A phosphodiesterase 2A isoform localized to mitochondria regulates respiration

Abstract

Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.

FIGURE 1.

cAMP-dependent respiratory chain regulation in brain mitochondria and enzymological and pharmacological characterization of mitochondrial PDE activity. A, cAMP-dependent regulation of oxidative phosphorylation in mitochondria isolated from mouse brain. Treatment with the membrane-transmissible cAMP analog 8-Br-cAMP increased the rate of ATP formation (left) and the activity of COX (right). The PKA inhibitor H89, in contrast, decreased both activities. n.t., non-treated. Error bars represent S.E. values. ***, p < 0.0001. B and C, sensitivities of the mitochondrial cAMP-hydrolyzing (B) and cGMP-hydrolyzing (C) activities to a panel of PDE inhibitors. White bars, brain mitochondria; gray bars, liver mitochondria. D, concentration-response curves for inhibition of mitochondrial cAMP-degrading activity from brain (white circles) and liver (gray circles) by BAY60. E, concentration-response curves for stimulation of mitochondrial cAMP-degrading activity from brain (white circles) and liver (gray circles) by cGMP. Error bars represent S.E. values.

FIGURE 2.

Identification of PDE2A in the mitochondrial matrix. A, Western blot of mouse liver and brain homogenates (hom) and crude mitochondria (mt). B, Western blot of mitochondrial compartments derived from purified mitochondria from mouse brain and liver. Mitochondria and mitoplasts (Mp; inner mitochondrial compartment) contain a PDE2A species, whereas it is absent from the post-mitoplast fraction (P-Mp; contains intermembrane space proteins). hsp60, marker for mitochondrial matrix proteins; COXI, marker for mitochondrial inner membrane proteins; cyt c, marker for intermembrane space proteins. C, mitochondria and mitoplasts are shown non-treated (n.t.) or after being subjected to PK or Triton plus proteinase K (T+PK) treatment. The β-actin control shows that the small cytoplasmic contamination present in the initial mitochondrial fraction is removed after PK treatment of mitochondria and during mitoplast preparation. hsp60 and grp75, markers for mitochondrial matrix proteins; COXI and Tim23, markers for mitochondrial inner membrane proteins; cyt c, marker for intermembrane space proteins. n.t., non-treated. Blots are representative of three independent experiments.

FIGURE 3.

The PDE2A2 N terminus acts as a mitochondrial localization signal. A, probability scores, calculated with SignalP, for the N termini of PDE2A isoforms 1 through 3 to act as mitochondrial localization sequences. A score combining this probability with the likelihood of a position to serve as a mitochondrial processing site (signal + cleavage) identifies three sites with a high probability to serve as proteolytic processing sites during import. aa, amino acid. B, a helical wheel representation of the 19 N-terminal residues of PDE2A2 reveals that these residues can form the amphipathic helix (top, hydrophobic; bottom, hydrophilic) typical for mitochondrial localization sequences. C, constructs used in the mitochondrial interaction experiments shown in panels D and E. Protein parts from the mitochondrial matrix protein Map are symbolized as white bars, residues from human PDE2A2 are symbolized as gray bars, and the 18 N-terminal residues unique for this PDE2A isoform (sequence on top) are symbolized as dark gray bars. D, in vitro tests for interaction with mitochondria. Radiolabeled samples (left) of PDE2A2(1–210) and PDE2A2(18–210) were incubated with rat liver mitochondria for 10 min at 25 °C. Mitochondria were reisolated, and associated proteins were analyzed by SDS-PAGE and autoradiography using a BAS-1800 II imager (left). For quantitation (right), the amount of protein in the absence of rat liver mitochondria was set to a value of 1 (control). Retic. lysate, reticulocyte lysate. E, in vitro tests for the interaction of Map(45–203) and PDE2A2(1–17)-Map(45–203) with mitochondria. The experiments were carried out as described in panel D. Error bars in panels D and E represent S.D. values. F, upper panel, confocal images of HEK cells overexpressing a mitochondrial marker (DsRed-mito, red) and the N termini of PDE2 isoforms fused to EGFP or CFP (green). The three PDE2A isoforms show distinct subcellular distributions, and considerable overlap with the mitochondrial marker, visible in yellow (see for example the arrow), is specifically observed for isoform 2 (see inset). Lower panel, graphical presentation of the intensity correlation analysis highlighting substantial overlap of the PDE2A2 isoform with DsRed-mito. Scale bar: 3 μm.

FIGURE 4.

Mitochondrial PDE2A regulates the respiratory chain. A, cAMP levels in intact mouse brain mitochondria after short treatment (10 min) with inhibitors for PDE2 (EHNA and BAY60), PDE4 (rolipram), and sAC (KH7) (n = 6). nt, non-treated. B, pyruvate plus malate-driven ATP synthesis in intact mouse brain mitochondria after treatment with the agonist 8-Br-cAMP or sAC and PDE inhibitors (n = 9). Values are represented as the percentage of control non-treated mitochondria. C, pyruvate plus malate-dependent respiration of isolated brain mitochondria after treatment with the agonist 8-Br-cAMP or sAC and PDE inhibitors (n = 2). Error bars represent S.E. values. *, p < 0.01; **, p < 0.001; ***, p < 0.0001.

FIGURE 5.

Model for the mitochondrial cyclic nucleotide signaling system. cAMP is formed inside the mitochondrial matrix by a sAC isoform and stimulates the respiratory chain. The cAMP signals are terminated through degradation by PDE2A isoform 2. Thus, formation of cAMP is stimulated by bicarbonate, which activates sAC, and degradation is stimulated by cGMP or a yet to be identified regulatory ligand for the PDE2A2 GAF domains. IMS, intermembrane space.