Department of Molecular Structural Biology, Max-Planck Institute of Biochemistry, 82152 Martinsried, Germany.
Science. 2015 Jan 23;347(6220):439-42. doi: 10.1126/science.1261197.
The 26S proteasome is a key player in eukaryotic protein quality control and in the regulation of numerous cellular processes. Here, we describe quantitative in situ structural studies of this highly dynamic molecular machine in intact hippocampal neurons. We used electron cryotomography with the Volta phase plate, which allowed high fidelity and nanometer precision localization of 26S proteasomes. We undertook a molecular census of single- and double-capped proteasomes and assessed the conformational states of individual complexes. Under the conditions of the experiment—that is, in the absence of proteotoxic stress—only 20% of the 26S proteasomes were engaged in substrate processing. The remainder was in the substrate-accepting ground state. These findings suggest that in the absence of stress, the capacity of the proteasome system is not fully used.
26S 蛋白酶体是真核生物蛋白质质量控制和众多细胞过程调节的关键因素。在这里，我们描述了在完整的海马神经元中对这种高度动态的分子机器进行定量原位结构研究。我们使用带有 Volta 相板的电子晶体断层扫描技术，该技术可以实现对 26S 蛋白酶体进行高保真和纳米精度的定位。我们对单帽和双帽蛋白酶体进行了分子普查，并评估了单个复合物的构象状态。在实验条件下（即在没有蛋白毒性应激的情况下），只有 20%的 26S 蛋白酶体参与底物加工。其余的处于接受底物的基础状态。这些发现表明，在没有应激的情况下，蛋白酶体系统的容量没有得到充分利用。
