Short communication Phenotypical consequences of expressing the dually targeted Presequence Protease, AtPreP, exclusively in mitochondria Beata Kmiec, Pedro F. Teixeira, Elzbieta Glaser * Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, SE-106 91 Stockholm, Sweden article info Article history: Received 10 October 2013 Accepted 16 December 2013 Available online 25 December 2013 Keywords: Presequence Protease Peptide degradation Dual targeting Mitochondria abstract Endosymbiotic organelles, mitochondria and chloroplasts, are sites of an intensive protein synthesis and degradation. A consequence of these processes is production of both free targeting peptides, i.e. mito- chondrial presequences and chloroplastic transit peptides, and other short unstructured peptides. Mitochondrial, as well as chloroplastic peptides are degraded by Presequence Protease (PreP), which is dually targeted to mitochondrial matrix and chloroplastic stroma. Elimination of PreP in Arabidopsis thaliana leads to growth retardation, chlorosis and impairment of mitochondrial functions potentially due to the accumulation of targeting peptides. In this work we analyzed the influence of the restoration of mitochondrial peptide degradation by AtPreP on plant phenotype. We showed that exclusive mito- chondrial expression of AtPreP results in total restoration of the proteolytic activity, but it does not restore the wild-type phenotype. The plants grow shorter roots and smaller rosettes compared to the plants expressing AtPreP1 in both mitochondria and chloroplasts. With this analysis we are aiming at understanding the physiological impact of the role of the dually targeted AtPreP in single type of destination organelle. Ó 2014 Elsevier Masson SAS. All rights reserved. 1. Introduction The vast majority of proteins localized in the endosymbiotic organelles, i.e. mitochondria and chloroplasts, are synthesized in the cytosol and have to be posttranslationally imported to their final destination. Proteins localized in the mitochondrial matrix or in the chloroplastic stroma usually possess targeting signals in a form of N-terminal extensions, termed presequences or transit peptides, respectively. Presequences and transit peptides are in most cases cleaved off in a reaction termed processing, generating the mature portion of the organellar proteins as well as free pre- sequences or transit peptides. Free targeting peptides are removed by the Presequence Pro- tease, PreP. PreP was initially characterized in plant mitochondria as the peptidase responsible for degradation of the presequence of ATPase beta subunit (pF 1 b) [1]. Further analyses showed that PreP degrades also the chloroplastic transit peptide of Rubisco small subunit as well as other unstructured peptides ranging between 10 and 65 amino acids (a.a) [2]. Recently, another system for mito- chondrial and chloroplastic peptide degradation has been described with Organellar Oligopeptidase, OOP, degrading short peptides ranging between 8 and 23 a.a in length, thus partially overlapping with PreP [3]. Arabidopsis thaliana has two PreP iso- forms, AtPreP1 and AtPreP2, displaying high sequence identity (86%) and recognizing the same substrates [4,5]. AtPreP1 and AtPreP2 are both dually localized in mitochondrial matrix and chloroplastic stroma [5,6], a fact consistent with their ability to cleave both mitochondrial and chloroplastic targeting peptides. Simultaneous knockout of both AtPreP isoforms results in both mitochondrial and chloroplastic abnormalities [7]. prep1 prep2 double knockout (prep DKO) plants display changes in ultrastruc- ture of mitochondria and chloroplasts and reduction of leaf pigment content. The chloroplast-related changes do not result in a lowered photosynthesis rate, but prep DKO plants manifest partial uncoupling of mitochondrial membranes, and a lowered mito- chondrial respiration rates. In consequence, prep DKO display growth retardation, growing smaller rosettes, and their develop- ment is delayed [7]. The wild-type phenotype can be restored by a stable expression of the dominant isoform, AtPreP1, in the prep DKO plants [7]. It has been proposed that the basis for the observed phenotypic changes might be accumulation of the organellar targeting pep- tides that are not efficiently degraded in prep DKO plants [7]. Indeed, it was shown by several independent studies that pre- sequences and presequence-like peptides impair the structural and the functional integrity of the mitochondrial membranes, causing * Corresponding author. Tel.: þ46 8 162457; fax: þ46 8 153679. E-mail address: e_glaser@dbb.su.se (E. Glaser). Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.12.012 Biochimie 100 (2014) 167e170