Abstract
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.
Highlights
Clp proteases are self-compartmentalized serine proteases present in most eubacteria and, as a consequence of endosymbiotic events, in the mitochondrion and chloroplast of eukaryotes
We show that IS1 is not a protein intron and that ClpP1L is the product of a complex proteolytic maturation of ClpP1H
ClpP1 IS1 Is Not a Protein Intron—To test the hypothesis that IS1 is a protein intron, we introduced at the C terminus of ClpP1 a His10 tag, a tandem affinity purification (TAP) tag (Fig. 1A), or a Strep1⁄7Tag
Summary
Clp proteases are self-compartmentalized serine proteases present in most eubacteria and, as a consequence of endosymbiotic events, in the mitochondrion and chloroplast of eukaryotes. In C. reinhardtii, clpP1 contains a large insertion sequence (IS1)3 translated in-frame with the conserved N- and C-terminal regions. This antibody recognized in Western blots all the bands that we propose represent C-terminal fragments of ClpP1 (Fig. 1C, f and Œ).
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