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The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features

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Abstract

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits.

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Abbreviations

ACN:

Acetonitrile

ADEP:

Acyldepsipeptide

MS:

Mass spectrometry

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Acknowledgments

We thank Adrain Clarke (Umea University) for providing the ClpP5 and ClpP6 antibodies and Francis-André Wollman for support and advice. This work was supported in part by the Agence Nationale de la Recherche (grant Algomics) and by the CNRS and Université Pierre et Marie Curie, UMR 7141 (BD, OV), by the National Science Foundation grant MCB-1021963 (WM, GF, KJVW), by the intramural research program of NIAMS (GE, ACS) and by the intramural program of the Center for Cancer Research, NCI (EJ, MRM).

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Correspondence to Olivier Vallon.

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File “Derrien Supplemental Table” provides detailed MS/MS results for three experiments. (XLS 43 kb)

11103_2012_9939_MOESM2_ESM.pdf

File “Derrien Supplemental figures” provides: the mapping of MS/MS data onto the protein sequences; an alignment of ClpT subunits and of Clp-N domains from various Hsp100 chaperones; an alignment of N-terminal domains in ClpR1 proteins (PDF 172 kb)

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Derrien, B., Majeran, W., Effantin, G. et al. The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features. Plant Mol Biol 80, 189–202 (2012). https://doi.org/10.1007/s11103-012-9939-5

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