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Dual targeting properties of the N-terminal signal sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts

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Abstract

thi1 has been recently isolated fromArabidopsis thaliana and is probably involved in both thiamine biosynthesis and as protection of organellar DNA from damage. Studies of thiamine biosynthesis in plants suggests a plastid location for the pathway, which is in agreement with the predicted THI1 N-terminal chloroplastic transit peptide (TP). On the other hand, thiamine is synthesized in mitochondria in yeast cells. Interestingly, A. thaliana thi1 cDNA complements a yeast strain disrupted for the homologous gene. Analysis of THI1 amino acid sequence revealed the presence of a putative amphiphilic α-helix, which is typical for mitochondrial presequences, located downstream of the chloroplast transit peptide. To define the putative role of the two predicted targeting sequences in tandem, we produced two chimeric genes encompassing the chloroplastic THI1 TP and either 4 or 27 (including the putative mitochondrial presequence) N-terminal residues of the mature THI1, both linked to the reporter (gusA) gene. Analysis of GUS distribution in subcellular fractions of transgenic plants revealed that in the construct retaining only 4 residues of mature THI1, GUS was found in the chloroplastic fraction. Extension of the THI1 transit peptide to 27 residues of the mature protein allowed import and processing of GUS into both mitochondria and chloroplasts. Direct analysis by immunogold-labeling with an anti-THI1 polyclonal antibody identified THI1 in both organelles in Arabidopsis. We also provide evidence that the precursors of both organellar isoforms are encoded by a single nuclear transcript. Thus, THI1 is targeted simultaneously to mitochondria and chloroplasts by a post transcriptional mechanism.

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Authors and Affiliations

  1. Departamento de Genética, Escola Superior de Agricultura `Luiz de Queiroz', Universidade de São Paulo, Caixa Postal 83, 13400-970, Piracicaba, SP, Brazil

    Sabrina M. Chabregas, Marcio C. Silva-Filho & Marcio C. Silva-Filho

  2. Departamento de Microbiologia, Instituto de Ciências Biomédicas 2, Universidade de São Paulo, Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil

    Douglas D. Luche & Carlos F.M. Menck

  3. Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11461, 05422-970, São Paulo, SP, Brazil

    Leonardo P. Farias & Marie-Anne van Sluys

  4. Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11461, 05422-970, São Paulo, SP, Brazil

    Alberto F. Ribeiro

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  1. Sabrina M. Chabregas
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  2. Douglas D. Luche
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Chabregas, S.M., Luche, D.D., Farias, L.P. et al. Dual targeting properties of the N-terminal signal sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts. Plant Mol Biol 46, 639–650 (2001). https://doi.org/10.1023/A:1011628510711

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