Figure 6. LiTXN3 localizes to the mitochondrion. A. Western blot analysis of 50 mg of total protein extract from wild type L. infantum and from promastigotes carrying the pTEX6HisLiTXN3 episome (see Material and Methods). The blot was hybridized with anti-His to detect the 6HisLiTXN3 chimera expressed by the transfected cell line. B. Immunolocalization of the 6HisLiTXN3 chimera in pTEX6HisLiTXN3- transformed promastigotes using the anti-His antibody (in red). The control mitochondrial LimTXNPx enzyme was detected with an antibody produced against this protein (marked in green). Also included is the merging of both channels plus the channel for DNA staining with DAPI. doi:10.1371/journal.pone.0012607.g006 enzyme LimTXNPx [26] (Figure 6B). However, these two enzymes did not entirely co-localize, looking as if LiTXN3 contours LimTXNPx. This might be due to the fact that LimTXNPx is a soluble enzyme, whereas LiTXN3 is predicted to be a membrane protein. LiTXN3 is an integral membrane protein with its redox- active domain exposed to the cytosol The confirmation that LiTXN3 is a membrane-bound protein was achieved by performing an alkaline carbonate extraction on 6HisLiTXN3-expressing parasites. The results in Figure 7A show that LiTXN3 was recovered in the insoluble fractions of parasite lysates prepared in low and high salt solutions, and only partially solubilized with the alkaline carbonate buffer, an elution profile that matches that of the membrane protein gp63, but not with that of a soluble molecule (LicTXNPx2). The association of LiTXN3 with cell membranes must occur through its single C-terminal stretch of hydrophobic residues, rendering it a C-tail anchored (TA) protein [27]. Mitochondrial TA proteins are always attached to the outer membrane and expose their functional N-terminal domains to the cytosol [27]. This was confirmed to be also the case with LiTXN3 by employing a digitonin/PK assay. As shown in Figure 7B, the N-terminal portion of LiTXN3 (detected with the anti-His antibody) was Mitochondrial Tryparedoxins digested at digitonin concentrations that expose cytosolic proteins to PK (at 0.8 mg digitonin/mg protein; controlled with the anti- LicTXNPx2 antibody), but that do not enable PK to reach the mitochondrial compartments. Access of PK to the mitochondrion could be tracked using an antibody specific for cytochrome c, a protein of the mitochondrial intermembrane space [28]. Altogether, this set of experiments demonstrates that LiTXN3 is an outer mitochondrial protein with its hydrophilic portion (containing the TXN active site) facing the cytosol. In other words, LiTXN3 cannot directly react with molecules that are located within the mitochondrial compartments, namely mTXNPx, nsGPX and UMSBP, and therefore sustain the biological processes driven by these proteins. Phylogenetic analysis of TXNs does not support the existence of a mitochondrial TXN in Trypanosoma LiTXN3 is a protein with sequence features that are not present in any of the active TXNs described to date (i.e. the cytosolic TXN1 enzymes and the mitochondrial TXN2 of L. infantum), but are maintained in the TXN2 enzymes of Trypanosoma. To gain insight into how these different TXNs relate to previously characterized enzymes at an evolutionary level, a phylogenetic analysis was performed using 30 DNA sequences, representative of 9 species of the genera Leishmania, Trypanosoma and Crithidia. Using the MEGA4 software [29], several methods were employed to construct various phylogenetic trees all of which, despite discrete differences, conserved the main features. The topology of one representative tree is shown in Figure 8. Remarkably, the 30 TXN ORFs analyzed separate into two distinct evolutionary branches which reflect the differences observed at the biochemical level and can, thus, be regarded as two different classes. The first of these classes contains the genes encoding the cytosolic TXNs of all trypanosomatids (TXN1), as well as the mitochondrial TXNs of Leishmania (TXN2). The second class, in which Leishmania TXN3 and Trypanosoma TXN2 are included, contains only sequences encoding proteins with a C-terminal hydrophobic extension typical of TA proteins and possessing alterations in amino acids required for classical TXN activity. The presence of members of these two classes in all trypanosomatids indicates that they were already contained within the trypanosomatid ancestor. Trypanoso- ma cruzi, maintains what is possibly the primitive situation, i.e. a single representative of each class in the same genomic locus, while T. brucei, for instance, have recently duplicated the TXN1 ORF and Leishmania have undergone further genetic rearrangements, such that, presently, they contain more than one member in each class (TXN1 and TXN2 in class I, TXN3 and TXN4 in class II). Noticeably, the tree represented in Fig. 8 further shows that the TXN2 of Leishmania (encoding the mitochondrial enzymes) and the TXN2 of Crithidia form a clear subgroup within class I whose origin must have preceded Leishmania and Crithidia speciation. Addition- ally, the tight definition of this subgroup (bootstrap support of 99%) and the fact that it contains no Trypanosoma sequences, strongly suggest that its precursor gene arose after Trypanosoma divergence or has been lost by the predecessor of this genus. In short, phylogenetic analysis of TXN sequences provides i) an explanation for the presence of fully active mitochondrial TXNs in Leishmania (e.g. LiTXN2), but not in Trypanosoma, and ii) demonstrates that Leishmania TXN3 genes group together with Trypanosoma TXN2 sequences in a separate class of TXNs whose distinctive feature is the presence of a C-terminal hydrophobic tail. The function of this class of TXNs is yet to be determined but characterization of the LiTXN3 member strongly suggests that these enzymes do not act as inner mitochondrial TXNs. PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e12607