Functional localization of two poly(ADP-ribose)-degrading enzymes to the mitochondrial matrix

Abstract

Recent discoveries of NAD-mediated regulatory processes in mitochondria have documented important roles of this compartmentalized nucleotide pool in addition to energy transduction. Moreover, mitochondria respond to excessive nuclear NAD consumption arising from DNA damage-induced poly-ADP-ribosylation because poly(ADP-ribose) (PAR) can trigger the release of apoptosis-inducing factor from the organelles. To functionally assess mitochondrial NAD metabolism, we overexpressed the catalytic domain of nuclear PAR polymerase 1 (PARP1) and targeted it to the matrix, which resulted in the constitutive presence of PAR within the organelles. As a result, stably transfected HEK293 cells exhibited a decrease in NAD content and typical features of respiratory deficiency. Remarkably, inhibiting PARP activity revealed PAR degradation within mitochondria. Two enzymes, PAR glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3), are known to cleave PAR. Both full-length ARH3 and a PARG isoform, which arises from alternative splicing, localized to the mitochondrial matrix. This conclusion was based on the direct demonstration of their PAR-degrading activity within mitochondria of living cells. The visualization of catalytic activity establishes a new approach to identify submitochondrial localization of proteins involved in the metabolism of NAD derivatives. In addition, targeted PARP expression may serve as a compartment-specific "knock-down" of the NAD content which is readily detectable by PAR formation.

FIG. 1.

PAR accumulation in mitochondria mediated by a targeted PARP1 construct. (A) Molecular architecture of the generated mitoPARP construct. The C-terminal 443 aa residues of full-length PARP1 (upper panel) harboring the catalytic domain were C-terminally fused to EGFP and endowed with the established N-terminal mitochondrial targeting sequence (MTS) of cytochrome c oxidase subunit VIII (lower panel). NLS, nuclear localization signal. (B) Images from confocal laser scanning of 293 cells and HeLaS3 24 h after transient transfection with mitoPARP-encoding vector in the absence (−; left panels) and presence (+; right panels) of 5 μM PJ34. The fluorescence revealed nuclei (DAPI), the mitoPARP protein (containing functional EGFP), and PAR (by indirect immunocytochemistry using 10H antibody). Bar, 20 μm.

FIG. 2.

Constitutive presence of PAR in mitochondria of stably transfected 293 cells. (A) Images from confocal scanning of 293 cells stably transfected with mitoPARP-encoding vector (293mitoPARP panels) after indirect immunocytochemistry using 10H antibody to detect PAR. Mitochondrial PAR accumulation colocalizing with the intrinsic EGFP fluorescence of the synthetic PARP construct was detectable in all cells. 293 cells stably transfected with a construct lacking the PARP portion of the construct (293mitoEGFP panels) were EGFP positive but PAR negative, whereas in parental 293 cells (parental 293 panels) neither green fluorescence nor immunoreactivity for PAR was detected. Bar, 20 μm. (B) Forty micrograms of total protein from the indicated cell lines was separated by 10% SDS-PAGE. Western blotting was performed using myc antibody. (C) Forty micrograms of total protein of the indicated cell lines was separated by 7% SDS-PAGE. PAR was revealed by Western blotting using the 10H antibody. β-Tubulin was used as a loading control in panels B and C.

FIG. 3.

Decrease of mitochondrial NAD by mitoPARP expression resembles respiratory deficiency compensated by enhanced glycolysis. (A) Cells were seeded into 96-well plates, and the viability was determined by MTT assays. Data are shown as means ± standard deviations of three experiments, each performed in triplicate. (B) Decrease in medium pH after long-term incubation. Data are presented as means ± standard deviations of three independent experiments. The inset shows representative culture dishes of the cell lines after 7 days in culture (DIC). (C) Increase in medium lactate levels after long-term incubation of the cell lines. Cell culture supernatants from panel B were subjected to determination of the lactate concentration. (D) 293mitoPARP cells exhibit a weaker mitochondrial membrane potential. Cells were loaded with JC-1, and fluorescence spectra were recorded before and after uncoupling the mitochondrial membrane potential by adding FCCP. Relative mitochondrial membrane potentials are expressed as the ratios between fluorescence intensities at 595 nm and 534 nm before and after FCCP treatment and were normalized to the data obtained for parental 293 cells. Data represent means ± standard deviations of three experiments.

FIG. 4.

PARP inhibition in 293mitoPARP cells reveals mitochondrial PAR degrading activity. (A) Images from confocal laser scanning of 293mitoPARP cells after incubation with 5 μM PJ34 for the indicated time periods and subsequent immunocytochemistry using 10H antibody. −PJ34, no PJ34. Bar, 50 μm. (B) Western blot analysis of 293 mitoPARP cells after incubation with 5 μM PJ34 for the indicated time periods. Cells before PJ34 incubation and after 24 h incubation in the absence of PJ34 were used as a control. The increase in PAR in the control cells is due to proliferation. Immunostaining of β-tubulin (used as a loading control) confirmed the steady increase of total protein amounts owing to cell proliferation.

FIG. 5.

A PARG isoform and ARH3 localize to mitochondria and are expressed in 293 cells. (A) Molecular architecture of the generated PARG and ARH3 constructs. The C-terminal 516 aa residues of full-length PARG (upper panel) harboring both the catalytic domain and an N-terminal predicted mitochondrial targeting sequence (MTS) were N-terminally fused to a FLAG epitope. Similarly, full-length ARH3 was endowed with a C-terminal FLAG tag (lower panel). As control, a PARG construct lacking the predicted targeting sequence was generated (middle panel). An additional methionine was added to the N terminus to enable translation. (B) PCR amplification products from HEK293 cDNA using specific primers for mitochondrial PARG isoforms (lane 1, exon Ia forward and exon IV reverse; lane 2, exon Ia forward and exon V reverse; lane 3, exon Ia forward and exon VI reverse; lane 4, exon Ia forward and exon V/VI reverse) and ARH3 (lane 5, exon V forward and exon VI reverse). The lack of specific amplicons in lanes 2 and 4 confirms the absence of exon V from the mitochondrial PARG isoform. Lanes 1a to 5a represent water controls (no DNA added) to lanes 1 to 5, respectively. (C) Western blot analyses of total cell lysates from 293 cells (lanes 1 and 4) and cells overexpressing PARG(Δ1-460) (lane 2), PARG(Δ1-477) (lane 3), and ARH3 (lane 5), using a FLAG antibody. (D) HeLaS3 cells were transiently cotransfected with mitoPARP-encoding vector and a vector encoding either PARG(Δ1-477) (panels in upper right quadrant), PARG(Δ1-460) (panels in lower left quadrant), or ARH3 (panels in lower right quadrant). As a control, empty plasmid was cotransfected (vehicle; panels in upper left quadrant). Cells were subjected to immunocytochemistry using FLAG antibody to stain PARG or ARH3. The mitoPARP protein was detected by its intrinsic EGFP fluorescence. Bar, 20 μm.

FIG. 6.

PARG(Δ1-460) and ARH3 degrade PAR in mitochondria. (A) Images from confocal laser scanning of HeLaS3 cells transiently cotransfected with vector encoding mitoPARP and either PARG(Δ1-460) harboring a predicted mitochondrial targeting sequence (panels in lower left quadrant) or ARH3 (panels in lower right quadrant). Control cells were cotransfected with mitoPARP-encoding vector and empty plasmid (vehicle; panels in upper left quadrant) and a vector encoding PARG(Δ1-477), which lacks the predicted targeting sequence (panels in upper right quadrant). Bar, 20 μm. (B) Stably transfected 293mitoPARP cells were either transfected with vectors encoding PARG(Δ1-460) (lane 2), PARG(Δ1-477) (lane 3), and empty plasmid (lane 4) or not transfected (lane 1) and subjected to Western blot analysis using 10H antibody. Fifty micrograms of total protein was separated by 7% SDS-PAGE; β-tubulin was used as loading control. (C) Doxycycline-induced (+) and noninduced (−) Flp-In T-REx 293 cells expressing ARH3 under control of a tetracycline-inducible promoter were transfected with the vector encoding mitoPARP (lane 3 and 4). Control cells were transfected with empty vector (lane 5 and 6) or left untransfected (lane 1 and 2). For Western blot analysis of PAR, 50 μg was separated by 4 to 12% SDS-PAGE. As a loading control eukaryotic translation initiation factor 2α was used.