Zinc metallothionein imported into liver mitochondria modulates respiration

doi:10.1073/pnas.041619198

Zinc metallothionein imported into liver mitochondria modulates respiration

Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Seeley G. Mudd Building, 250 Longwood Avenue, Boston, MA 02115

Contributed by Bert L. Vallee

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 β-domain of MT; in this system, the isolated C-terminal α-domain is inactive. Free zinc inhibits respiration at concentrations commensurate with the zinc content of either MT or the isolated β-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.

Footnotes

↵ * To whom reprint requests should be sent. E-mail: bert_vallee{at}hms.harvard.edu.
↵ † In the absence of ADP, incubation of mitochondria with zinc activates oxygen consumption (data not shown; ref. 7). Apparently, incubation allows enough time for zinc to reach mitochondrial sites where it can affect the rate of succinate oxidation. Alternatively, activation by zinc or MT could be caused by an effect on mitochondrial swelling and ensuing uncoupling, which occurs on a time scale longer (>5 min) than that of the effects on the respiratory chain (28,29). In the absence of ADP, the effect of MT depends on the order of addition. In succinate-powered mitochondria, MT inhibits the rate of oxygen consumption. However, if added before succinate, MT substantially activates this rate at concentrations below 4 μM (data not shown). Such an activation by Cd,Zn-MT was also reported by Simpkins et al. (7), who did not specify the order of additions, however.
↵ ‡ Little effect was noticed with zinc sulfate from a 500 μM stock solution prepared in incubation buffer that contained phosphate, presumably because of the formation of insoluble zinc phosphate. The chemical form of zinc is critical and may be the reason for the wide variations noted when zinc was tested for its effects on mitochondrial respiration under different assay conditions (7, 30).
Abbreviations:

MT,

metallothionein;

IMS,

intermembrane space;

T,

thionein;

TPEN,

N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine;

m-Cl-CCP,

carbonyl cyanide m-chlorophenylhydrazone