Here we show that in contrast with YiiP, ZnT3 forms Labetalol hydrochloride covalent dimers mediated by intermolecular dityrosine bonds. Dityrosine bond formation occurs both spontaneously and induced by oxidative stress. Using site-directed mutagenesis we identified two critical tyrosines in the carboxyterminal of ZnT3, one that prevented and one that increased dimerization. Our studies indicate that covalent tyrosine dimerization of a SLC30A family 4-(Benzyloxy)phenol member modulates its subcellular localization and zinc transport capacity. One modification that could explain formation of covalent dimers regulated by oxidative stress is dityrosine bond formation. Based on the chemistry of dityrosine synthesis, we selected two criteria to identify dityrosine bonds in the 80 kDa ZnT3 species. First, dityrosine formation is induced by oxidative stress and second, is abrogated by tyrosine to phenylalanine mutation of critical residues. Dityrosine formation has been described mainly as a product of oxidative stress and as a normal extracellular posttranslational modification only in a limited group of secreted proteins, such as collagen and elastin. We used crosslinking and sucrose sedimentation to evaluate whether dimer formation in tyrosine mutants occurs spontaneously in the absence of H2O2 as we observed 
previously for wild type ZnT3. Until now, dimerization of polytopic transmembrane proteins has been shown to occur by covalent and non-covalent interaction mainly through transmembrane domains. Covalent cysteine-based dimer formation has been extensively described for neurotransmitter transporters, such as the dopamine transporter, DAT and the glycine transporter as well as receptors. The fact that dimeric forms of ZnT3 were resistant to reducing agents and increased in response to oxidative stress, lead us to investigate tyrosine-mediated dimerization. Since its discovery in 1959, dityrosine formation has been described as a post-translational modification related with cellular stress and disease. Dityrosine modifications are produced in response to oxidative stress, aging, UV and c irradiation. Increased levels of dityrosine have been found in atheromatous plates, cataracts, acute inflammation, systemic bacterial infection and recently associated with a-synuclein fibrillogenesis and Ab amyloid oligomerization. Only in a limited group of structural proteins of the bacteria cell wall and insect ligaments.