Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Biomolecules. 2023 Jun 15;13(6):992. doi: 10.3390/biom13060992.
Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multi-subunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generates the 26S proteasome, which is the major proteasomal complex in cells. Native gel protocols are used to investigate the 26S/20S ratio. However, a simple method for detecting these proteasome complexes in cells is missing. To this end, using CRISPR technology, we YFP-tagged the endogenous PSMB6 (β1) gene, a 20S CP subunit, and co-tagged endogenous PSMD6 (Rpn7), a 19S RP subunit, with the mScarlet fluorescent protein. We observed the colocalization of the YFP and mScarlet fluorescent proteins in the cells, with higher nuclear accumulation. Nuclear proteasomal granules are formed under osmotic stress, and all were positive for YFP and mScarlet. Previously, we have reported that PSMD1 knockdown, one of the 19 RP subunits, gives rise to a high level of "free" 20S CPs. Intriguingly, under this condition, the 20S-YFP remained nuclear, whereas the PSMD6-mScarlet was mostly in cytoplasm, demonstrating the distinct subcellular distribution of uncapped 20S CPs. Lately, we have shown that the PSMA3 (α7) C-terminus, a 20S CP subunit, binds multiple intrinsically disordered proteins (IDPs). Remarkably, the truncation of the PSMA3 C-terminus is phenotypically reminiscent of PSMD1 knockdown. These data suggest that the PSMA3 C-terminal region is critical for 26S proteasome integrity.
蛋白酶体通过蛋白质降解来严格调控蛋白质稳态。蛋白酶体是由 20S 蛋白酶体核心颗粒（20S CP）组成的多亚基复合物，与一个或两个 19S 调节颗粒（19S RPs）结合，形成 26S 蛋白酶体，它是细胞中主要的蛋白酶体复合物。天然凝胶方案用于研究 26S/20S 比值。然而，目前还缺乏一种简单的方法来检测细胞中的这些蛋白酶体复合物。为此，我们使用 CRISPR 技术对内源性 PSMB6（β1）基因（20S CP 亚基）进行了 YFP 标记，并与内源性 PSMD6（Rpn7）（19S RP 亚基）进行了 mScarlet 荧光蛋白的共标记。我们观察到细胞中 YFP 和 mScarlet 荧光蛋白的共定位，并且核内积累更高。在渗透胁迫下形成核蛋白酶体颗粒，所有颗粒均对 YFP 和 mScarlet 呈阳性。此前，我们报道过，19RP 亚基之一 PSMD1 的敲低会导致高水平的“游离”20S CP。有趣的是，在这种情况下，20S-YFP 仍留在核内，而 PSMD6-mScarlet 主要在细胞质中，表明未加帽的 20S CP 具有明显不同的亚细胞分布。最近，我们已经表明，20S CP 亚基 PSMA3（α7）的 C 端结合多个固有无序蛋白（IDP）。值得注意的是，PSMA3 C 端的截断在表型上类似于 PSMD1 的敲低。这些数据表明 PSMA3 C 端区域对于 26S 蛋白酶体的完整性至关重要。
