Fig. 2
From: Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus
dUTPase-GFP fusion proteins localized to mitochondria. a Sequences of expressed D. discoideum dUTPase-green fluorescent protein (GFP) fusions. The full-length D. discoideum dUTPase polypeptide had GFP fused to the C-terminal Asn (N) 179 to produce dUTPase-GFP. Residues 1–40 of dUTPase were fused to GFP with a (Gly-Ala)5 linker; in bold are residues 1–20 used in the helical wheel drawn in Panel e. In both constructs, the Thr-Ser-Ser (TSS) tripeptide sequence arose from the cloning process. The underlined linker sequence was identified by N-terminal sequencing of protein immunoprecipitated by anti-GFP from Ax2 cells expressing N1–40-dUTPase-GFP. b Confocal microscopy images of live Ax2 cells expressing dUTPase-GFP stained with Mitotracker; merged signals showed co-localization. Scale bar, 5 µm. c Ax2 cells expressing dUTPase-GFP were fixed and stained with Hoechst 33342 to identify the nuclear DNA. dUTPase-GPF was absent from the nucleus. Scale bar, 5 µm. d Confocal microscopy images of live Ax2 cells expressing N1–40-dUTPase-GFP stained with Mitotracker; merged signals showed co-localization of the GFP signal with mitochondria. Scale bar, 5 µm. e Helical wheel of residues 1-20 of the N-terminus of D. discoideum dUTPase shows its amphipathic character. Symbols represent the following: circles, hydrophilic residues; diamonds, hydrophobic residues; triangles, potentially negatively charged; and pentagons, potentially positively charged. Color code: the most hydrophobic residue is green, and the amount of green is decreasing proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow; hydrophilic residues are coded red with pure red being the most hydrophilic (uncharged) residue, and the amount of red decreasing proportionally to the hydrophilicity; potentially charged residues are light blue [13]. f Immunoblots showed N1–40-dUTPase-GFP in mitochondria. A mouse monoclonal specific for D. discoideum porin (upper panel; 30.1 kDa) [18] or a rabbit polyclonal anti-GFP (lower panel) was used to probe blots of whole cell lysates (WCL; ~ 100,000 cells) and enriched mitochondria preparations (mt; ~ 9 × 106 cell equivalents) from Ax2 cells expressing N1–40-dUTPase-GFP. Positions of prestained markers are shown to the right of each blot