title: The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features.
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.
authors: Institut de Biologie Physico-Chimique, UMR7141 CNRS/UPMC, 13 rue Pierre et Marie Curie, 75005 Paris, France.
authors: Benoît
authors: B
authors: Derrien
authors: Wojciech
authors: W
authors: Majeran
authors: Grégory
authors: G
authors: Effantin
authors: Joseph
authors: J
authors: Ebenezer
authors: Giulia
authors: G
authors: Friso
authors: Klaas J
authors: KJ
authors: van Wijk
authors: Alasdair C
authors: AC
authors: Steven
authors: Michael R
authors: MR
authors: Maurizi
authors: Olivier
authors: O
authors: Vallon
mesh_terms: D020418:Algal Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics*; D060365:Chloroplast Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010802:Phylogeny; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
chemical_list: D020418:Algal Proteins; D060365:Chloroplast Proteins; D021122:Protein Subunits; D014357:Trypsin; D049071:Endopeptidase Clp