title: Subunit stoichiometry, evolution, and functional implications of an asymmetric plant plastid ClpP/R protease complex in Arabidopsis.
abstract: The caseinolytic protease (Clp) protease system has been expanded in plant plastids compared with its prokaryotic progenitors. The plastid Clp core protease consists of five different proteolytic ClpP proteins and four different noncatalytic ClpR proteins, with each present in one or more copies and organized in two heptameric rings. We determined the exact subunit composition and stoichiometry for the intact core and each ring. The chloroplast ClpP/R protease was affinity purified from clpr4 and clpp3 Arabidopsis thaliana null mutants complemented with C-terminal StrepII-tagged versions of CLPR4 and CLPP3, respectively. The subunit stoichiometry was determined by mass spectrometry-based absolute quantification using stable isotope-labeled proteotypic peptides generated from a synthetic gene. One heptameric ring contained ClpP3,4,5,6 in a 1:2:3:1 ratio. The other ring contained ClpP1 and ClpR1,2,3,4 in a 3:1:1:1:1 ratio, resulting in only three catalytic sites. These ClpP1/R1-4 proteins are most closely related to the two subunits of the cyanobacterial P3/R complex and the identical P:R ratio suggests conserved adaptation. Furthermore, the plant-specific C-terminal extensions of the ClpP/R subunits were not proteolytically removed upon assembly, suggesting a regulatory role in Clp chaperone interaction. These results will now allow testing ClpP/R structure-function relationships using rationale design. The quantification workflow we have designed is applicable to other protein complexes.
authors.affiliation: Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Paul Dominic B
authors.initials: PD
authors.lastname: Olinares
authors.forename: Jitae
authors.initials: J
authors.lastname: Kim
authors.forename: Jerrold I
authors.initials: JI
authors.lastname: Davis
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: van Wijk
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D010450:Endopeptidases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D018087:Plastids / Q000201:enzymology* / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D029681:Arabidopsis Proteins; D046912:Multiprotein Complexes; D010455:Peptides; D021122:Protein Subunits; D010450:Endopeptidases