Journal Article

Catalysis, Subcellular Localization, Expression and Evolution of the Targeting Peptides Degrading Protease, <i>At</i>PreP2

Shashi Bhushan, Annelie Ståhl, Stefan Nilsson, Benoit Lefebvre, Motoaki Seki, Christian Roth, David McWilliam, Sarah J. Wright, David A. Liberles, Kazuo Shinozaki, Barry D. Bruce, Marc Boutry and Elzbieta Glaser

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 6, pages 985-996
Published in print June 2005 | ISSN: 0032-0781
Published online June 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci107
Catalysis, Subcellular Localization, Expression and Evolution of the Targeting Peptides Degrading Protease, AtPreP2

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  • Biochemistry
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  • Plant Sciences and Forestry

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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.

Keywords: Chloroplasts; Dual targeting; Mitochondria; Presequence protease; Protein import; Zinc metalloprotease; ESI, electrospray ionization; EST, expressed sequence tag; GB, grinding buffer; GFP, green fluorescent protein; GST, glutathione-S-transferase; HPLC, high-performance liquid chromatography; IP, import buffer; MPP, mitochondrial processing peptidase; MS, mass spectrometry; PK, proteinase K; PreP, presequence protease; RT–PCR, reverse transcription–polymerase chain reaction; SPP, stromal processing peptidase

Journal Article.  8276 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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