Biochemical characterization of the chloroplast ClpP peptidase of Chlamydomonas
B. Derrien1, W. Majeran2, J. Ebenezer3, G. Effantin4, C. Raynaud1, D. Saint-Marcoux1, C. de Vitry1, Y. Choquet1, M.R. Maurizi3, A.C. Steven4, F.-A. Wollman1, and O. Vallon1
1) UMR7141, IBPC, Paris, France
2) Cornell University, Ithaca, New York USA
3) NCI, NIH, Bethesda, USA
4) NIAMS, NIH, Bethesda, USA
The chloroplast ClpP complex is a hetero-oligomeric peptidase composed of homologous ClpP and ClpR subunits. In C. reinhardtii, the ClpP1 subunit, encoded in the chloroplast genome, cannot be deleted nor can its catalytic triad be mutated. It contains a large insertion sequence (IS1) initially proposed to be a new type of protein intron. By studying transformants harboring mutations at the borders of IS1 or a tag at the C-terminus of ClpP1or within IS1, we show that IS1 is not a protein intron and that maturation of ClpP1 involves 3 endoproteolytic cleavage events within IS1. IS1 is essential. Mutations around the cleavage sites hamper but do not prevent uts processing. Using TAPtag or Strep-tag affinity chromatography, we have purified the C. reinhardtii ClpP complex containing all the ClpP/R subunits, plus two new subunits (ClpT3 and ClpT4) that are homologous to the N-terminal domain of ClpC and may participate in substrate binding. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like structure of homo-oligomeric bacterial ClpP, with side extensions likely representing IS1 and the additional subunits. The complex shows no acticity towards a fluorigenic Leu-Tyr dipeptide, and low activity towards the model peptide FAPHMALVPV. The functions of the ClpP complex has been studied using strains in which its accumulation is reduced by translation attenuation. We now show that ClpP participates in the control of chloroplast gene expression. MCA1, the mRNA stability factor for petA (the chloroplast gene encoding cytochrome f) over-accumulates in ClpP-depleted strains, and its degradation during nitrogen starvation is retarded. ClpP also appears to control the CCB proteins which are responsible for assembly of the c'-heme onto cytochrome b6.
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