Multistep Processing of an Insertion Sequence in an Essential Subunit of the Chloroplast ClpP Complex

S In Chlamydomonas reinhardtii, the clpP1 chloroplast gene encoding one of the catalytic subunits of the ClpP protease com- plex contains a large in-frame insertion sequence (IS1). Based on the Escherichia coli ClpP structure, IS1 is predicted to pro- trude 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: ClpP1H (59 kDa) and ClpP1L (25 kDa). It has been proposed that IS1 is a new type of protein intron (different from inteins). By studying transformants har- boring mutations at the predicted borders of IS1 and tags at the C terminus of ClpP1 (tandem affinity purification tag, His tag, StrepTag) or within the IS1 sequence (3-hemagglutinin tag), we show that IS1 is not a protein intron and that ClpP1L 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.