Fig. 5.

(a) The putative mitochondrial targeting sequence (MTS) of TfR2 targets red fluorescent protein (RFP) to mitochondria. Top row, HEK 293 cells were co-transfected with cyan fluorescent protein (CFP) ligated to a known mitochondrial targeting sequence (MTS-CFP) and with RFP. The MTS-CFP protein labeled mitochondria, where as RFP was seen diffusely in the cytosol. A merged image reveals no colocalization of the MTS-CFP and RFP signals. Bottom row, when RFP was ligated to the putative MTS of TfR2 (TfR2-RFP), RFP was directed to mitochondria rather than cytosol, and the merged image showed extensive colocalization with MTS-CFP. The signal of MTS-CFP is represented in the pseudo-color green to better illustrate the yellow-colored overlapping regions in the merge. (b) In dopamine neurons from control animals (top row), there was relatively little expression of Tf and TfR2, but there was substantial colocalization (arrows) – and there was FRET between the two proteins, indicating a physical interaction (molecular proximity) of the proteins. After rotenone treatment (bottom row), there was an increase in the levels of both proteins and the amount of FRET between them also increased. Images from control and rotenone-treated animals were collected using identical parameters. As a result, in the control animals (top row), the Tf and TfR2 signals appear artificially low, so as to avoid saturation of the images from rotenone-treated animals.