In vivo trapping of proteins interacting with the chloroplast CLPC1 chaperone; potential substrates and adaptors

ABSTRACT: 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 possible 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 hydrolysis of ATP (CLPC1-TRAP). Based on homology to non-plant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released, i.e. they are trapped. When expressed in 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 to affinity purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates and/or new adaptors. Several of these trapped proteins over-accumulated in clp mutants and/or were found as interactions for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR and CLPT subunits, indicating stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Photosynthetic Cell, Leaf

SUBMITTER: Giulia Friso  

LAB HEAD: van Wijk Klaas

PROVIDER: PXD013424 | Pride | 2019-04-10

REPOSITORIES: Pride