thi1 has been recently isolated from Arabidopsis 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 alpha-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.