FIG. 3.
Intracellular targeting of PB1-F2-EGFP fusion constructs. HeLa cells were labeled with the ΔΨm-sensitive mitochondrial dye TMRE, and images were acquired live at 16 h posttransfection with plasmids expressing PB1-F2-EGFP fusion proteins as indicated. Fluorescence images are presented as unmerged EGFP (left column), TMRE (middle column), and merged fluorescence (right column; EGFP is shown in green, and TMRE is shown in red). The full-length (1 to 87) chimeric protein (A to C) localizes to mitochondria (arrowheads) but also to the nucleus, nuclear membrane, and cytoplasm (double arrowheads). A deletion mutant (1 to 72) lacking the last 15 amino acids (D to F) displays uniform cytoplasmic and nuclear localization and is clearly absent from mitochondria. Among all the chimeric proteins, that consisting of PB1-F2 residues 65 to 87 exhibits the most complete mitochondrial localization (G to I). Replacing the 5 positively charged residues in the MTS of the full-length construct with Ala (1-87-5A) targeted the fusion protein to the nuclear membrane and another cytoplasmic structure (J to L). Replacing the MTS with the MTS of p13II in the full-length construct (1-71-p13 h-EGFP) targeted the fusion protein to mitochondria (M to O). Bars, 10 μm.