Zinc is an essential trace element in all forms of life. Despite its critical importance in maintaining such basic functions as proliferation and cell growth, little is known about how zinc is acquired, stored, and utilized by the cell. Recent pioneering studies in the area of zinc homeostasis have started to uncover the mysteries of subcellular distribution and much less on the proteins that maintain it. Furthermore, the manner in which zinc is incorporated into the proper enzymatic or regulatory metalloprotein sites are not clear. Before these questions can be addressed, the fundamental mechanisms of Zn-homeostasis need to be established.

A central component of metal ion homeostasis systems is regulation of ion flow across lipid membranes. Whether it is the plasma membrane that separates the cell from external environment or intracellular organelle membrane that spatially partition reagents into specific domains, these lipids serve as barriers to the diffusion of charged and hydrophilic particles. Compounds or small peptides that bind tightly to zinc may partially shield the charge and act to ferry the ion across membranes. Ion-specific channels that form gated pores through which ions can easily traverse may allow fort the rapid and mass transport of the charged species. Until recently, little was known about how eukaryotic cells take up metal ions or how they regulate accumulation in response to ion availability and metabolic demand. Over the past five years, pioneering studies in Saccharomyces cerevisiae (Bakers yeast) have identified several genes involved in metal homeostasis (Eide et al, 1994; Palmiter, 1994). Clearly, metal ion uptake is remarkably more complex than was originally portrayed.

Separate high-affinity systems for Cu, Fe, Mn, and Zn are responsible for providing the element to the cell when it is in short supply. Each system is controlled by metal responsive regulatory proteins. In addition, low-affinity systems play a "housekeeping" role, supplying the metal when they are more abundant in the environment. In the case of zinc, ZRT-1 encodes the high-affinity transporter, while ZRT-2 codes for the low-affinity system (Figure 1). Zrc-1 is thought to be responsible for the transport of zinc into the storage vesicles. Recently, three more zinc transporters have been cloned from mammals by Palmiter et al. ( 1995; 1995; 1996; 1996). The first, ZnT-1, encodes a membrane efflux protein that localizes to the plasma membrane. The other two, ZnT-2 and ZnT-3, encode membrane transporters that move zinc form the cytosol into vesicles. Other proteins in the cellular machinery that may be responsible for storage, shuttling, and metal insertion into nascent proteins remain unidentified (Suhy and O'Halloran 1996).

Figure 1: Zinc Homeostasis Model (2)

Figure 2: Mouse fibroblast cells loaded with 25 uM Zinquin for 30 min. The picture was recorded with a Zeiss fluorescence microscope and UV excitation light source.

 

To identify possible mechanisms of zinc homeostasis, we have synthesized and structurally characterized the Zn(II) complex of zinquin, a fluorescent probe specific for zinc (Figure 3) (Nasir et al, 1999). This probe, first reported by Mahadevan et al (1996) is cell permeable and fluoresces upon binding zinc. zinquin binds Zn(II) ions with high selectivity. Among all metal cations only Cd(II) was observed to substantially increase the fluorescence intensity.

We and others have shown that zinquin fluorescence is an useful probe that reveals zinc fluxes within the cell. Through utilizing zinquin we are able to visually localize compartments of zinc within intact, living cells (Figure 4). We have shown that these compartements are vesicles that can be loaded or purged of Zn depending upon the levels of zinc in the growth media. To determine the nature of the vesicular structures observed with zinquin staining, a variety of immunofluorescent experiments were employed. The failure of these vesicles to correspond to any of the organelles tested, leads to the hypothesis that these vesicles are a new, compartment and are essential aspect of zinc storage and homeostasis. Further studies confirmed that cells starved for zinc will deplete Zn-levels in the vesicles but then resupply them as Zn(II) is restored to the media.

In our proposed zinc homeostasis model (Suhy and O'Halloran, 1996), the vesicles containing zinc act as intracellular storage or buffer sites. It is apparent that cells utilize these vesicles not only to avoid zinc toxicity but also and more importantly, as a defense against zinc deficiency.

References

• D. Eide, M. L. Guerinot, ASM News, 63(4), 199 (1997); R. D. Palmiter, Proc. Natl. Acad. Sci., 91, 1219 (1994) .

• R. D. Palmiter and S. D. Findley, EMBO J., 14, 639 (1995); R. D. Palmiter, Tox. Appl. Pharm., 135, 139 (1995); R. Palmiter, T. Cole, S. Findley, EMBO J., 15, 1784 (1996); R. D. Palmiter, T. B. Coby, C. J. Quaife, S. D. Findley, Proc. Natl. Acad. Sci., 93, 14934 (1996).

• D. A. Suhy, T. V. O'Halloran, in 'Metal Ions in Biological Systems: Interactions of Metal Ions with Nucleotides, Nucleic Acids and Their Constituents ', Vol. 32, Marcel Dekker, Inc. Basel, Switzerland, 1996, pp. 558.

• I. B. Mahadevan, M. C. Kimber, S. F. Lincoln, E. R. Tiekink, A. D. Ward, W. H. Betts, I. J. Forbes, P. D. Zalewski, Aust. J. Chem., 49, 561 (1996)

• Nasir, M.S., Fahrni, C.J., Suhy, D.A., Kolodisck, K.J., Singer, C.P. and O'Halloran, T.V. "The Chemical Cell Biology of Zinc: Structure and Intracellular Fluorescence of Zinc-Quinolinesulfonamide Complex" J. Biol. Inorg. Chem. - in press.

• Fahrni, C.J. and O'Halloran, T.V. "Aqueous Coordination Chemistry of Quinoline-based Fluorescence Probes for the Biological Chemistry of Zinc" J. Amer. Chem. Soc. - in press.

• Suhy, D.A., Simon, K.D., Linzer, D.I.H., O'Halloran, T.V. "Metallothionein is Part of a Zinc-Scavenging Mechanism for Cell Survival Under Conditions of Extreme Zinc Deprivation" J. Bio. Chem. 1999, 274, 9183-9192.