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Abstract The chloroplast chaperone CLPC1 aids to select, unfold, and deliver hundreds of proteins to the CLP protease for degradation. Through in vivo CLPC1, trapping we previously identified dozens of proteins that are (potential) substrate adaptors or substrates for the CLP chaperone–protease system. In this study, we show that two of these highly trapped proteins, DUF760-1 and DUF760-2, are substrates for the CLP protease in Arabidopsis (Arabidopsis thaliana). Loss-of-function mutants and transgenic plants were created for phenotyping, protein expression, and localization using immunoblotting and confocal microscopy. In planta BiFC, cycloheximide chase assays, and yeast 2-hybrid analyses were conducted to determine protein interactions and protein half-life. Both DUF760 proteins directly interacted with the N-domain of CLPC1 and both were highly enriched in clpc1-1 and clpr2-1 mutants. Accordingly, in vivo cycloheximide chase assays demonstrated that both DUF760 proteins are degraded by the CLP protease. The half-life of DUF760-1 was 4 to 6 h, whereas DUF760-2 was highly unstable and difficult to detect unless CLP proteolysis was inhibited. Null mutants for DUF760-1 and DUF760-2 showed weak but differential pigment phenotypes and differential sensitivity to protein translation inhibitors. This study demonstrates that DUF760-1 and DUF760-2 are substrates of the CLP chaperone–protease system and excellent candidates for the determination of CLP substrate degrons.
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This content will become publicly available on February 19, 2026
An improved variant of tobacco etch virus ( TEV) protease that does not need reducing agents
Abstract Here we show that a combination of previously suggested mutations for tobacco etch virus (TEV) protease results in a TEV protease mutant that maintains the same catalytic efficiency as previously described mutants but has enhanced stability and solubility. Another advantage of this new variant of TEV protease is that it does not need the inclusion of a reducing agent to maintain its effectiveness, making it easier to generate, store, and use in cleavage reactions compared to previous TEV protease mutants and, in particular, makes it a good choice for cleaving proteins that contain disulfide bonds that would otherwise be altered by the inclusion of a reducing agent. We also provide a straightforward purification protocol for generating this new version of TEV protease.
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- Award ID(s):
- 2003557
- PAR ID:
- 10573923
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 34
- Issue:
- 3
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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