Zinc trafficking 1. Probing the roles of proteome, metallothionein, and glutathione

The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that nonspecific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 10(10-11), that are dependent on sulfhydryl ligands to achieve high-affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase, and also considered to be insignificant in cells. Adding the lowmolecularweight fraction of cell supernatant to the proteomemarkedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione (GSH). In agreement, inclusion of GSH accelerated the reaction in a concentration-dependent manner. The implications of abundant high-affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with GSH in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.

Automated summary

This study examined the role of nonspecific proteomic binding sites in zinc trafficking within cells. The results indicate that the proteome competes with metallothionein for Zn2+ binding, and the presence of GSH enhances the reaction speed. Overall, the study highlights the importance of high-affinity binding sites within the proteome for efficient zinc delivery to functional binding sites.