Abstract
Serine: pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide.
In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, STPp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.
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Noguchi, T., Minatogawa, Y., Takada, Y., Okuno, E., and Kido, R. (1978) Subcellular distribution of pyruvate (glyoxylate) amino-transferases in rat liver. Biochem. J., 170, 173–175.
Oda, T., Yanagisawa, M., and Ichiyama, A. (1982) induction of serine: pyruvate amino-transferase in rat liver organelles by glucagon and a high-protein diet. J. Biochem. 91, 219–232.
Oda, T., Funai, T., and Ichiyama, A. (1990) Generation from a single gene of two mRNAs that encode the mitochondrial and peroxisomal serine:pyruvate aminotransferase of rat liver. J. Biol. Chem. 265, 7513–7519.
Mori, M., Oda, T., Nishiyama, K., Serikawa, T., Yamada, J., and Ichiyama, A. (1992) A single serine:pyruvate aminotransferase gene on rat chromosome 9q34-36. Genomics, 13, 686–689.
Oda, T., Ichiyama, A., Miura, S., and Mori, M. (1984) Uptake and processing of serine:pyruvate aminotransferase precursor by rat liver mitochondria in vitro and in vivo. J. Biochem. 95, 815–824.
Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J., and Subramani, S. (1989) A conserved tripeptide sorts proteins to peroxisomes. J. Cell Biol., 108, 1657–1664.
Swinkels, B. W., Gould, S. J., Bodnar, A. G., Rachubinski, R. A., and Subramani, S. (1991) A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J., 10, 3255–3262.
Osumi, T., Tsukamoto, T., Hata, S., Yokota, S., Miura, S., Fujiki, Y., et al. (1991) Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem. Biophys. Res. Commun., 181, 947–954.
Motley, A., Lumb, M. J., Oatey, P. B., Jennings, P. R., De Zoysa, P. A., Wanders, R. J. A., Tabak, H. F., and Danpure, C. J. (1995) Mammalian alanine/gloxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1. J. Cell Biol., 131, 95–109.
Yokota, S., Funai, T., and Ichiyama, A. (1991) Organelle localization of rat liver serine: pyruvate aminotransferase expressed in transfected COS-1 cells. Biomed. Res., 12, 53–59.
Funai, T. and Ichiyama, A. (1995) Fidelity of translation initiation of mRNA for the precursor of rat mitochondrial serine: pyruvate/alanine: glyoxylate aminotransferase. J. Biochem., 117, 1008–1016.
Deng, W. P. and Nickoloff, J. A. (1992) Sitedirected mutagenesis of virtually any plasmid by eliminating a unique site. Anal. Biochem., 200, 81–88.
Conley, E. C., and Saunders, J. R. (1984) Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli. Mol. Gen. Genet., 194, 212–218.
Higuchi, R., Krummel, B., and Saiki, R. K. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nuc. Acids Res., 16, 7351–7367.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Expression of cloned genes in cultured mammalian cell in Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 16.30–16.36.
Miura, S., Kasuya-Arai, I., Mori, H., Miyazawa, S., Osumi, T., Hashimoto, T., and Fujiki, Y. (1992) Carboxy-terminal consensus Ser-Lys-Leu-related tripeptide of peroxisomal proteins functions in vitro as a minimal peroxisome-targeting signal. J. Biol. Chem., 267, 14405–14411.
McNew, J. A. and Goodman, J. M. (1996) The targeting and assembly of peroxisomal proteins: some old rules do not apply. Trends Biochem. Sci., 21, 54–58.
Purdue, P. E. and Lazarow, P. B. (1996) Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence. J. Cell Biol., 134, 849–862.
Oda, T., Funai, T., and Miura, S. (1996) In vitro association with peroxisomes and conformational change of peroxisomal serine: pyruvate/alanine: glyoxylate aminotransferase in rat and human livers. Biophys. Biochem. Res. Commun., 228, 341–346.
Terada, K., Ueda, I. Ohtsuka, K., Oda, T., Ichiyama, A., and Mori, M. (1996) The requirement of heat shock cognate 70 protein for mitochondrial import varies among precursor proteins and depends on precursor length. Mol. Cell. Biol., 16, 6103–6109.
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Oda, T., Mizuno, T., Ito, K. et al. Peroxisomal and mitochondrial targeting of serine: Pyruvate/alanine: Glyoxylate aminotransferase in rat liver. Cell Biochem Biophys 32, 277–281 (2000). https://doi.org/10.1385/CBB:32:1-3:277
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DOI: https://doi.org/10.1385/CBB:32:1-3:277