Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions. We identified a single promoter region for the clpP1P2 operon; no promoter was detected upstream of clpP2 demonstrating that independent expression of clpP1 and clpP2 was highly unlikely. Promoter activity was not induced by heat shock or oxidative stress. We identified a regulatory region upstream of the promoter with a consensus sequence matching the ClgR regulator motif; we determined the limits of the region by mutagenesis and confirmed that positive regulation of the promoter occurs in M. tuberculosis. We developed a reporter system to monitor ClpP1 and ClpP2 enzymatic activities based on LacZ incorporating ssrAtag sequences. We showed that whilst both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, ClpP2 (but not ClpP1) degrades untagged proteins. Our data suggest that the two proteolytic subunits display different substrate specificities and therefore have different, but overlapping roles in M. tuberculosis.     

The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperone rings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation. Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Mycobacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2, and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently, this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobic chaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clp proteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core, and it frees the ClpP1 interaction interface for putative new binding partners.     

Validation of the essential ClpP protease in Mycobacterium tuberculosis as a novel drug target

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.     

The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring

Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N-blocked dipeptides that stimulate dramatically (>1000-fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re-associate to form the active ClpP1P2 2-ring mixed complex. No analogous small molecule-induced enzyme activation mechanism involving dissociation and re-association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase-like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis.     

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram‐positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl‐leucyl‐leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP‐activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP‐dependent protein degradation.     

Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase

Mycobacterial Clp‐family proteases function via collaboration of the heteromeric ClpP1P2 peptidase with a AAA+ partner, ClpX or ClpC1. These enzymes are essential for M. tuberculosis viability and are validated antibacterial drug targets, but the requirements for assembly and regulation of functional proteolytic complexes are poorly understood. Here, we report the reconstitution of protein degradation by mycobacterial Clp proteases in vitro and describe novel features of these enzymes that distinguish them from orthologues in other bacteria. Both ClpX and ClpC1 catalyse ATP‐dependent unfolding and degradation of native protein substrates in conjunction with ClpP1P2, but neither mediates protein degradation with just ClpP1 or ClpP2. ClpP1P2 alone has negligible peptidase activity, but is strongly stimulated by translocation of protein substrates into ClpP1P2 by either AAA+ partner. Interestingly, our results support a model in which both binding of a AAA+ partner and protein‐substrate delivery are required to stabilize active ClpP1P2. Our model has implications for therapeutically targeting ClpP1P2 in dormant M. tuberculosis, and our reconstituted systems should facilitate identification of novel Clp protease inhibitors and activators.     

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X₃X₂X₁-aminomethylcoumarin) and by determining specificity constants (k_cat/K_m), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X₁ position, basic residues or Trp in the X₂ position, and Pro ≫ Ala > Trp in the X₃ position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.     

Inactivation of the clpP1 gene for the proteolytic subunit of the ATP-dependent Clp protease in the cyanobacterium Synechococcus limits growth and light acclimation

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 µmol photons m^-2 s^-1. A clpP1 inactivation strain (clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.     

Multistep Processing of an Insertion Sequence in an Essential Subunit of the Chloroplast ClpP Complex

In Chlamydomonas reinhardtii, the clpP1 chloroplast gene encoding one of the catalytic subunits of the ClpP protease complex contains a large in-frame insertion sequence (IS1). Based on the Escherichia coli ClpP structure, IS1 is predicted to protrude at the apical surface of the complex, likely influencing the interaction of the catalytic core with ClpC/HSP100 chaperones. Immunoblotting with an anti-ClpP1 antibody detected two immunoreactive forms of ClpP1: ClpP1_H (59 kDa) and ClpP1_L (25 kDa). It has been proposed that IS1 is a new type of protein intron (different from inteins). By studying transformants harboring mutations at the predicted borders of IS1 and tags at the C terminus of ClpP1 (tandem affinity purification tag, His tag, Strep·Tag) or within the IS1 sequence (3-hemagglutinin tag), we show that IS1 is not a protein intron and that ClpP1_L results from endoproteolytic cleavage inside IS1. Processing sites have been identified in the middle of IS1 and near its C terminus. The sites can be mutated without abolishing processing.Clp proteases are self-compartmentalized serine proteases present in most eubacteria and, as a consequence of endosymbiotic events, in the mitochondrion and chloroplast of eukaryotes. In Escherichia coli, the organism in which they have been best characterized, Clp proteases associate a homo-oligomeric peptidase (ClpP) and a chaperone (ClpA or ClpX) that belongs to the Clp/HSP100 family, itself part of the large group of AAA⁺ ATPases (1–4). ClpP is composed of 14 identical subunits arranged in two heptameric rings related by central symmetry. They form a barrel-like structure with the 14 active sites facing an inner proteolytic chamber (5). ClpP alone is able to degrade only small peptides (6), and the recognition and unfolding of protein substrates are carried out by the Clp/HSP100 chaperone. The chaperone docks on the apical surfaces of ClpP and uses ATP hydrolysis to unfold and feed substrates through the ClpP axial pore into the proteolytic chamber (7–10).In chloroplasts, ClpP is present as a hetero-oligomer associating up to eight different types of subunit. This is the result of a gene diversification process that has begun in cyanobacteria and continues in the chloroplast of land plants. Not only has the number of clpP genes grown, but clpR genes have appeared that carry mutations in at least one residue of the catalytic triad and are thus presumed catalytically inactive. In the green alga Chlamydomonas reinhardtii, three clpP genes (clpP1, CLPP4, and CLPP5) and five clpR genes (CLPR1–CLPR4 and CLPR6) code for the subunits of the chloroplast ClpP complex (11). An additional CLPP2 gene codes for the homo-oligomeric mitochondrial ClpP.ClpP1 is the only subunit that is encoded in the chloroplast and probably the best conserved. In C. reinhardtii, clpP1 contains a large insertion sequence (IS1)3 translated in-frame with the conserved N- and C-terminal regions. This results in a protein about twice as large (∼59 kDa) as in other organisms. Chlamydomonas ClpP1 can be divided into two sequence domains, SD1 and SD2 (the latter containing the catalytic residues), corresponding to the conserved sequence, and one insertion sequence, IS1 (12). In C. reinhardtii, antisera raised against the entire open reading frame (ORF) recognize two products of clpP1 in Western blot: ClpP1_H (59 kDa) and ClpP1_L (21 kDa) (13). As the clpP1 mRNA does not undergo splicing (12), it has been proposed that IS1 could be a protein intron. Protein introns such as inteins (14) are defined as in-frame intervening sequences that disrupt a host gene and are post-translationally excised by a self-catalytic mechanism. In the case of clpP1, ClpP1_H would be the precursor protein and ClpP1_L the spliced form. However, IS1 lacks the sequence motifs characteristic of inteins. In addition, both ClpP1_L and ClpP1_H are stable, and both associate in the 540-kDa ClpP complex (11). Thus, if IS1 were a protein intron, it would be an unusual type. In the related species Chlamydomonas eugametos, clpP1 contains, in addition to IS1, another insertion sequence (IS2) displaying most of the sequence features of inteins. Indeed, IS2 can be induced to self-splice in E. coli by changing a single residue (15).In this study, we show that IS1 is not a protein intron and that ClpP1_L is the product of a complex proteolytic maturation of ClpP1_H. We have found similar insertion sequences in the clpP1 genes of other green algae from the group Chlorophyceae. Green algae accumulate such insertion sequences in many of their chloroplast genes, probably as a result of a high frequency of genome rearrangements.     

A ClpP protein model as tuberculosis target for screening marine compounds

ATP-dependent Clp protease (ClpP) is a core unit of a major bacterial protease complex employing as a new attractive drug target for that isolates, which are resistant to antibiotics. Mycobacterium tuberculosis, a gram-positive bacterium, is one of the major causes of hospital acquired infections. ClpP in Mycobacterium tuberculosis is usually tightly regulated and strictly requires a member of the family of Clp-ATPase and often further accessory proteins for proteolytic activation. Inhibition of ClpP eliminates these safeguards and start proteolytic degradation. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death. In order to inhibit Clp protease, at first three dimensional structure model of ClpP in Mycobacterium tuberculosis was determined by comparative homology modeling program MODELLER based on crystal structure of the proteolytic component of the caseinolytic Clp protease (ClpP) from E. coli as a template protein and has 55%sequence identity with ClpP protein. The computed model's energy was minimized and validated using PROCHECK to obtain a stable model structure and is submitted in Protein Model Database (PMDB-ID: PM0075741). Stable model was further used for virtual screening against marine derived bioactive compound database through molecular docking studies using AutoDock 3.05. The docked complexes were validated and enumerated based on the AutoDock Scoring function to pick out the best marine inhibitors based on docked Energy. Thus from the entire 186 Marine compounds which were Docked, we got best 5 of them with optimal docked Energy (Ara-A: -14.31 kcal/mol, Dysinosin C: - 14.90kcal/mol, Nagelamide A: -20.49 kcal/mol, Strobilin: -8.02 kcal/mol, Manoalide: -8.81 kcal/mol). Further the five best-docked complexes were analyzed through Python Molecular Viewer software for their interaction studies. Thus from the Complex scoring and binding ability its deciphered that these Marine compounds could be promising inhibitors for ClpP as Drug target yet pharmacological studies have to confirm it.     

Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis

In contrast with the model Escherichia coli Clp protease, the ATP-dependent Clp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple ClpP and ClpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded ClpP subunit, ClpP6, we show that the Arabidopsis thaliana Clp protease is vital for chloroplast development and function. Repression of ClpP6 produced a proportional decrease in the Clp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the ClpP1 and ClpR1-4 proteins, the other containing ClpP3-6. Proteomic analysis revealed several stromal proteins more abundant in clpP6 antisense lines, suggesting that some are substrates for the Clp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal Clp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the clpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, and quality control, rather than in metabolic activities such as photosynthesis.     

The ClpP component of Clp protease is the sigma 32-dependent heat shock protein F21.5.

The genes that encode the subunits of the Clp protease of Escherichia coli, clpA and clpP, appear to be regulated differently from each other. The clpA gene does not seem to be under heat shock control (Y. S. Katayama, S. Gottesman, J. Pumphrey, S. Rudikoff, W. P. Clark, and M. R. Maurizi, J. Biol. Chem. 263:15226-15236, 1988). In contrast, the level of ClpP protein was increased in rpoH+ cells but not in null rpoH cells after an upshift in temperature from 17 to 43 degrees C. The level of ClpP protein in a null dnaK strain was also elevated relative to the level of ClpP protein in an otherwise isogenic dnaK+ strain. In two-dimensional gels, the ClpP protein was located in the position of the previously unidentified heat shock protein F21.5. No protein spot corresponding to F21.5 was present in two-dimensional gels of a null clpP strain. The clpP gene, therefore, appears to be a heat shock gene, expressed in a sigma 32-dependent manner and negatively regulated by DnaK; the product of clpP is the previously unidentified heat shock protein F21.5.     

The ATP-dependent Clp protease is essential for acclimation to UV-B and low temperature in the cyanobacterium Synechococcus

ClpP is the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. Cyanobacterial ClpP protein is encoded by a multigene family, producing up to four distinct isozymes. We have examined the importance of the first ClpP protein (ClpP1) isolated from the cyanobacterium Synechococcus sp. PCC 7942 for acclimation to ecologically relevant UV-B and low-temperature regimens. When the growth light of 50 μmol photons m^?2 s^?1 was supplemented with 0.5 W m^?2 UV-B for 8 h, the constitutive level of ClpP1 rose eightfold after an initial lag of 1 h. Wild-type cells readily acclimated to this UV-B level, recovering after the initial stress to almost the same growth rate as that before UV-B exposure. Growth of a clpP1 null mutant (ΔclpP1), however, was severely inhibited by UV-B, being eight times slower than the wild type after 8 h. In comparison, ClpP1 content increased 15-fold in wild-type cultures shifted from 37°C to 25°C for 24 h. Wild-type cultures readily acclimated to 25°C after 24 h, whereas the ΔclpP1 strain did not and eventually lost viability with prolonged cold treatment. During acclimation to either UV-B or cold, photosynthesis in the wild type was initially inhibited upon the shift but then recovered. Photosynthesis in ΔclpP1 cultures, however, was more severely inhibited by the stress treatment and failed to recover. Acclimation was also monitored by examining the exchange of photosystem II reaction centre D1 proteins that occurs in wild-type Synechococcus during conditions of excitation stress. During both cold and UV-B shifts, wild-type cultures replaced the acclimative form of D1 (D1:1) with the alternative D1 form 2 (D1:2) within the first hours. Once acclimated to either 25°C or 0.5 W m^?2 UV-B, D1:2 was exchanged back for D1:1. In ΔclpP1 cultures, this second exchange between D1 forms did not occur, with D1:2 remaining the predominant D1 form. Our results demonstrate that the ATP-dependent Clp protease is an essential component of the cold and UV-B acclimation processes of Synechococcus.     

Role of Clp protease subunits in degradation of carbon starvation proteins in Escherichia coli.

When deprived of a carbon source, Escherichia coli induces the synthesis of a group of carbon starvation proteins. The degradation of proteins labeled during starvation was found to be an energy-dependent process which was inhibited by the addition of KCN and accelerated when cells were resupplied with a carbon source. The degradation of the starvation proteins did not require the ATP-dependent Lon protease or the energy-independent proteases protease I, protease IV, OmpT, and DegP. During starvation, mutants lacking either the ClpA or ClpP subunit of the ATP-dependent Clp protease showed a partial reduction in the degradation of starvation proteins. Strains lacking ClpP failed to increase degradation of starvation proteins when glucose was added to starving cells. The clpP mutants showed a competitive disadvantage compared with wild-type cells when exposed to repeated cycles of carbon starvation and growth. Surprisingly, the glucose-stimulated, ClpP-dependent degradation of starvation proteins did not require either the ClpA or ClpB protein. The patterns of synthesis of starvation proteins were similar in clpP+ and clpP cells. The clpP mutants had reduced rates of degradation of certain starvation proteins in the membrane fraction when a carbon source was resupplied to the starved cells.     

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.     

Subunit stoichiometry, evolution, and functional implications of an asymmetric plant plastid ClpP/R protease complex in Arabidopsis

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.