Involvement of N-terminal region in mitochondrial targeting of rice RPS10 and RPS14 proteins Nakao Kubo a,1 , Shin-ichi Arimura b , Nobuhiro Tsutsumi b , Atsushi Hirai b , Koh-ichi Kadowaki a, * a Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2 Tsukuba, 305-8602, Japan b Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku 113-8657, Japan Received 5 December 2002; received in revised form 3 February 2003; accepted 24 February 2003 Abstract We have investigated targeting signal of rice mitochondrial ribosomal proteins, RPS10 and RPS14. Their predicted protein structures show that RPS14 has a cleavable N-terminal extension whereas RPS10 does not, and that the both proteins contain several a-helix structures. To know which regions are involved in protein targeting into mitochondria, subcellular localization of the two proteins was examined in vivo by using a green fluorescent protein (GFP). A portion of RPS10 or RPS14 was fused to the GFP and introduced into tobacco BY-2 cells. Localization of fusion proteins was visualized by GFP fluorescence. When the N-terminal part of RPS10 (amino acid position 1  /56) was fused to GFP, resultant GFP fusion proteins were detected specifically in mitochondria. In contrast, no such localization was found when the C-terminal part of RPS10 was fused to GFP. GFP fusion proteins were clearly localized to mitochondria when the N-terminal region of RPS14 (amino acid position 1  /48) was fused to GFP. Introduction of sequence alterations into their N-terminal regions abolished the specificity of mitochondrial targeting. These results strongly suggest that the N-terminal region plays an important role for the targeting of the ribosomal proteins into plant mitochondria irrespective of the N-terminal extension. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Green fluorescent protein; Plant mitochondria; Ribosomal protein; Rice; Targeting signal 1. Introduction Most of the mitochondrial proteins in eukaryotes are encoded by nuclear genes, synthesized in the cytosol and imported into mitochondria. Concerning the origin of these nuclear-encoded proteins, it has been proposed that they had been transferred from the mitochondrion to the nucleus during the course of cell evolution [1] but some proteins may have been acquired from other origins in plants (e.g. see [2  /4]). These nuclear-encoded genes require specific targeting signals for effective transport of the proteins into mitochondria. It has been reported that targeting signals are often encoded in the N-terminal extended region as a presequence [5]. To date, a number of mitochondrial genes have been isolated from nuclear genomes and it is generally accepted that presequences have typical characteristics: for example, no similarity in the primary amino acid sequence among different proteins, relatively rich in basic and hydroxylated amino acid residues while rare in acidic residues, capable of forming amphiphilic a-helix structure, and importance of positively charged residues for import and subsequent processing [5]. Most of the evidences have been obtained mainly by intensive studies of yeast and Neurospora . Although such char- acteristics have also been found to be applicable to plant mitochondrial presequences [6,7], there are some excep- tions in plants. Significant homology in presequences has been found between different mitochondrial pro- teins [8  /10]. Similar findings have also been reported in targeting sequences of chloroplast proteins [11]. More- * Corresponding author. Tel.:  /81-298-38-7449; fax:  /81-298-38- 7408. E-mail address: kadowaki@affrc.go.jp (K. Kadowaki). 1 Present address: Graduate School of Agriculture, Kyoto Prefectural University/Kyoto Prefectural Institute of Agricultural Biotechnology, Soraku-gun 619-0244, Japan. Plant Science 164 (2003) 1047  /1055 www.elsevier.com/locate/plantsci 0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00108-0