Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2009 Aug;8(8):1789-1810.
doi: 10.1074/mcp.M900104-MCP200.

Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism

Boris Zybailov  1 , Giulia Friso, Jitae Kim, Andrea Rudella, Verenice Ramírez Rodríguez, Yukari Asakura, Qi Sun, Klaas J van Wijk
Affiliations
Comparative Study

Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism

Boris Zybailov et al. Mol Cell Proteomics. 2009 Aug.

Abstract

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.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Comparative proteome analysis of wt and clpr2-1 and CLPB3 mutant analysis. A, outline of the comparative proteome analysis of clpr2-1 and wt. Total leaves were harvested at growth stages 1.07 and 1.14 at, respectively, 18 and 23 days for wt and 34 and 46 days for clpr2-1 for the comparison of total cellular proteomes. Soluble stromal proteins were collected from chloroplasts isolated from fully developed wt and clpr2-1 plants at growth stage 3.90 at 42 and 90 days, respectively, for wt and clpr2-1. The seedling proteomes were compared using the spectral counting technique from data obtained by nano-LC-ESI-LTQ-Orbitrap, whereas the stromal proteomes were compared using spectral counting and ICAT using data obtained by nano-LC-ESI-Q-TOF. B, example of 1-D electrophoresis gels of extracted seedling proteomes at stage 1.07 and stage 1.14. Gels were stained with Coomassie Brilliant Blue, and each lane was cut in 12 slices followed by manual in-gel digestion and extraction of peptides. Samples were analyzed in duplicate on the LC-LTQ-Orbitrap following the gradient and injection scheme as depicted. Two blanks were injected after each sample to prevent carryover between samples. C, comparison of the wt, clpr2-1, clpb3-1, and clpb3-1xclpr2-1 (b3-1 x r2-1) mutants grown on Murashige and Skoog medium + 2% sucrose for 10 weeks under a 10-h light/14-h dark cycle at 60 μmol photons·m−2·s−1. DDA, data-dependent acquisition.
Fig. 2.
Fig. 2.
Quantification of clpr2-1 and wt seedling proteomes using spectral counting. Cross-correlation between the number of spectral counts in clpr2-1 and wt and significant changes for the 768 quantified proteins using log-scaled scatter plots are shown. A, 711 proteins quantified in the 1.07 growth stage seedlings, biological replicate 1. B, 711 proteins quantified in the 1.07 growth stage seedlings, biological replicate 2. C, 537 proteins quantified in the 1.14 growth stage. Horizontal and vertical coordinates correspond to the SPCs measured in clpr2-1 and wt, respectively. Closed dark circles correspond to proteins that showed significant changes in accumulation levels as determined by the G-test (95% confidence) and passed the requirement of consistent direction of change across both technical replicates. Open gray circles correspond to the quantified proteins that either showed no statistical evidence for differential accumulation or failed the consistency requirement. All proteases are indicated by squares with black filled squares indicating those that are significant. Two gray lines marking 2 and 0.5 clpr2-1/wt ratios are shown. Names of selected proteins are indicated. Abbreviations not provided in the main text are: MDAR, monodehydroascorbate reductase; P5CS A, Δ1-pyrroline-5-carboxylate synthetase A; EF1Bα2, elongation factor 1β subunit α-2; EDGP, extracellular dermal glycoprotein; NDH-H, NADPH dehydrogenase complex subunit H; PPR, pentapeptide repeat protein At5g46580; CYS-synth, cysteine synthase; RBCL, Rubisco large subunit; PC, plastocyanin.
Fig. 3.
Fig. 3.
Bar diagram displaying the clpr2-1/wt protein accumulation ratios (in log scale) for the significantly affected proteins involved in protein homeostasis (A), photosynthetic electron transport located in the thylakoid (B), and primary and secondary metabolism as well proteins located in plastoglobules (C). The bars represent quantifications for the different biological replicates for the seedlings at growth stages 1.07 and 1.14 and for chloroplast stroma isolated from mature leaves. The standard deviations show the variation between the technical replicates. Bars marked with an asterisk (*) indicate a significant change for that biological replicate as determined by the G-test with 95% confidence. RBCL, Rubisco large subunit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Fd-GOGAT, ferredoxin-dependent glutamate synthase; RBCS, Rubisco small subunit; LHC, light-harvesting complex; GGDR, geranylgeranyl reductase; WDK, water dikinase.
Fig. 4.
Fig. 4.
rRNA processing is affected in clpr2-1 seedlings. To determine whether the clpr2-1 mutant showed any defects in 70 S chloroplast rRNA processing and ribosome assembly, rRNA populations from stages 1.07 or 1.08 and stage 1.14 clpr2-1 and wt plants were determined. A, sucrose density centrifugation and RNA analysis by ethidium bromide staining. The two cytosolic 18 and 25 S rRNAs are clearly visible in 40 and 60 S particles and assembled 80 S particles. Two additional, high molecular mass bands (between the 18 and 25 S bands) are visible in the clpr2-1 seedlings and represent unprocessed rRNA species. B, Northern blot analysis of total RNA extracted from wt and clpr2-1 seedling leaves. RNA was blotted to membrane and stained by methylene blue or analyzed with specific probes against chloroplast ribosomal RNA molecules derived from genes rrn23, rrn4.5, rrn16, and rrn5 as indicated above each panel. RNA species accumulating in clpr2-1 are indicated by asterisks and arrows.
Fig. 5.
Fig. 5.
Changes in the plastid-localized MEP and chlorophyll biosynthetic pathways in the clpr2-1 mutant. A, schematic overview of the isoprenoid and chlorophyll synthesis pathways in chloroplasts Protein names that are boxed (solid line) were significantly up-regulated in clpr2-1 seedlings: HDS, 9-fold; GUN5, 11-fold up; CHLI-1, 3-fold up; and GGR, 1.9-fold. 1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) (boxed dashed line) was on average 2-fold increased in both replicates at stage 1.07, but it did not pass our significance test. B, Western blot analysis of young clpr2-1 and wt seedlings showed that 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT) levels are increased 2-fold in clpr2-1. The upper panel shows the immune response, and the lower panel shows a control for protein loading from the Ponceau red stain of the transfer blot prior to immunodecoration. Proteins are loaded as a dilution series with 1 corresponding to 20 μg of protein. Metabolite names are written in italics. DXS, 1-deoxy-d-xylulose-5-phosphate synthase; HDR, hydroxymethylbutenyl-diphosphate reductase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; PP, diphosphate; GGPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate; GG, geranylgeranyl; chllide, chlorophyllide; CMK, 4-diphosphocytidyl 2-C-methyl-d-erythritol kinase; MDS, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; FLU, fluorescent in blue light.
Fig. 6.
Fig. 6.
Summarizing overview of the consequences of reduced accumulation of the ClpPR complex. The nucleus-encoded proteins are imported through the Toc/Tic translocon in the chloroplast envelope followed by removal of the N-terminal chloroplast transit peptide (cTP), folding, and assembly (61, 117). Proteins destined for the thylakoid membrane system, including the thylakoid lumen, are targeted following different pathways and involving different sorting components (118). The chloroplast-encoded proteins are synthesized on 70 S ribosomes either in the chloroplast stroma or at the thylakoid surface (118). Proteins that were significantly up-regulated in clpr2-1 seedlings are: Tic110 (1); PreP1,2 (2); cpHSP70 and CPN60 (3); cpHSP90 (4); ClpB3 (5); EF-BipA, EF-TU, and DEAD box helicase RH3 (6); FIB1a,B (8); GGR (9); HDS (10); THI1 (11); GUN5 and CHLI-1 (12); and water dikinase (WDK)/SEX1 (13). Proteins that were significantly down-regulated in clpr2-1 seedlings are components of PSII, PSI, and ATP synthase (7). Chl, chlorophyll; PPP, triphosphate; GG, geranylgeranyl; GGPP, geranylgeranyl diphosphate; vit, vitamin.
See this image and copyright information in PMC

References

    1. Sakamoto W. ( 2006) Protein degradation machineries in plastids. Annu. Rev. Plant Biol. 57, 599– 621 - PubMed
    1. Richter S., Lamppa G. K. ( 1998) A chloroplast processing enzyme functions as the general stromal processing peptidase. Proc. Natl. Acad. Sci. U.S.A. 95, 7463– 7468 - PMC - PubMed
    1. Ståhl A., Nilsson S., Lundberg P., Bhushan S., Biverståhl H., Moberg P., Morisset M., Vener A., Mäler L., Langel U., Glaser E. ( 2005) Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different. J. Mol. Biol. 349, 847– 860 - PubMed
    1. Walling L. L. ( 2006) Recycling or regulation? The role of amino-terminal modifying enzymes. Curr. Opin. Plant Biol. 9, 227– 233 - PubMed
    1. Giglione C., Boularot A., Meinnel T. ( 2004) Protein N-terminal methionine excision. Cell. Mol. Life Sci. 61, 1455– 1474 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources