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

thi1 has been recently isolated from Arabidopsis thaliana and is probably i nvolved in both thiamine biosynthesis and as protection of organellar DNA f rom damage. Studies of thiamine biosynthesis in plants suggests a plastid l ocation for the pathway, which is in agreement with the predicted THI1 N-te rminal chloroplastic transit peptide (TP). On the other hand, thiamine is s ynthesized 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 amphiphili c alpha -helix, which is typical for mitochondrial presequences, located do wnstream 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 T HI1, both linked to the reporter (gusA) gene. Analysis of GUS distribution in subcellular fractions of transgenic plants revealed that in the construc t retaining only 4 residues of mature THI1, GUS was found in the chloroplas tic fraction. Extension of the THI1 transit peptide to 27 residues of the m ature protein allowed import and processing of GUS into both mitochondria a nd chloroplasts. Direct analysis by immunogold-labeling with an anti-THI1 p olyclonal antibody identified THI1 in both organelles in Arabidopsis. We al so provide evidence that the precursors of both organellar isoforms are enc oded by a single nuclear transcript. Thus, THI1 is targeted simultaneously to mitochondria and chloroplasts by a post transcriptional mechanism.