Fig. 3.

Submitochondrial localization of PARP1cd fusion proteins based on PAR accumulation in the matrix, but not the intermembrane space. a GDH-PARP1cd and AIF-PARP1cd fusion proteins localize to mitochondria. HeLa S3 cells were transiently transfected with vectors encoding the indicated mitochondrial PARP1cd fusion proteins. After 24 h, cells were stained with MitoTracker (MT) and subjected to myc-immunocytochemistry. The fluorescence micrographs show nuclei (DAPI), expressed recombinant proteins (myc), and mitochondria (MT). Bar 10 μm. b Robust PAR accumulation is restricted to the mitochondrial matrix. After transient transfection of vectors encoding GDH-PARP1cd and AIF-PARP1cd, HeLa S3 cells were subjected to immunocytochemistry using myc- and PAR-specific antibodies. The fluorescence micrographs show nuclei (DAPI), expressed recombinant proteins (myc), and generated PAR. Bar 10 μm. c Artificial re-targeting of mitochondrial PARP1cd fusion proteins confirms matrix localization to be essential for PAR accumulation. HeLa S3 cells were transfected with vectors encoding the indicated PARP1cd fusion constructs (matrix-targeted mAIF-PARP1cd and GDHΔ53-PARP1cd lacking the mitochondrial targeting sequence) and subjected to immunocytochemistry as in (b). Bar 10 μm. d PAR accumulation requires PARP1cd catalytic activity. HeLa S3 cells transiently transfected with a GDH-PARP1cd encoding plasmid were cultured in presence (+) or absence (−) of the PARP1 inhibitor PJ34 (5 μM). Cells were subjected to PAR- and myc-immunocytochemistry 24 h post transfection as in (b). Bar 10 μm. e Co-expression of matrix-targeted PARG (mPARG) eliminates GDH-PARP1cd-mediated PAR accumulation. HeLa S3 cells were co-transfected with vectors encoding GDH-PARP1cd and a FLAG-tagged mitochondrial PARG construct (mPARG). The fluorescence micrographs show nuclei (DAPI), GDH-PARP1cd (myc), mPARG (FLAG), and polymers (PAR). Bar 10 μm. f Schematic representation of the principle to distinguish between mitochondrial matrix and intermembrane space protein localization. If PARP1cd fusion proteins are directed into the matrix, PAR accumulation (aggregates of red spheres) is readily observed, whereas PAR is virtually not detectable (dotted spheres) for fusion proteins residing in the intermembrane space