In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1 Chaperone: Potential Substrates and Adaptors

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.