Deletion analysis of rat GFG mitochondrial targeting. (A) Schematic representation of GFG:GFP fusion proteins. The grey, black and hatched boxes represent the GFP, nudix and mitochondrial targeting signal peptide (MTS) domains, respectively. rGFGΔ2 indicates the rGFGΔ2 isoform in which amino acids (aa) 77–145 encoded by exon 2 are deleted by alternative splicing. rGFGΔ2/3, an alternative splice form lacking exons 2 and 3, encodes a GFG isoform containing only the N-terminal 76 aa of full-length GFG and a unique C-terminal tail consisting of 26 aa. rGFGΔ2/C56 is the GFP fusion of rGFGΔ2 mutant in which the C-terminal 56 aa were deleted. rGFGΔNudix is the GFP fusion of full length GFG from which the conserved nudix domain (23 aa) was deleted. rGFGN41, rGFGN145, rGFGC168, rGFGC146 and rGFGC102 indicate the GFP fusions of the N-terminal 41aa and 145aa, and C-terminal 168 aa, 146 aa and 102 aa of rat GFG, respectively. Their subcellular localizations are summarized at the right side of the diagram. C/N indicates the fusion protein localized in the cytoplasm and nucleus. M indicates the fusion protein targeted to mitochondria. (B) and (C) Subcellular localization of GFG:GFP fusion proteins transiently expressed in the rat C6 glioma cells. C6 cells were seeded in the wells of the chambered coverslips containing DMEM supplemented with 10% FBS and incubated overnight, then transfected with the plasmids expressing the indicated GFG:GFP. The subcellular localization of the GFG:GFP fusions in the live cells was visualized with confocal laser scanning microscopy and a single slice of the stacks taken from a cell is shown here. The green channel shows the fluorescence of GFG:GFP fusion proteins (left panels), and the red channel shows the fluorescence of the MitoTracker indicating the subcellular patterns of mitochondria (middle panels), The yellow color in the merged images (right panels) indicates the GFG fusion proteins targeted to mitochondria.