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.

Modified Clp Protease Complex in the ClpP3 Null Mutant and Consequences for Chloroplast Development and Function in Arabidopsis

“…The homozygous clpp3-1 could be fully complemented with genomic CLPP3 (Fig. 1D) and to a lesser extent also with 1335S-cDNA-CLPP3-StrepII (Olinares et al, 2011a). PCR of genomic DNA (data not shown) and RT-PCR (Fig.…”

“…Furthermore, three Clp AAA+ chaperones (C1, C2, and D) similar to the Escherichia coli ClpA and the adaptor ClpS (homologous to the E. coli ClpS) likely serve to deliver protein substrates to the core complex. Attached to the ClpPR core are ClpT1 and ClpT2, which have similarity to the N-terminal domain of the ClpC/ ClpD chaperones (Peltier et al, 2004;Olinares et al, 2011a). These ClpT subunits are unique to chloroplasts and have been hypothesized to regulate chaperone binding and/or substrate selection (Peltier et al, 2004;Olinares et al, 2011b) or to aid in the assembly of the ClpPR core complex (Sjögren and Clarke, 2011).…”

“…The quantitative subunit compositions for the intact ClpPR core and each heptameric ring were recently determined (Olinares et al, 2011a). The chloroplast ClpP/ ClpR protease was affinity purified from clpr4 and clpp3 Arabidopsis (Arabidopsis thaliana) null mutants complemented with C-terminal StrepII-tagged versions of CLPR4 and CLPP3, respectively.…”

“…This showed that the ClpPR core consisted of one heptameric ring containing ClpP3, ClpP4, ClpP5, and ClpP6 in a 1:2:3:1 ratio (designated the P-ring) and the other ring containing ClpP1 and ClpR1, ClpR2, ClpR3, and ClpR4 in a 3:1:1:1:1 ratio (designated the R-ring). Moreover, based on biochemical and phylogenetic analysis, it was suggested that ClpT1 and ClpT2 bind to the adaxial side of the P-ring (Olinares et al, 2011a;Sjögren and Clarke, 2011). This Clp core complexity is puzzling and is very different from the much simpler Clp composition in photosynthetic and nonphotosynthetic bacteria.…”

“…Thus, in contrast to CLPP4 and CLPP5 null mutants, clpp3-1 is the only null mutant in a ClpP protease subunit that can germinate, likely because other ClpPR subunits can partially substitute for ClpP3. Using the QconCAT technique (Olinares et al, 2011a), we also determined the composition of the ClpPR core in clpp3-1 in an effort to understand how the Clp system can function (even if suboptimally) without ClpP3. Furthermore, we quantified ClpT1/ClpT2 in the various Clp assemblies using immunodetection.…”