Plant and Cell Physiology Advance Access originally published online on April 11, 2005
Plant and Cell Physiology 2005 46(6):985-996; doi:10.1093/pcp/pci107
JSPP © 2005
Catalysis, Subcellular Localization, Expression and Evolution of the Targeting Peptides Degrading Protease, AtPreP2
Shashi Bhushan1,
Annelie Ståhl1,
Stefan Nilsson1,
Benoit Lefebvre2,
Motoaki Seki3,
Christian Roth4,
David McWilliam5,
Sarah J. Wright6,
David A. Liberles4,
Kazuo Shinozaki3,
Barry D. Bruce5,6,
Marc Boutry2 and
Elzbieta Glaser1,7
1 Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden
2 Unité de Biochimie Physiologique, Institut des sciences de la vie, Université Catholique de Louvain de Louvain, Croix du Sud, 2-20, B-1348 Louvain-la-Neuve, Belgium
3 Plant Functional Genomic Research Group, RIKEN GSC, Japan
4 Computational Biology Unit, BCCS, University of Bergen, 5020 Bergen, Norway
5 Graduate Program in Genome Science and Technology, University of Tennessee, Knoxville, TN 37996, USA
6 Center of Excellence in Structural Biology, Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37996 USA
7 Corresponding author: E-mail, e_glaser{at}dbb.su.se; Fax, +46-81-53679.
We have previously identified a zinc metalloprotease involved in the degradation of mitochondrial and chloroplast targeting peptides, the presequence protease (PreP). In the Arabidopsis thaliana genomic database, there are two genes that correspond to the protease, the zinc metalloprotease (AAL90904 and the putative zinc metalloprotease (AAG13049. We have named the corresponding proteins AtPreP1 and AtPreP2, respectively. AtPreP1 and AtPreP2 show significant differences in their targeting peptides and the proteins are predicted to be localized in different compartments. AtPreP1 was shown to degrade both mitochondrial and chloroplast targeting peptides and to be dual targeted to both organelles using an ambiguous targeting peptide. Here, we have overexpressed, purified and characterized proteolytic and targeting properties of AtPreP2. AtPreP2 exhibits different proteolytic subsite specificity from AtPreP1 when used for degradation of organellar targeting peptides and their mutants. Interestingly, AtPreP2 precursor protein was also found to be dual targeted to both mitochondria and chloroplasts in a single and dual in vitro import system. Furthermore, targeting peptide of the AtPreP2 dually targeted green fluorescent protein (GFP) to both mitochondria and chloroplasts in tobacco protoplasts and leaves using an in vivo transient expression system. The targeting of both AtPreP1 and AtPreP2 proteases to chloroplasts in A. thaliana in vivo was confirmed via a shotgun mass spectrometric analysis of highly purified chloroplasts. Reverse transcription–polymerase chain reaction (RT–PCR) analysis revealed that AtPreP1 and AtPreP2 are differentially expressed in mature A. thaliana plants. Phylogenetic evidence indicated that AtPreP1 and AtPreP2 are recent gene duplicates that may have diverged through subfunctionalization.
(Received February 23, 2005; Accepted April 7, 2005)
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