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

Abstract

In mammalian cells the cytosolic concentration of free Zn²⁺ 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 Zn²⁺ ions. Since metallothionein (MT) is an important ligand form of cellular zinc, we determined if Zn₇-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 Zn₇-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, Zn₇-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.

1. Introduction

We previously reported the kinetic identification of a putative zinc uptake transporter in prostate and liver mitochondria [1]. The transporter is associated with the inner mitochondrial membrane, exhibits the characteristics of a facilitative transport process, does not require a free Zn²⁺ ion pool for transport; and effectively transports zinc from zinc ligands with log K_f values up to ~11, but not from ligands with log K_f values equal to or greater than 13. The transporter exhibits an apparent log K_f ~12. The Km ranges from about 30–80 μM zinc, depending upon the ligand and the source of mitochondria. Because the free Zn²⁺ ion concentration of cytosol is negligible (nanomolar to femtomolar range) [2], we postulated that low molecular weight zinc ligands (ZnLigands) comprise the major cytosolic pool of zinc for delivery to and transport into the mitochondria by this putative zinc uptake transporter.

It was essential to determine if zinc metallothionein might also be an important donor form for mitochondrial zinc uptake. Recently we obtained two different Zn₇-MT sources with which we determined zinc transport into mitochondria. Rabbit liver Zn₇-MT-2, prepared as described in Halthout et al. [3], was kindly provided by Dr. C. Fenselau at the University of Maryland, College Park, MD. Recombinant human Zn₇-MT, described in Hong and Maret [4], was generously provided by Dr. Maret at the University of Texas Medical Branch, Galveston, Texas. Both preparations were devoid of free Zn²⁺ ions. A poly-clonal antibody against human and rat MT-1 and -2 proteins was generously provided by Dr. P.C. Huang at Johns Hopkins University, Baltimore, MD [5]. The methods for prostate and liver mitochondria preparation, the conditions for zinc accumulation, and all other assays were the same as previously described [1]. Although the experimental protocol was limited by the availability Zn₇-MT, the results obtained were apparent, conclusive and consistent with our earlier report [1].

Zinc uptake from 30 μM purified rabbit liver Zn₇-MT-2 (MT-2: 30 μM; total bound zinc: 210 μM) by intact mitochondria was compared to that from 30 μM ZnCysteine (zinc: 30 μM; Cysteine: 90 μM). The latter is included because we previously showed that it is as effective as free Zn²⁺ ions as a donor form for mitochondrial uptake and because its log K_f ~10 is close to Zn₇-MT-2 (~11–12). Fig. 1(a) shows that mitochondrial zinc accumulation from rabbit liver Zn₇-MT-2 was as effective as from ZnCysteine in both liver and ventral prostate mitochondrial preparations. The results with ZnCysteine also confirms our previous observation that the liver and prostate mitochondria have the capacity to accumulate similar levels of zinc. In order to determine if zinc accumulation was due to the mitochondrial accumulation of Zn₇-MT-2, the levels of the mitochondrial MT were determined by Western blot. Fig. 1(b) shows that the presence of endogenous ~12 kDa MT in liver mitochondria, but no significant MT change was observed during incubation with rabbit liver Zn₇-MT-2. In the ventral prostate mitochondria, no detectable MT was found in the presence or absence of 30 μM rabbit liver Zn₇-MT-2. Therefore, zinc accumulation involves the transfer of zinc from the ligand, which confirms our previous observation [1] with other ZnLigands.

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.

Zinc accumulation was also determined from recombinant Zn₇-MT (Maret preparation) in liver mitoplast preparation. Fig. 2(a) shows that significant zinc accumulation resulted from Zn₇-MT; but, unlike in Fig. 1(a), somewhat less than the accumulation from ZnCysteine. The absence of zinc accumulation from ZnEDTA shows that the inner membrane is in tact, and that ZnEDTA is not a zinc donor form due to its high binding affinity (log K_f ~16) as we previously reported [1]. The absence of MT accumulation in the mitoplasts (Fig. 2(b)) confirms that zinc is transferred from the ligand in the transport process, and confirms that the putative transporter is associated with the inner membrane. An apparent difference in the recombinant preparation is its detection as a ~40 kDa doublet for which no explanation can be provided at this time (personal communication with Dr. Maret). This might also account for the lower zinc accumulation with this preparation. Nevertheless, when combined with the results in Fig. 1, it is apparent that Zn₇-MT-2 is an effective zinc donor for the putative zinc uptake transporter involved in the transport of zinc across the inner membrane and into the mitochondrial matrix. The results are also consistent with our earlier conclusion that the putative zinc uptake transporter has an apparent log K_f ~ 12.

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.

Previous studies indicated that oxidants can induce zinc release from MT by oxidizing the sulfur ligands in zinc/thiolate cluster [6–9]. Ye et al. [10] reported that cytosolic Zn₇-MT-2 could enter the intermembrane space where oxidative conditions and/or lower pH cause the release of zinc that inhibits terminal oxidation. This raises the possibility that released free Zn²⁺ ions from Zn₇-MT-2 traversed the inner membrane into the mitochondrial matrix. However, our zinc uptake studies employed non-respiring oxidatively inactive in tact mitochondria and mitoplasts. Therefore the uptake of zinc from Zn₇-MT-2 did not require a release of free Zn²⁺ ions for transport into the mitochondria, as we previously reported [1] for other ligands. This is consistent with the observation that zinc is directly transferred from Zn₇-MT-2 to acceptor protein without the release of free Zn²⁺ ion [3]. Although a total of seven zinc binds to one MT molecule, previous studies demonstrated that Zn₇-MT does not transfer all of its zinc to other proteins, but one zinc in the Zn₃-β cluster is more prone to transfer than the others[3,8,11]. This might account for the observation (Fig. 1) that the accumulation of zinc from Zn₇-MT-2 that contains 210 μM zinc/30 μM MT-2 was similar to zinc accumulation from 30 μM ZnCysteine.

In the rat ventral prostate cells, the high levels of citrate and aspartate are the apparent important ZnLigands that serve as cytosolic zinc-donors for the characteristic high mitochondrial uptake and accumulation of zinc [1] since metallothioneins are not detectable in these cells [unpublished information; 11–14]. In liver, and likely other mammalian cells, Zn₇-MT-2 could be an important chaperone for delivery to and transport of zinc by the mitochondria. Most importantly, the mounting evidence supports the concept that, in the absence of a sufficient cytosolic free Zn²⁺ pool, loosely bound ZnLigands (including Zn₇-MT-2) are effective donors of zinc for the mitochondrial transport of zinc.