Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism

The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a “systems view” of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.Intracellular proteolysis is important for regulation of metabolic and signaling pathways as well as protein homeostasis and viability of cells and organelles. Chloroplasts contain multiple soluble and membrane-bound proteases and processing peptidases (1) presumably with partially overlapping substrates. These include stromal processing peptidase (2) and stromal PreP1,2 involved in degradation of cleaved transit peptides (3); various amino peptidases (4, 5); the thylakoid processing peptidases cTPA (6), TPP (7), and thylakoid/envelope signal peptidase I (8); and thylakoid-bound proteases SppA (9) and Egy1 (10) as well as stromal and thylakoid members of the Deg, FtsH, and Clp families (11–13). Together with several chaperone systems, including CPN60/CPN10, HSP70/DnaJ, HSP90, and ClpB3 (14), these proteases are part of the chloroplast protein homeostasis network. Importantly the connectivity and overlap of proteins within this homeostasis network is poorly understood; in particular it is unclear how protease substrates are recognized by the different proteolytic systems. Several suppressors of variegated FtsH protease mutants in Arabidopsis have elegantly demonstrated that the balance between protein synthesis and degradation plays an important role in chloroplast homeostasis (15–17). Comparative proteome analysis of chloroplast homeostasis mutants will provide insights in this homeostasis network as we recently showed for a protein sorting mutant (18), and it will identify candidate protease substrates.The Clp proteins form the largest plastid localized protease family with five serine-type ClpP (P1,P3–6) proteases, four non-catalytic ClpR (R1–4) proteins, three Clp AAA⁺ chaperones (C1,C2, and D), and several additional members (ClpT1,T2,S) with unknown functions (1, 13, 19). We note that we renamed Arabidopsis ClpS1,S2 and ClpT as ClpT1,T2 and ClpS to be consistent with the Escherichia coli nomenclature for ClpS (20). The ClpR proteins lack the three catalytic amino acid residues that are conserved across ClpP proteins (21). All proteins of the Arabidopsis Clp proteolytic system have been identified by mass spectrometry (13), including a potential substrate affinity regulator, ClpS.¹Recent evidence shows that the Clp proteolytic system plays a critical role in plant growth, development, and protein homeostasis. ClpP1 is plastid-encoded and was shown to be essential for shoot development in tobacco (22, 23). Down-regulation of the plastid-encoded CLPP1 gene in the green algae Chlamydomonas reinhardtii suggested that ClpP1 is involved in the degradation of the thylakoid-bound subunits of cytochrome b₆f and photosystem II (PSII)² complex (24, 25). Arabidopsis mutant clpr1-1 carries a premature stop codon in the CLPR1 gene and showed a virescent phenotype and delayed chloroplast development and differentiation (26). Maturation of 23 and 4.5 S chloroplast ribosomal RNA (rRNA) is delayed in clpr1-1 (26), but it is not clear how this is related to the loss of ClpR1 protein. Phenotypes of Arabidopsis antisense lines against CLPP4 (27) and CLPP6 (28) also showed delayed chloroplast and plant development as well as reduced accumulation of other ClpP,R subunits. Based on two-dimensional gel analysis, several chloroplast proteins were suggested to be substrates of the Clp machinery (28–30), but direct evidence is lacking. A null mutant for the CLPC1 chaperone (also named HSP93-V) resulted in reduced plant growth, chloroplast development, and protein import rates, but homozygous plants are autotrophic and seeds are viable (31–33). A null mutant for chaperone CLPC2 has no visible phenotype, whereas lack of both CLPC1 and CLPC2 prevents embryogenesis (34). Interestingly ClpC1 is also involved in accumulation of chlorophyll a oxygenase, which is responsible for conversion of chlorophyll a to chlorophyll b (35).In a previous study, we identified and characterized a T-DNA-tagged Arabidopsis thaliana mutant with reduced expression of CLPR2; this mutant was named clpr2-1 (36). Accumulation of the assembled 325-kDa ClpPRT complex was 2–3-fold reduced and resulted in delayed chloroplast and plant development with small chloroplasts and a pale green phenotype. The clpr2-1 mutant shows the strongest visible phenotype when seedlings are young. To better understand the role of the Clp machinery in chloroplast biogenesis and homeostasis and to discover potential Clp substrates, a comprehensive proteome analysis at different points in leaf development of the clpr2-1 mutant is presented in the current study. The methods to quantitatively analyze differences in protein accumulation have greatly improved over the last decade and have shifted from gel image-based quantification to quantification within the mass spectrometer (37–39). Taking advantage of these new developments and opportunities, we compared the leaf proteome of clpr2-1 and wt seedlings early in development using spectral counting. This was complemented with a comparative analysis of the chloroplast soluble proteome of fully developed leaf rosettes. The seedling proteome analysis showed that the strongest effects occurred within the chloroplast. The functional significance of one of the most up-regulated proteins, ClpB3, was confirmed by additional mutant analysis. Putative substrates for the Clp system suggested in recent studies (28–30, 35) are reviewed in the context of our findings. This study provides testable hypotheses to further determine the significance of Clp-driven proteolysis and provides new insights in the plastid protein homeostasis network and how secondary metabolism is intertwined with photosynthetic capacity. We show that a systems view of chloroplast biogenesis and proteome homeostasis is needed to identify putative protease substrates and to understand the role of proteolysis in chloroplast biology. Finally we believe that the experimental setup described in this study provides an attractive template for comparative proteome analysis of other (chloroplast) mutants.