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
. 2011 Jan;23(1):322-32.
doi: 10.1105/tpc.110.082321. Epub 2011 Jan 25.

Assembly of the chloroplast ATP-dependent Clp protease in Arabidopsis is regulated by the ClpT accessory proteins

Lars L E Sjögren  1 , Adrian K Clarke
Affiliations

Assembly of the chloroplast ATP-dependent Clp protease in Arabidopsis is regulated by the ClpT accessory proteins

Lars L E Sjögren et al. Plant Cell. 2011 Jan.

Abstract

The ATP-dependent caseinolytic protease (Clp) is an essential housekeeping enzyme in plant chloroplasts. It is by far the most complex of all known Clp proteases, with a proteolytic core consisting of multiple catalytic ClpP and noncatalytic ClpR subunits. It also includes a unique form of Clp protein of unknown function designated ClpT, two of which exist in the model species Arabidopsis thaliana. Inactivation of ClpT1 or ClpT2 significantly reduces the amount of Clp proteolytic core, whereas loss of both proves seedling lethal under autotrophic conditions. During assembly of the Clp proteolytic core, ClpT1 first binds to the P-ring (consisting of ClpP3-6 subunits) followed by ClpT2, and only then does the P-ring combine with the R-ring (ClpP1, ClpR1-4 subunits). Most of the ClpT proteins in chloroplasts exist in vivo as homodimers, which then apparently monomerize prior to association with the P-ring. Despite their relative abundance, however, the availability of both ClpT proteins is rate limiting for the core assembly, with the addition of recombinant ClpT1 and ClpT2 increasing core content up to fourfold. Overall, ClpT appears to regulate the assembly of the chloroplast Clp protease, revealing a new and sophisticated control mechanism on the activity of this vital protease in plants.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Null Mutations Affecting the Arabidopsis CLPT1 and CLPT2 Genes. (A) Schematic representation of the two CLPT genes (with accession number underneath each) and the location of the T-DNA insertion (triangle) in the corresponding clpT1 and clpT2 mutants. Protein-coding exons and untranslated regions (UTRs) are represented by black and white boxes, respectively, with introns drawn as thin lines between the boxes. (B) Immunoblot analysis of ClpT1 and ClpT2 in the wild type (WT) and clpT mutant lines. Total leaf protein was extracted from each plant and separated by denaturing-PAGE on the basis of equal chlorophyll content. ClpT1 (19.5 kD) and ClpT2 (19 kD) were detected by immunoblotting with specific antibodies as indicated below each panel. Molecular mass markers (kD) are shown on the left. (C) A typical, mature silique from a heterozygous F1 clpT1 clpT2 double-knockout line containing both normal (green) and abnormal (white; arrowheads) seeds. [See online article for color version of this figure.]
Figure 2.
Figure 2.
Relative Amounts of Chloroplast Clp Proteins in Wild-Type Arabidopsis and clpT Null Mutants. Stromal proteins from 3-week-old plants were separated by denaturing-PAGE on the basis of equal protein content. The various chloroplast Clp proteins were detected by immunoblotting using specific polyclonal antibodies (A) and the relative amounts of each were then quantified (B). Values shown are averages ± se (n = 3) with the wild-type (WT) values set to 100%. The molecular masses in kilodaltons of the proteins detected are as follows: 20.5 (ClpP1), 28.5 (ClpP3), 27 (ClpP4), 22.5 (ClpP5), 21.5 (ClpP6), 28 (ClpR1), 25 (ClpR2), 28.5 (ClpR3), 24.5 (ClpR4), 19.5 (ClpT1), 19 (ClpT2), 93 (ClpC), and 96 (ClpD).
Figure 3.
Figure 3.
Clp Protein Complexes in Wild-Type Arabidopsis and clpT Null Mutants. (A) Schematic representation of the chloroplast Clp proteolytic core and its various subcomplexes as previously identified, indicating relative size and subunit composition. (B) Clp proteolytic core complexes in stromal fractions from 3-week-old wild type (WT) and clpT null mutants were separated by native-PAGE on the basis of equal protein content. The different complexes of the Clp proteolytic core (indicated on the left) were visualized by immunoblotting with antibodies specific for selected marker subunits of each as indicated below the panels (ClpP6 for the core, P-ring, and P/T1-ring; ClpR3 for the core and R-ring; ClpT1 for the core, P/T1-ring, and unknown T1 oligomer; ClpT2 for the core and unknown T2 oligomer). (C) Quantification of the relative amount of the Clp proteolytic core and other Clp subcomplexes in the clpT null mutants based on the immunoblots with the ClpP6 and ClpR3 antibodies. Values shown are averages ± se (n = 6) and plotted relative to the wild-type value for the core complex, which was set to 100%. (D) Detection of the 200-kD ClpT2 oligomer in stromal fractions from 3-week-old wild-type Arabidopsis and a clpP6 antisense line in which the Clp core, P-ring, and ClpP/T1-ring are reduced to ~10% wild-type levels (as confirmed using the ClpP3 antibody). [See online article for color version of this figure.]
Figure 4.
Figure 4.
Purification of Recombinant ClpT1 and ClpT2. Soluble C-terminally His-tagged Arabidopsis ClpT1 (A) and ClpT2 (B) were overexpressed in E. coli upon induction with IPTG. Each ClpT protein was purified from cell lysates by sequential Ni2+ affinity chromatography and gel filtration. Fractions before (−IPTG) and after (+IPTG) induction in E. coli and after each chromatography step were analyzed by denaturing-PAGE and Coomassie blue staining. The molecular masses of the recombinant ClpT1 (rClpT1) and ClpT2 (rClpT2) were then compared with those of the native stromal ClpT proteins (stroma) isolated from wild-type Arabidopsis by immunoblotting with specific ClpT antibodies.
Figure 5.
Figure 5.
Size Determination of the ClpT Oligomers. Native and recombinant ClpT1 (A) and ClpT2 (B) were separated by a modified form of native-PAGE designed specifically to size slow migrating protein complexes accurately. The rClpT oligomers were visualized by Coomassie blue staining (Stain), the sizes of which were then compared with those of native ClpT1 and ClpT2 in stromal fractions (Stroma) from wild-type Arabidopsis by immunoblotting with ClpT-specific antibodies. Molecular mass standards in kilodaltons are shown on the left.
Figure 6.
Figure 6.
Restored Assembly of the Clp Proteolytic Core by Addition of Recombinant ClpT Proteins. Stromal fractions from wild-type (WT) Arabidopsis and the two clpT mutants were isolated from 3-week-old plants. Different amounts of rClpT (0.03 to 3% of total stromal protein content) were then added to the stroma from the clpT mutants and left for 1 min at 22°C. Stromal fractions were then separated by native-PAGE and the various complexes of the Clp proteolytic core identified by immunoblotting with specific marker antibodies (ClpP6 and ClpR3). (A) rClpT1 was added to stroma from the clpT1 mutant and rClpT2 to stroma from clpT2 mutant. (B) rClpT1 was added to stroma from clpT2 and rClpT2 added to stroma from clpT1.
Figure 7.
Figure 7.
Increased Clp Proteolytic Core Content in Wild-Type Arabidopsis by Addition of Recombinant ClpT. Stroma was extracted from 3-week-old wild-type leaves and then incubated with excess rClpT1 for 1 min at 22°C and then excess rClpT2 for a further 1 min (both ClpT proteins were added at 1% of total stromal protein content). Aliquots of stroma were taken prior to rClpT1 addition (WT), after 1 min incubation with added rClpT1 (WT + rClpT1), and after 1 min incubation with rClpT2 (WT + rClpT1 + rClpT2). The reverse experiment was then performed adding first rClpT2 and then rClpT1 following the same procedure as above. Aliquots were separated by native-PAGE, and complexes of the Clp proteolytic core identified by immunoblotting with specific marker antibodies (ClpP6 and ClpR3) (A) with the relative amount of the Clp proteolytic core then quantified (B). Values shown are averages ± se (n = 3) with the wild-type values set to 100%.
Figure 8.
Figure 8.
Model for the Assembly Pathway of the Chloroplast Clp Protease in Arabidopsis. The bulk of ClpT1 and ClpT2 in the stroma exists as homogeneous dimers. Prior to association with the Clp proteolytic core, both ClpT dimers undergo monomerization by an unknown mechanism. Both ClpT monomers presumably have high affinity for the P-ring since they do not accumulate within the wild-type stroma (as indicated by parentheses). ClpT1 binds first to the P-ring followed by ClpT2. It is the P/T1/T2-ring that has high affinity for the R-ring to form the intact Clp proteolytic core complex. Later association of the ClpC chaperone partner completes the assembly of the active Clp protease. [See online article for color version of this figure.]
See this image and copyright information in PMC

References

    1. Adam Z., Adamska I., Nakabayashi K., Ostersetzer O., Haussuhl K., Manuell A., Zheng B., Vallon O., Rodermel S.R., Shinozaki K., Clarke A.K. (2001). Chloroplast and mitochondrial proteases in Arabidopsis. A proposed nomenclature. Plant Physiol. 125: 1912–1918 - PMC - PubMed
    1. Adam Z., Clarke A.K. (2002). Cutting edge of chloroplast proteolysis. Trends Plant Sci. 7: 451–456 - PubMed
    1. Alonso J.M., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657 - PubMed
    1. Andersson F.I., Blakytny R., Kirstein J., Turgay K., Bukau B., Mogk A., Clarke A.K. (2006). Cyanobacterial ClpC/HSP100 protein displays intrinsic chaperone activity. J. Biol. Chem. 281: 5468–5475 - PubMed
    1. Andersson F.I., et al. (2009). Structure and function of a novel type of ATP-dependent Clp protease. J. Biol. Chem. 284: 13519–13532 - PMC - PubMed

Publication types

MeSH terms