In regards to animals several studies have reported the

In regards to animals, several studies have reported the peroxisomal localization of FPS (Krisans et al., 1994, Olivier et al., 2000, Kovacs et al., 2007). Transfections of mammalian cells with the rat short FPS isoform followed by immunodetection localized the enzyme to the peroxisome (Olivier et al., 2000, Kovacs et al., 2007). Furthermore, the authors have shown that the rat FPS requires the PTS2 import pathway. They identified, within the N-terminal amino PDK1 inhibitor sequence, a degenerated PTS2-like motif and demonstrated that this motif is necessary for efficient targeting of rat FPS to the peroxisome (Olivier et al., 2000). By contrast, no PTS1 located at the C-terminal end could be found within the amino acid sequence of CrFPS as well as no canonical PTS2 consensus sequence. However, other proteins that do not contain classic PTS have been shown to be targeted to peroxisomes via internal PTS-like motifs or poorly characterized internal targeting signals. Furthermore, proteins have been shown to be targeted to the peroxisome via a mechanism known as ‘piggy-backing’ in which they form a complex with a protein containing a PTS (van der Klei and Veenhuis, 2006, Oshima et al., 2008, Penha et al., 2009). In our experiments, the fact that only the YFP-CrFPS construct is targeted to peroxisomes could be explained by the presence of a putative non-PTS domain near the CrFPS C-terminus necessary for peroxisome targeting. In turn, the cytosolic localization of the CrFPS-YFP construct could be a consequence of the physical obstruction of the C-terminal domain of CrFPS caused by the fluorescent protein. The yeast S. cerevisiae contains only one FPS (ERG20) generally regarded as cytosolic, although this was not confirmed by cell imaging studies. Furthermore, no studies have reported a peroxisomal localization of the yeast FPS. The mitochondrial polyprenyl diphosphates, such as the prenyl side chain of ubiquinone, are synthesized by a mitochondrial hexaprenyl diphosphate synthase (COQ1), which could accept FPP or longer-chain allelic substrates (Ashby and Edwards, 1990), suggesting that they are transported from the cytosol to the mitochondria. Note that in the above complementation experiment, CrFPS, which is targeted to peroxisome in C. roseus, can complement the likely cytosolic deficient FPS from S. cerevisiae. The general view of mammalian FPS is that a single functional gene is present in the genome. This gene encodes more than one isoform, since for example, the Genbank database contains the annotated short (Genbank accession number: EDM00669) and long (Genbank accession number: EDM00670) versions of rat FPS. The long mammalian isoform, bearing an N-terminal transit peptide, is targeted to the mitochondria (Martin et al., 2007) while the short isoform was detected in the peroxisomes (Olivier et al., 2000, Kovacs et al., 2007, Martin et al., 2007) as well as in the cytosol (Martin et al., 2007). In plants, it appears that there are at least two FPS genes (Cunillera et al., 1997, Gaffé et al., 2000) encoding several trans-type isoforms. One gene encodes a long isoform targeted to mitochondria (Cunillera et al., 1997) and a short isoform, for which no localization has been reported, thereby raising the question of its cytosolic or peroxisomal nature. The second gene encodes a short isoform potentially targeted to the peroxisome as shown in this study for CrFPS (Fig. 3E–G). Furthermore, in addition to the punctuated peroxisomal pattern, CrFPS-transformed C. roseus cells display a diffuse pattern that can be associated with the cytosol (Fig. 3E). Therefore, we cannot totally exclude the possibility that a certain proportion of the enzyme is also localized within the cytosol as in mammals, although such result could also be due to over-expression of the fusion-protein.
Acknowledgements We thank “Le STUDIUM” (Agency for Research and Hosting Foreign associated Researchers in the Centre region, France) for the financial support of Andrew J. Simkin. Grégory Guirimand is the recipient of a PhD fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche (France). Insaf Thabet is supported by a cotutelle PhD fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche Scientifique (Tunisie).