Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland.
EMBO J. 2010 Apr 7;29(7):1262-71. doi: 10.1038/emboj.2010.23. Epub 2010 Mar 4.
Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin-like protein (Pup) that is recognized by the N-terminal coiled-coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa-proteasome complex unfolds and degrades Pup-tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N-terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.
结核分枝杆菌与其他放线菌一样，除了具有一般细菌降解复合物的成员外，还具有蛋白酶体。与真核生物中的泛素化类似，用原核泛素样蛋白（Pup）对蛋白酶体降解的底物进行标记，该蛋白被 ATP 酶 Mpa（也称为 ARC）的 N 端卷曲螺旋结构域识别。在这里，我们重新组装了整个分枝杆菌蛋白酶体降解系统，用于 pupylated 底物，并确定了其在底物募集、展开和降解方面的机制特征。我们表明，Mpa-蛋白酶体复合物可展开和降解 Pup 标记的蛋白质，并且该活性需要 ATP 酶与蛋白酶体的物理相互作用。此外，我们确定了 Pup 的 N 端区域是将 pupylated 底物纳入 Mpa 孔所需的结构元件。在此过程中，Mpa 拉动 Pup 以启动底物蛋白的展开，并将其拖向蛋白酶体腔。与真核生物的泛素不同，Pup 不会被回收，而是与底物一起降解。这赋予了 Pup 双重功能，既是 Mpa 的识别元件，也是穿线决定因素。
