Mitochondrial Tryparedoxins Figure 7. LiTXN3 is an integral membrane protein with the N-terminal domain exposed to the cytosol. A. Alkaline carbonate extraction performed on 6HisLiTXN3-expressing L. infantum promastigotes. Parasites were disrupted in either 10 mM Tris-HCl pH 7.4, 10 mM Tris-HCl 0.5 M NaCl pH 7.4, or 0.1 M Na2CO3 pH 11.5 and fractionated by ultracentrifugation. Total protein extracts (T), as well as supernatants (S) and membrane pellets (P) resulting from fractionation were analyzed by western blot using anti-His antibody to detect 6HisLiTXN3. Anti-gp63 and anti-LicTXNPx2 antibodies were used to control for membrane and hydrophilic proteins, respectively. B. Protein samples resulting from permeabilization of 6HisLiTXN3- expressing promastigotes with increasing concentrations of digitonin in the presence of proteinase K were analyzed by western blot using an anti-His antibody. Other antibodies were used to detect the cytosolic protein LicTXNPx2, as well as a mitochondrial intermembrane space molecule (cytochrome c, detected with anti-TbCytc). doi:10.1371/journal.pone.0012607.g007 Discussion In the cytosol of trypanosomatid parasites (e.g. L. infantum, T. brucei and T. cruzi) TXNs enzymes transfer reducing equivalents from trypanothione to redox pathways involving thiol/disulfide exchange. Several TXN-dependent enzymes have also been identified within the mitochondrion of different trypanosomatids and so it is generally accepted that likewise a fully active TXN must also be present within the mitochondrion of these organisms. Such TXN would fuel essential pathways such as peroxide detoxification and mitochondrial DNA replication, and could also participate in iron-sulfur cluster biosynthesis [1]. The actual need for an active TXN operating within the mitochondrion was challenged in this report, by demonstrating that the mitochondrial LiTXN2 enzyme of L. infantum is not vital for parasite viability or successful infectivity. Moreover, evidence was provided that no other L. infantum TXN was capable of replacing LiTXN2 functions. Importantly, as discussed next, the non-requirement for an inner mitochondrial TXN should not be exclusive of Leishmania, but instead, a feature common to all trypanosomatids. The Trypanosoma genomes contain, apart from TXN1 (that codes for the cytosolic TXN1 enzyme), an extra TXN coding sequence, TXN2. It is generally accepted that Trypanosoma TXN2 genes encode the mitochondrial TXN of these organisms [1,16,17]. Still, the only evidence for this is a report where overexpression of the T. brucei TbTXN2 reversed a defective phenotype induced by excess of mitochondrial enzymes involved in redox reactions, either a cytochrome b5 reductase-like protein (CBRL) or the mitochondrial TXN-dependent peroxidase mTXNPx [17], even though no such defect was evident when mTXNPx was overexpressed in T. cruzi [30] and Leishmania [26,31,32]. The data presented in this manuscript raises serious doubts regarding the participation of Trypanosoma TXN2 enzymes in intramitochondrial redox processes. Phylogenetic analysis shows that Trypanosoma TXN2 and Leishmania TXN2 sequences, despite sharing the same nomenclature, are not orthologues. Instead, Trypanosoma TXN2 genes cluster together with Leishmania TXN3 in a class of TXNs (class II) that is distinct from the one containing the TXN1 of all trypanosomatids and the TXN2 sequence of Leishmania and Crithidia (class I). Contrary to class I members, class II TXNs are unlikely to function as TXNs owing to the presence of a number of non-identical amino acid substitutions. Although direct evidence for this was only obtained for LiTXN3, Trypanosoma TXN2 share with LiTXN3 all sequence alterations likely to impair TXN activity, the exception being a Ser residue at position 40 (LiTXN3 numbering), preserved in Trypanosoma TXN2, but replaced by an Arg in LiTXN3. By showing that this amino acid substitution is not responsible for the defective activity of LiTXN3 (see Text S2 and Figure S2), the hypothesis that Trypanosoma TXN2 enzymes behave differently from LiTXN3, i.e. that they retain TXN activity, has no sustainability. An additional reason precluding class II TXNs from reducing molecules present in the mitochon- drial compartments is their subcellular compartmentalization. All class II TXNs possess a single C-terminal hydrophobic domain PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e12607