In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1 Chaperone: Potential Substrates and Adaptors
| Autor: | Klaas J. van Wijk, Cyrille Montandon, Giulia Friso, Junsik Choi, Jui-Yun Rei Liao |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Chloroplasts Proteolysis Mutant Arabidopsis Biochemistry Chloroplast Proteins 03 medical and health sciences Affinity chromatography ATP hydrolysis medicine Heat-Shock Proteins 030102 biochemistry & molecular biology medicine.diagnostic_test biology Arabidopsis Proteins Chemistry Wild type Endopeptidase Clp General Chemistry Cell biology Chloroplast 030104 developmental biology Chaperone (protein) biology.protein Mutant Proteins CLPB Molecular Chaperones |
| Zdroj: | Journal of Proteome Research. 18:2585-2600 |
| ISSN: | 1535-3907 1535-3893 |
| Popis: | The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2, or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possibly also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for the hydrolysis of ATP (CLPC1-TRAP). On the basis of homology to nonplant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released; that is, they are trapped. When expressed in the wild type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared with affinity-purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates or new adaptors. Several of these trapped proteins overaccumulated in clp mutants or were found as interactors for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, the affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR, and CLPT subunits, indicating the stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction. |
| Databáze: | OpenAIRE |
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