32. Evaluation of the genetic diversity and subcellular localization of mitochondrial ribosomal protein S10

1)Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba, 305-8602 Japan
2)Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113-8657 Japan

Most of mitochondrial proteins in eucaryotes are encoded by nuclear genes, synthesized in the cytosol, and imported into mitochondria. These nuclear genes seem to have been transferred from mitochondrion to the nucleus during the course of evolution. In general, nuclear-encoded genes have N-terminal extension for protein targeting to mitochondria. Previously, we have found that a gene coding for ribosomal protein S10 (rps10) had been lost from rice mitochondrial genome and been transferred to the nucleus (Kubo et al. 2000). Comparison of

rps10 gene-structure among plant species shows that there are great variations in the size of their N-terminal extensions. Interestingly, the rice rps10 gene has no obvious N-terminal extension regarding to mitochondrial targeting signal (Fig. 1). This observation suggests that mitochondrial targeting signal is located in the internal region of the rice rps10 gene.

In order to evaluate whether or not rice RPS10 protein, having no apparent N-terminal extension, can be sorted into mitochondria, subcellular localization of RPS10 protein was examined in vivo using green fluorescent protein (GFP). A part of the rice rps10 gene was amplified by PCR and cloned into SalI and NcoI sites of the GFP expression vector S65TGFP, kindly provided from Dr. Y. Niwa (Chiu et al. 1996). Ten micrograms of the plasmid DNA was precipitated onto 1.0-microm gold beads, and bombarded into suspension-cultured tobacco BY-2 cells. GFP-fluorescence was visualized as described previously (Nakazono et al. 2000). Fluorescence of GFP fusion proteins coincided with that of mitochondrial specific dye, Mito Tracker Red (Molecular Probes, USA) when N-terminal portion of the rice RPS10 was fused upstream of GFP (Fig. 2). This result confirms successful import and localization of the RPS10 protein into mitochondria, and strongly suggests that mitochondrial targeting signal is located in the N-terminal portion of the rice rps10 gene. Prediction of protein secondary structure shows that this region has the potential to fold into an amphiphilic alpha-helix, which may be important for protein import into mitochondria (Tamm 1991). There are several amino acid alternations in the rice RPS10 peptide when compared with mitochondrial-encoded rps10 sequences of potato, pea and liverwort (Zanlungo et al. 1995; Knoop et al. 1995; Oda et al. 1992) (data not shown). These alternations may have been involved in creation of targeting signal for the rice rps10 gene.


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