Multistep processing of an insertion sequence in an essential subunit of the chloroplast ClpP complex.
In Chlamydomonas reinhardtii, the clpP1 chloroplast gene encoding one of the catalytic subunits of the ClpP protease complex contains a large in-frame insertion sequence (IS1). Based on the Escherichia coli ClpP structure, IS1 is predicted to protrude at the apical surface of the complex, likely influencing the interaction of the catalytic core with ClpC/HSP100 chaperones. Immunoblotting with an anti-ClpP1 antibody detected two immunoreactive forms of ClpP1: ClpP1(H) (59 kDa) and ClpP1(L) (25 kDa). It has been proposed that IS1 is a new type of protein intron (different from inteins). By studying transformants harboring mutations at the predicted borders of IS1 and tags at the C terminus of ClpP1 (tandem affinity purification tag, His tag, Strep.Tag) or within the IS1 sequence (3-hemagglutinin tag), we show that IS1 is not a protein intron and that ClpP1(L) results from endoproteolytic cleavage inside IS1. Processing sites have been identified in the middle of IS1 and near its C terminus. The sites can be mutated without abolishing processing.
Derrien
Benoit
B
Institut de Biologie Physico-Chimique, UMR7141 CNRS/Université Pierre et Marie Curie, 75005 Paris, France.
Majeran
Wojciech
W
Wollman
Francis-André
FA
Vallon
Olivier
O
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics; D004251:DNA Transposable Elements; D049071:Endopeptidase Clp / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D007438:Introns; D010446:Peptide Fragments; D021122:Protein Subunits / Q000235:genetics; D017384:Sequence Deletion
D004251:DNA Transposable Elements; D010446:Peptide Fragments; D021122:Protein Subunits; D049071:Endopeptidase Clp