A Ca2+-induced mitochondrial permeability transition causes complete release of rat liver endonuclease G activity from its exclusive location within the mitochondrial intermembrane space. Identification of a novel endo-exonuclease activity residing within the mitochondrial matrix

Abstract

Endonuclease G, a protein historically thought to be involved in mitochondrial DNA (mtDNA) replication, repair, recombination and degradation, has recently been reported to be involved in nuclear DNA degradation during the apoptotic process. As a result, its involvement in mtDNA homeostasis has been called into question and has necessitated detailed analyses of its precise location within the mitochondrion. Data is presented localizing rat liver endonuclease G activity exclusively to the mitochondrial intermembrane space with no activity associated with either the interior face of the inner mitochondrial membrane or with the mitochondrial matrix. Additionally, it is shown that endonuclease G can be selectively released from the mitochondrion via induction of a Ca2+-induced mitochondrial permeability transition and that, upon its release, a further nuclease activity loosely associated with the interior face of the inner mitochondrial membrane and distinct in its properties from that of endonuclease G can be detected.

Figure 1

Size exclusion chromatography and endonuclease activity of salt-extracted mitochondrial inner membrane proteins. Separation of proteins extracted from the inner mitochondrial membrane was achieved by means of size exclusion chromatography. The elution of known standards was used to identify the approximate size of the proteins in the active fractions: 1, β-amylase (200 kDa); 2, alcohol dehydrogenase (150 kDa); 3, bovine serum albumin (66 kDa); 4, carbonic anyhdrase (29 kDa); 5, cytochrome c (12.4 kDa). The relative activity of each fraction was determined by the loss of 20 ng of linearized pBR322 incubated in 20 µl of buffer containing proteins extracted from the inner mitochondrial membrane at a concentration of 0.02 mg/ml. (A) Sephacryl HR-200 separation; proteins were incubated with DNA for 1 h at 37°C. (B) Sephacryl HR-100 separation; proteins and DNA were incubated for 10 min at 37°C. (C) pH dependence of endonuclease activity.

Figure 2

Endonuclease digestion of supercoiled pBR322 plasmid DNA and analysis of proteins associated with open circular intermediate. (A) Supercoiled pBR322 plasmid DNA (1 µg) was incubated at 37°C in the presence of the active 48–64 kDa endonuclease preparation (0.01 mg/ml) from Figure 1B. Aliquots (100 ng) were removed at the time points indicated and analyzed by agarose gel electrophoresis. Control lane indicates plasmid DNA incubated in the absence of protein for 60 min. (B) SDS–PAGE analysis of proteins associated with open circular plasmid DNA separated in (A).

Figure 3

Analysis of endonuclease-mediated cleavage of PCR-amplified mtDNA D-loop fragment. (A) Time course of D-loop fragment cleavage. PCR amplified mtDNA D-loop fragment was incubated in the presence of a 48–64 kDa (see Fig. 1B) active endonuclease fraction (0.05 mg/ml) at 37°C for the times indicated. Aliquots (100 ng) were removed and analyzed by agarose gel electrophoresis. Lanes 1–5, time course; lane 6, mtDNA D-loop incubated for 120 min in the absence of protein; lane 7, SspI cleavage of mtDNA D-loop fragment; lane 8, 1 kb DNA ladder (Life Technologies, Rockville, MA). (B and C) D-loop fragment was amplified using primer 1 5′-end-labeled with fluorescein. Lane 1, 1 kb DNA ladder; lane 2, unlabeled D-loop fragment; lane 3, unlabeled D-loop fragment incubated with active endonuclease; lane 4, labeled D-loop fragment incubated with active endonuclease; lane 5, fluorescein-labeled λ/HindIII fragments. (B) Agarose gel, ethidium bromide stained. (C) DNA transferred to membrane and developed using anti-fluorescein antibody.

Figure 4

EndoG activity associated with inner mitochondrial membrane detected by degradation of 403 bp fragment of mtDNA. Mitoplasts (0.25 mg/ml) were incubated with amplified D-loop fragment (100 ng) for the times indicated at 25°C. Lane 1, molecular weight markers; lanes 2–4, time course using sucrose-washed mitoplasts; lanes 5–7, time course using salt-washed mitoplasts.

Figure 5

Effect of a MPT upon the retention of endoG activity and cytochrome c protein by rat liver mitochondria. (A) Mitochondria (75 µg/ml) were suspended in 2 mM HEPES, pH 7.5, 0.25 M sucrose, 10 mM succinate and 1 mM potassium phosphate at 25°C, in the presence or absence of cyclosporin A (CyA, 4.5 µg/ml), and a MPT induced by the addition of Ca²⁺ (1 µM final concentration). Absorbance was monitored at 540 nm. (B and D) EndoG activity released and retained by the mitochondria upon induction of a MPT. (C and E) Cytochrome c protein released and retained by the mitochondria upon induction of a MPT. Lane 1, control mitochondria; lane 2, Ca²⁺-induced MPT; lane 3, Ca²⁺-induced MPT following pre-treatment (5 min) with CyA.

Figure 6

Western blotting of endoG activity gel. An endoG activity gel was run, transferred to nitrocellulose and probed with an antibody to endoG as described in Materials and Methods. (A) Activity gel. Lane 1, proteins released during induction of a MPT; lane 2; molecular weight markers (MW). (B) Western blot of lane 1. (C) Coomassie blue stained MW.

Figure 7

Characterization of mitochondrial endo-exonuclease activity. (A) Time course of amplified mitochondrial D-loop degradation by an endo- exonuclease extract isolated from SMP that had been freeze–thawed and the membranes removed by centrifugation. DNA (100 ng) was incubated in the presence of endo-exonuclease extract (0.25 µg/µl) at 37°C for the times indicated. (B) Dose–response degradation of linearized pBR322. All incubations proceeded for 15 min. C1, control DNA; C2, control DNA incubated for 15 min at 37°C. (C) Time course of supercoiled mtDNA cleavage and subsequent degradation. Mitochondrial protein (0.15 µg/µl) was incubated with DNA (100 ng) at 37°C for the times indicated. C1, control mtDNA; C2, control mtDNA incubated for 60 min at 37°C. (D) pH dependence of endonuclease activity. Supercoiled mtDNA (100 ng) was incubated in the presence of endo-exonuclease extract (0.15 µg/µl) for 3 min.