Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Jun 5;284(23):15408-15.
doi: 10.1074/jbc.M109.002733. Epub 2009 Apr 3.

Multistep processing of an insertion sequence in an essential subunit of the chloroplast ClpP complex

Benoit Derrien  1 , Wojciech Majeran, Francis-André Wollman, Olivier Vallon
Affiliations

Multistep processing of an insertion sequence in an essential subunit of the chloroplast ClpP complex

Benoit Derrien et al. J Biol Chem. .

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.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Immunoblot analysis of C-terminally tagged strains (ClpP1-His and ClpP1-TAP) and IS1 border mutants (S60A and S365A). Western blots reacted with antisera raised against ClpP1 (A and B) and an IS1 peptide epitope (C). The products of the clpP1 gene are indicated: ClpP1H (●), ClpP1N (♦), ClpP1C (■), and ClpP1C′ (▲).
FIGURE 2.
FIGURE 2.
Model for the maturation of ClpP1. SD1 and SD2 are colored in black, and IS1 in light gray. The excised portion of IS1 is shown as a cross-hatched box. The positions of the various tags used in this study are indicated by flags. The peptide used to raise the anti-IS1 antibody (N325NESGRSLYRKQTER339) is shown as a white box.
FIGURE 3.
FIGURE 3.
Insertion of a 3-HA tag in IS1. Immunoblots reacted with antibodies to ClpP1 and the HA tag. The symbols are the same as indicated in the legend to Fig. 1. A, analysis of ClpP1HA85; B, analysis of ClpP1HA176 and ClpP1ΔNHA176.
FIGURE 4.
FIGURE 4.
Purified Strep·Tag-tagged ClpP complex. Lane 1, Coomassie Blue; lanes 2 and 3, immunoblots probed with antibodies to ClpP1 and the IS1 peptide, respectively.
FIGURE 5.
FIGURE 5.
Immunoblot analysis of cleavage site C, C′, and N mutants. Immunoblots reacted with antibodies to ClpP1. A, site C and C′ mutants. Note the new band at ∼40 kDa (*). In the WT lane, the band indicated by an exclamation point is not ClpP1C′ but ClpP1C without a tag. B, site N mutant.
FIGURE 6.
FIGURE 6.
IS1 in Chlorophyceae. A, shown is an alignment of IS1 sequences from various Chlorophyceae. Cleavage sites are marked by arrows. Six conserved sequence blocks are marked by brackets. B, whole cells protein extracts from S. obliquus (S.o.) reacted with the anti-ClpP1 antibody. The two bands marked by an asterisk likely correspond to ClpP1H and ClpP1N. C.r., C. reinhardtii.
See this image and copyright information in PMC

References

    1. Katayama Y., Gottesman S., Pumphrey J., Rudikoff S., Clark W. P., Maurizi M. R. ( 1988) J. Biol. Chem. 263, 15226– 15236 - PubMed
    1. Maurizi M. R., Clark W. P., Katayama Y., Rudikoff S., Pumphrey J., Bowers B., Gottesman S. ( 1990) J. Biol. Chem. 265, 12536– 12545 - PubMed
    1. Gottesman S., Clark W. P., de Crecy-Lagard V., Maurizi M. R. ( 1993) J. Biol. Chem. 268, 22618– 22626 - PubMed
    1. Ortega J., Lee H. S., Maurizi M. R., Steven A. C. ( 2004) J. Struct. Biol. 146, 217– 226 - PubMed
    1. Wang J., Hartling J. A., Flanagan J. M. ( 1997) Cell 91, 447– 456 - PubMed

Publication types

LinkOut - more resources