Subcellular localization of rat GFG and deletion mutants. (A) Schematic representation of GFG:GFP fusion proteins. The grey, black and hatched boxes represent the GFP, nudix and mitochondrial targeting signal peptide domains, respectively. rGFG indicates full-length rat GFG fused to GFP, ΔN28 indicates the GFG mutant lacking the N-terminal 28 amino acids encoding the predicted mitochondrial targeting peptide (MTS) of the rat full-length GFG. The N76 mutant encodes only the N-terminal 76 amino acids of GFG, including the MTS. The molecular weights and subcellular localizations are indicated at the right side of the diagram. Cyt//Nuc indicates that the fusion protein was localized in the cytoplasm and nucleus. Mt indicates the fusion protein was localized to mitochondria. The numbers in brackets indicate the predicted molecular weight of the fusion protein after cleavage of the N-terminal MTS. (B, C) Subcellular fractionation and immunodetection of GFG:GFP fusions in stably transfected C6 cells. Wild type (WT) C6 cells or cells stably transfected with the indicted GFP-GFG constructs were harvested and subjected to isolation of mitochondrial (M) vs. non-mitochondrial (cytoplasm + nuclei; C/N) fractions (panel B) or nuclear vs. non-nuclear (cytoplasm + mitochondria; C/M) fractions (panel C). Following SDS-PAGE proteins were immunodetected with a monoclonal anti-GFP antibody or a polyclonal antibody to heat shock protein Hsp60 as a molecular marker of mitochondria in the fractions. Arrowheads indicate putative GFG dimers. (D) Subcellular localization of GFG:GFP fusion proteins stably expressed in rat C6 glioma cells. Stable transfectants were seeded in the wells of the chambered coverslips containing DMEM supplemented with 10% FBS and incubated overnight. Mitochondrial staining of stable cell lines was as described in Materials and Methods. The subcellular localization of the GFG:GFP fusions in the live cells was monitored 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), the red channel shows the fluorescence of the MitoTracker indicating the subcellular patterns of mitochondria (middle panels), the yellow fluorescence in the merged images (right panels) indicates the GFG fusion proteins targeted to mitochondria. (E) Subcellular localization of GFG:GFP fusion proteins transiently expressed in rat C6 glioma cells. Cells were transiently transfected with the indicated constructs. The images were taken as described in (D).