Metallothionein can function as a chaperone for zinc uptake transport into prostate and liver mitochondria

Abstract

In mammalian cells the cytosolic concentration of free Zn(2+) ions is extremely low (nM-fM range) and unlikely to provide an adequate pool for the uptake and accumulation of zinc in mitochondria. We previously identified a mitochondrial uptake transport process that effectively transports zinc directly from low molecular weight zinc ligands independent of and in the absence of available free Zn(2+) ions. Since metallothionein (MT) is an important ligand form of cellular zinc, we determined if Zn(7)-MT was an effective chaperone and donor for delivery and uptake of zinc by prostate and liver mitochondria. The results reveal that both intact mitochondria and mitoplasts effectively accumulated zinc from Zn(7)-MT. The study confirms and extends our previous report that the putative zinc transporter is associated with the inner mitochondrial membrane and involves a direct exchange of zinc from the ligand to the transporter. The ventral prostate cells contain no detectable MT; so that ligands (such as citrate, aspartate) other than MT are zinc donors for mitochondrial zinc accumulation. However, in liver and perhaps other cells, Zn(7)-MT is probably important in the cytosolic trafficking of zinc to the mitochondria for the uptake of zinc into the mitochondrial matrix by the putative zinc uptake transporter.

Fig. 1

Liver and VP mitochondrial zinc accumulation from rabbit liver Zn₇-MT-2: (a) comparison of zinc accumulation from ZnCys and Zn₇-MT-2 in liver and ventral prostate mitochondria by atomic absorption assay. ZnCys contains 30 μM zinc and 90 μM ligand. Zn-MT contains 30 μM MT and 210 μM zinc. Zinc uptake was performed for 10 min incubation at room temperature. (VP Zn₇-MT: n = 2; liver Zn₇-MT: n = 1; ZnCys: n = 3); (b) western blot detection of MT-2 imported into isolated liver and ventral prostate mitochondria. Aliquots of 50 μg mitochondrial protein were used for western blot. Purified rabbit liver MT-2 (0.1 μg) served as a MT standard.

Fig. 2

Zinc accumulation from recombinant human Zn₇-MT-2 by liver mitoplast: (a) zinc accumulation from 30 μM ZnCys, Zn₇-MT-2 and ZnEDTA by liver mitoplast by atomic absorption assay. Conditions were the same as Fig. 1(a). (Zn₇-MT: n = 2; others: n = 3); (b) western blot detection of Zn₇-MT-2 imported into liver mitoplast. Aliquots of 50 μg mitoplast protein were used for western blot. Fifty microgram liver cell protein extract and 0.1 μg recombinant human MT-2 served as MT standards.