Steinberger, S.; Adler, J.; Shaul, Y. Method of Monitoring 26S Proteasome in Cells Revealed the Crucial Role of PSMA3 C-Terminus in 26S Integrity. Biomolecules2023, 13, 992.
Steinberger, S.; Adler, J.; Shaul, Y. Method of Monitoring 26S Proteasome in Cells Revealed the Crucial Role of PSMA3 C-Terminus in 26S Integrity. Biomolecules 2023, 13, 992.
Steinberger, S.; Adler, J.; Shaul, Y. Method of Monitoring 26S Proteasome in Cells Revealed the Crucial Role of PSMA3 C-Terminus in 26S Integrity. Biomolecules2023, 13, 992.
Steinberger, S.; Adler, J.; Shaul, Y. Method of Monitoring 26S Proteasome in Cells Revealed the Crucial Role of PSMA3 C-Terminus in 26S Integrity. Biomolecules 2023, 13, 992.
Abstract
Proteasomes critically regulate proteostasis via protein degradation. Proteasomes are multisubunit complexes composed of the 20S proteolytic core particle (20S CP) that, in association with one or two 19S regulatory particles (19S RPs), generate the 26S proteasome. 26S is the major proteasomal complex in the cells. Native gel protocols are used to investigate the 26S/20S ratio. However, a simple method for detecting these proteasome complexes in the cells is missing. To this end, using CRISPR technology, we YFP tagged the endogenous PSMB6 (b1) gene, a 20S CP subunit, and co-tagged endogenous PSMD6 (Rpn7), a 19S RP subunit, with mScarlet fluorescent protein. We observed colocalization of 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 CP. Intriguingly, under this condition, the 20S-YFP remained nuclear, whereas the PSMD6-mScarlet is mostly in cytoplasm, demonstrating the distinct subcellular distribution of uncapped 20S CP. Lately, we have shown that the PSMA3 (a7) C-termininus, a 20S CP subunit, binds multiple intrinsically disordered proteins (IDPs). Remarkably, truncation of the PSMA3 C-terminus is phenotypically reminiscent of PSMD1 knockdown. These data suggest the PSMA3 C-terminal region is critical for the 26S proteasome integrity.
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
