的主要蛋白，这表明它在去除锌离子后26S蛋白酶体重组过程中发挥作用。通过化学交联检测到的另一种与Rpn10/p54相互作用的蛋白——Smt3小泛素相关修饰物1（SUMO）激活酶的生理相关性，已通过酵母双杂交分析得到证实。除了Smt3 SUMO激活酶外，Ubc9 SUMO结合酶在酵母细胞中也与Rpn10/p54的5'端表现出体内相互作用。ATP耗尽后26S蛋白酶体的分解机制与锌离子诱导的机制明显不同。在ATP依赖的组装 - 拆卸循环中，Rpn10/p54永久与RP结合，但在锌离子循环中，它在与RP结合和游离状态之间可逆地穿梭。
