Zinc metallothionein imported into liver mitochondria modulates respiration

Abstract

Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiological, micromolar concentrations of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the N-terminal beta-domain of MT; in this system, the isolated C-terminal alpha-domain is inactive. Free zinc inhibits respiration at concentrations commensurate with the zinc content of either MT or the isolated beta-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apoform of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]/[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are critical for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apocytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homology to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biological modulating activity of MT in cellular respiration and energy metabolism in a tissue-specific manner.

Figure 1

Western blot detection of MT in subcellular and submitochondrial fractions from rat liver. Lane 1, rat Zn₇-MT-1; lanes 2–5, isolated submitochondrial fractions; lane 6, total mitochondrial protein obtained in the presence of 1% (vol/vol) Triton X-100; lane 7, supernatant from the centrifugation of mitochondria. Proteins (20 μg of total protein in each lane except for MT-1, which was 4.5 μg) were separated on 15% SDS/PAGE and transferred to a nitrocellulose membrane. The relative amounts of MT were determined with the monoclonal antibody II-10a.

Figure 2

Western blot detection of MT-3 imported into isolated rat liver mitochondria. Lane 1, IMS fraction (200 μg); lane 2, IMS fraction (200 μg) after incubating mitochondria with human MT-3; lane 3, MT-3 (0.2 μg). For details see Materials and Methods.

Figure 3

Inhibition of state 3 mitochondrial respiration by MT isoforms and zinc-reconstituted MT-domain peptides. Mitochondria corresponding to 500 μg of protein were injected into the electrochemical cell (450 μl) and incubated for 3 min. Succinate and ADP were then added to final concentrations of 3 mM and 0.5 mM, respectively. MT (up to 15 μl) was added when about half of the ADP was consumed (after about 1 min). The data for rabbit Zn₇-MT-1 (●) and Zn₇-MT-2 (○) were from two preparations of mitochondria. Incubation buffer (2–15 μl) or BSA (15 μg in 10 μl of buffer) had no effect. Zn-reconstituted α-domain (■) and β-domain (□) of human MT-2 were also equilibrated with incubation buffer. Addition of 10 μl of buffer served as a control. Standard errors are based on at least two measurements.

Figure 4

Inhibition of uncoupled mitochondrial respiration by rabbit MT-1 (▾), zinc-reconstituted β-domain (■), and zinc (●). The order of addition was mitochondria, succinate, m-Cl-CCP, and MT, MT-domain peptide, or zinc. The final concentration of m-Cl-CCP was 2 μM.

Figure 5

(A) Reactivation of zinc-inhibited state 3 respiration by T (●), TPEN (▴), and DTT (■). Mitochondrial respiration was first inhibited by zinc (10 μM). Respiration rates were normalized to that of a buffer control. The order of addition was mitochondria, succinate, ADP, zinc, and then T, TPEN, or DTT. (B) Reactivation of MT-2-inhibited state 3 respiration by T. Respiration was about 55% inhibited by 6.5 μM rabbit Zn₇-MT-2 and then fully activated by T (2 μM). Buffer as a control had no effect. (Bars: x = 1 min, y = 22 nmol of oxygen atoms.)

Figure 6

Western blot detection of MT in mitochondria of rat liver and heart. Mitochondria isolated by differential centrifugation were purified by density gradient centrifugation as described in Materials and Methods. Ubiquinol–cytochrome c oxidoreductase core 1 protein served as a control for the presence of the same amount of total mitochondrial protein on the membrane.