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

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, Strep⅐Tag) 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.

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control

“…In C. reinhardtii, the plastid-encoded ClpP1 protein contains a 30-kD insertion that can be removed by endoproteolytic cleavage to give rise to a short ClpP1 isoform (Wang et al, 1997;Majeran et al, 2005). Both the long and short isoforms are essential and can be detected within the ClpP1 core (Huang et al, 1994;Derrien et al, 2009). Moreover, chaperones that are orthologs of the bacterial AAA+ proteins (ClpC1/C2 and ClpD) and ClpS adaptors (ClpS1 and ClpS1-like) have been identified (Peltier et al, 2004;Derrien et al, 2012).…”

Activation of an Endoribonuclease by Non-intein Protein Splicing

“…This model need not be limited to a splicing activity, since this would presumably happen only when the insertion is folded such that the future junction site residues are in close enough proximity to be joined together. One possible example of an alternative NIPS activity is the C. reinhardtii ClpP1 subunit of the chloroplast ClpP protease complex, which contains a large insertion that is removed by an unknown endoprotease(s) (29). This is reminiscent of the "excision, no splicing" activity that we predicted for the insertion repair function of NIPS machinery in Fig.…”

“…Given that the insertion of inteins, transposons, and recombination events are, in combination, an almost universal phenomenon, a critical question becomes how cells mitigate such events when they are deleterious, for example when inactivating insertions arise within essential genes. In the case of Chlamydomonas, the chloroplast genome encodes several essential proteins involved in transcription, translation, or proteolysis, which possess internal inframe DNA insertions or extensions (27)(28)(29), which would seemingly make the resulting proteins non-functional. A NIPS machinery may have evolved to post-translationally repair and restore functionality to such genes (Fig.…”

Conditional repression of essential chloroplast genes: Evidence for new plastid signaling pathways

“…It contains a proteolytic chamber made of two stacked heptameric rings of the ClpP subunit and an antechamber consisting of a hexameric ring of the ATP-dependent chaperone ClpP or ClpA. In addition, ClpC1/ C2 and ClpD chaperones that are orthologous to the bacterial AAA + proteins and ClpS adaptors have been identified [49,54]. However, the ClpP proteolytic system is more complex in plastids than in bacteria [49] as it contains several ClpP isoforms in the holoenzyme, some of which have lost the catalytic residues.…”