Zinc trafficking pathways from its entry into cells to the formation of native Zn-Proteins are poorly understood. Studies have reported that Zn2+ binds to apo-metallothionein to make Zn-metallothionein (Zn-MT), which then transfers Zn2+ to apo-proteins, thereby constituting native Zn-proteins. However, other studies revealed the existence of a substantial concentration of protein binding sites for Zn2+, suggesting that the proteome may compete for binding intracellular Zn2+ and also act as an intermediate along the pathway to constitute Zn-proteins. The present study focuses on the hypothesis that proteomic binding sites for Zn2+ are involved in the trafficking mechanism related to the formation of native Zn-proteins. First, the Zn2+ buffering capacity of proteome was reexamined using a weak Zn2+ sensor, Zincon. The titration curve yielded by the addition of Zn2+ to proteome (LLC-PK1 cells) in presence of Zincon (ZI, Kd ~ 10 -5 M) shows that initially this sensor could not compete for mobile Zn2+ with the proteome’s higher affinity Zn2+ binding sites. This region of the titration was followed by an increase in absorbance which was previously thought to represent free Zn-ZI. However, the absorbance spectrum of Zn-ZI was red shifted. This part of the titration revealed the presence of lower affinity binding sites for Zn2+ in the proteome. Preincubation of the proteome with N-ethyl-maleimide (NEM) abolished the first phase of the titration. Fractionation of the resultant reaction mixture showed that absorbance due to Zn-ZI was confined to the proteome, suggesting the formation of ternary adducts, proteome*Zn-ZI. Therefore, proteome contains large concentration of both high and low affinity, nonspecific binding sites that compete with ZI for Zn2+ or participate in ternary complex formation. To further investigate the role of proteome in Zn2+ trafficking, Zn-MT was induced in LLC-PK1 cells by exposure to Zn2+. Besides binding to MT, added Zn2+ was also nonspecifically bound to proteome, showing that proteome binding sites have high affinity for Zn2+. When apo-carbonic anhydrase (apo-CA) was titrated with Zn2+ in the presence of proteome, the formation of Zn-CA occurred but was retarded. These results show that proteomic non-specific zinc binding sites can play a significant role in Zn2+ trafficking to form native Zn-proteins and possibly in Zn2+ signaling.

Understanding proteome dependent cellular zinc trafficking to form native Zn-proteins

2019 Spring National Meeting

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