NEURL4 is a mitochondrial protein with a functional ART domain. (A) Graphical representation of NEURL4 known and predicted structural domains and alignment of the putative NEURL4 catalytic domain with the PARP1 catalytic domain showing conservation in the H-Y-E triad and D-loop. (B) NEURL4 localization to the mitochondria by IHF staining in HeLa cells as shown by costaining with the mitochondrial marker ATP5B. (C) Proteinase K protection assay showing NEURL4 localization to the mitochondrial matrix. Degradation patterns of TOM20, AIF1, and mtHSP70 are representative of proteins in the OMM, IMM, and matrix, respectively. (D) Mitochondria labeling by Immunogold EM for NEURL4 in HeLa cells. (E) Increased FLAG-NEURL4 stability upon down-regulation of MMPs (both MMPα and MMPβ) by specific siRNA. (F–H) In vitro PARylation assays showing NEURL4 ability to produce poly-ADP-ribosylation chains (F) via its Ct domain (G and H). Immuno-purified endogenous NEURL4, FLAG-NEURL4-ΔCt, or FLAG-NEURL4-Ct was incubated with biotin-NAD⁺ in the presence or the absence of activated DNA. ADP-ribosylation was detected by WB anti-biotin. Loading controls for IP proteins were run on a different gel and tested with anti–FLAG-HRP antibody. Recombinant PARP1/ARTD1 was used as the positive control. The PARP1 reaction was run on the same gel and is shown here at lower exposure (F). MLS, mitochondria localization signal; Nt, N terminus; Pk, Proteinase K; ng, nanograms; siMMP, siRNA against MMPs (defined in E); FL, full length; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane.