Fig 1.

Subcellular localization analysis of T. brucei aaRSs in bloodstream forms expressing C-terminal 3V5-tagged aaRS (except as indicated). The cells were fixed in 4% paraformaldehyde and stained with FITC-conjugated anti-V5 monoclonal antibody (V5-FITC), mitochondria were stained with Texas Red-conjugated anti-HSP70 monoclonal antibody (HSP70) or MitoTracker, and DNA was stained with DAPI or by GFP fluorescence. The images were then merged, as indicated. Differential interference contrast (DIC) images are also shown. (a) Parasites expressing 3V5-tagged proteins, as indicated. GlnRS has a predicted MTS from M1 to I51 and a trans-splicing site at nucleotide 80 (45), which would remove most of the MTS. Expression of the longer variant resulted in proteins localized in both the cytoplasm and mitochondrion. MCP2 is not an aaRS. (b) IleRS splicing variants. See reference 45 for alternative splicing information. The longer variant, IleRS-3V5 (IleRS-V5), starts from M1 and contains a predicted MTS from M1 to G66, and the shorter variant, IleRS-3V5 with the deletion of amino acids M1 to G66 (IleRS-V5 Δ^M1-G66), starts at M67 and lacks the predicted MTS. (c) The IleRS MTS (M1 to G66) was cloned in frame with GFP (^M1-G66-GFP) and is sufficient to target GFP to the mitochondrion. Parasites expressing GFP alone were used as a control. (d) ProRS splicing variants. See reference 45 for alternative splicing information. The longer variant, ProRS-3V5 (ProRS-V5), starts with M1 and contains a predicted MTS, and the shorter splicing variant, ProRS-3V5 with a deletion from amino acids M1 to T93 (ProRS-V5 Δ^M1-T93), starts at M94 and has the predicted MTS region from M1 to T93 removed.