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. 2000 Jan;12(1):137-50.
doi: 10.1105/tpc.12.1.137.

Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex

W Majeran  1 , F A Wollman, O Vallon
Affiliations

Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex

W Majeran et al. Plant Cell. 2000 Jan.

Abstract

In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO(2) levels. It also affected the rate of degradation of the cytochrome b(6)f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron-sulfur protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b(6)f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b(6)f is controlled by the Clp protease.

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Figures

Figure 1.
Figure 1.
Map of the Inserts in the Plasmids Used for Mutagenesis. The positions of the genes and their translation direction are indicated, together with those of the primers used for PCR. The sequence surrounding the translation initiation site is indicated for the pWBI (control) and pdBATTK and pdBACCK (mutant) plasmids. Mutations introduced are indicated in bold, and the PvuI site is underlined.
Figure 2.
Figure 2.
Attenuation of ClpP by the clpP-AUU Mutation. (A) Immunoblot of total cell proteins taken from exponential-phase TAP cultures of the wild-type (WT), BI, clpP-AUU, and clpP-AUU2 (an independent transformant carrying the clpP-AUU mutation) strains. (B) Temperature-dependent attenuation of ClpP. Samples were taken from cultures of the wild type (WT) or clpP-AUU in exponential phase and grown at 18, 25, or 32°C. The amount of LMM-ClpP and HMM-ClpP was determined by immunoblotting with a ClpP antibody and PhosphorImager analysis. The value 100 represents the total immunoreactivity in the wild type at 18°C. AU, arbitrary units.
Figure 3.
Figure 3.
Growth Curves of the BI and clpP-AUU Strains. Cells grown in TAP medium at 60 μE m−2 sec−1 were diluted at 2 × 105 cells per mL in fresh medium and placed at 320 μE m−2 sec−1 with (clpP-AUU, open triangles; BI, X's) or without (clpP-AUU, filled diamonds; BI, filled squares) CO2 bubbling (5% in air).
Figure 4.
Figure 4.
Immunoblot Analysis of ccs1-ac206 clpP-AUU and ccb4-2 clpP-AUU Double Mutants. Cells were harvested during the exponential phase of growth (0.5 to 1 × 106 cells per mL). Blots were reacted with antibodies to cytochrome (Cyt) f and subunit (Su) IV.
Figure 5.
Figure 5.
Analysis of petC-ac21 clpP-AUU Double Mutants. (A) Immunoblot of total cell proteins from parental strains ac21 and clpP-AUU, and two double mutant progeny from the cross, harvested during exponential phase. (B) Stability of cytochrome f, subunit IV, and the Rieske protein in the ac21 (X's) and petC-ac21 clpP-AUU mutant (open circles) strains. Chloramphenicol was added at time 0 in early exponential- phase cultures. Immunoreactivity is plotted as the percentage of the initial level in each culture. Cyt f, cytochrome f; Su IV, subunit IV.
Figure 6.
Figure 6.
Analysis of the petC-Δ1 clpP-AUU Double Mutant. Cells were harvested during the exponential phase of growth (A) or in stationary phase, 3 days later (B). Blots were reacted with antibodies to cytochrome f (Cyt f), subunit IV (Su IV), and the Rieske protein.
Figure 7.
Figure 7.
The clpP-AUU Mutation Retards Loss of Cytochrome b6f in Nitrogen Starvation. (A) Chlorophyll fluorescence induction kinetics, in the presence (+) or absence (−) of DCMU, of TAP-grown cells (top) and cells starved for nitrogen for 61 hr (bottom). Solid lines, clpP-AUU; dotted lines, BI. (B) Evolution of the (Fmax − Fs)/(Fmax − F0) parameter during nitrogen starvation. Solid line, clpP-AUU; dotted line, BI. (C) Immunoblot of total cell proteins as given for (A) and (B), reacted with an antibody to cytochrome f (Cyt f). (D) Protein chase experiment under nitrogen starvation conditions with antibodies to cytochrome f (Cyt f), subunit IV (Su IV), and the Rieske protein. Chloramphenicol was added at time 0.
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