Specific Modification of Aged Proteasomes Revealed by Tag-Exchangeable Knock-In Mice

ABSTRACT

The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α′ subunit of casein kinase II (CK2α′) as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

KEYWORDS:

• knock-in mice
• molecular aging
• proteasome
• protein turnover
• whole-genome siRNA screen

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00426-18.

ACKNOWLEDGMENTS

We thank Hidenori Ichijo and Isao Naguro for advice on the generation of phosphorylation-specific antibodies and the siRNA screen. We are also grateful to Hiroshi Nishina for Cre recombinase expression vectors.

This study was supported by JSPS KAKENHI grants JP25221102 and JP26111704 and the AMED-CREST from the Japan Agency for Medical Research and Development (JP18gm1110003).