Abstract
The hypothesis was tested that a kinetical flow equilibrium of uptake and efflux reactions is responsible for balancing the cellular zinc content. The experiments were done with the metal-resistant bacterium Cupriavidus metallidurans. In pulse-chase experiments, the cells were loaded with radioactive 65Zn and chased with the 100-fold concentration of non-radioactive zinc chloride. In parallel, the cells were loaded with isotope-enriched stable 67Zn and chased with non-enriched zinc to differentiate between zinc pools in the cell. The experiments demonstrated the existence of a kinetical flow equilibrium, resulting in a constant turnover of cell-bound zinc ions. The absence of the metal-binding cytoplasmic components, polyphosphate and glutathione, metal uptake, and metal efflux systems influenced the flow equilibrium. The experiments also revealed that not all zinc uptake and efflux systems are known in C. metallidurans. Cultivation of the cells under zinc-replete, zinc-, and zinc-magnesium-starvation conditions influenced zinc import and export rates. Here, magnesium starvation had a stronger influence compared to zinc starvation. Other metal cations, especially cobalt, affected the cellular zinc pools and zinc export during the chase reaction. In summary, the experiments with 65Zn and 67Zn demonstrated a constant turnover of cell-bound zinc. This indicated that simultaneously occurring import and export reactions in combination with cytoplasmic metal-binding components resulted in a kinetical flow equilibrium that was responsible for the adjustment of the cellular zinc content. Importance: Understanding the biochemical action of a single enzyme or transport protein is the pre-requisite to obtain insight into its cellular function but this is only one half of the coin. The other side concerns the question of how central metabolic functions of a cell emerge from the interplay of different proteins and other macromolecules. This paper demonstrates that a flow equilibrium of zinc uptake and efflux reactions is at the core of cellular zinc homeostasis and identifies the most important contributors to this flow equilibrium: the uptake and efflux systems and metal-binding components of the cytoplasm.