In these gradients, changes in the targeting of membrane proteins to SLMVs is frequently seen as modification in the amount but not sedimentation of a marker. We hypothesized that the inability of the Y372F mutant to support zinc storage in vesicular compartments would affect cell viability when cells are challenged with toxic extracellular zinc concentrations. To test this hypothesis, PC12 cells lines expressing wild type human ZnT3 and tyrosine mutants were cultured in media containing increasing concentrations of ZnSO4 during 24 h. Cells expressing human ZnT3 carrying the Y372F mutation, which impairs ZnT3 dimerization,MG132 were sensitive to extracellular zinc. Cell viability was reduced when compared with cells expressing wild type human ZnT3. In contrast, cells expressing human ZnT3 carrying the Y357F mutant, which enhances ZnT3 dimerization, showed a modest but significant increase in cell viability compared with human wild type ZnT3. These findings indicate that, although modest, a gain-of-function phenotype in human ZnT3 carrying the Y357F mutation becomes evident in cell challenged for a prolonged time with toxic zinc concentrations. Predicted structural changes induced by ZnT3 dityrosine bond oligomerization Dityrosine bonded ZnT3 supports efficient zinc accumulation in intracellular organelles. This functional change predicts that ZnT3 oligomers containing trans dityrosine bridges between residues 357 and 372 should modify cytoplasmic determinants involved in MK-1775 zinc binding. We explored the structural changes induced by bridging tyrosines 357 and 372 in ZnT3 dimers using AMMP molecular modeling. We modeled human ZnT3 primary sequence using as a backbone the crystal structure coordinates of the bacterial ZnT3 homologue YiiP bound to zinc atoms. ZnT3 dimers lacking dityrosine bonds closely resembled the crystal structure of YiiP. Zinc atoms in human ZnT3 bound to the cytosolic domain were exposed to water. However, ZnT3 dimers carrying a dityrosine bond between residues 357 and 372 acquired a closed conformation with zinc atoms bound to the cytosolic domain completely buried and away from solvent.