SLC30A3 (ZnT3) Oligomerization by Dityrosine Bonds Regulates Its Subcellular Localization and Metal Transport Capacity

Abstract

Non-covalent and covalent homo-oligomerization of membrane proteins regulates their subcellular localization and function. Here, we described a novel oligomerization mechanism affecting solute carrier family 30 member 3/zinc transporter 3 (SLC30A3/ZnT3). Oligomerization was mediated by intermolecular covalent dityrosine bonds. Using mutagenized ZnT3 expressed in PC12 cells, we identified two critical tyrosine residues necessary for dityrosine-mediated ZnT3 oligomerization. ZnT3 carrying the Y372F mutation prevented ZnT3 oligomerization, decreased ZnT3 targeting to synaptic-like microvesicles (SLMVs), and decreased resistance to zinc toxicity. Strikingly, ZnT3 harboring the Y357F mutation behaved as a “gain-of-function” mutant as it displayed increased ZnT3 oligomerization, targeting to SLMVs, and increased resistance to zinc toxicity. Single and double tyrosine ZnT3 mutants indicate that the predominant dimeric species is formed between tyrosine 357 and 372. ZnT3 tyrosine dimerization was detected under normal conditions and it was enhanced by oxidative stress. Covalent species were also detected in other SLC30A zinc transporters localized in different subcellular compartments. These results indicate that covalent tyrosine dimerization of a SLC30A family member modulates its subcellular localization and zinc transport capacity. We propose that dityrosine-dependent membrane protein oligomerization may regulate the function of diverse membrane protein in normal and disease states.

In Situ Dimerization of Multiple Wild Type and Mutant Zinc Transporters in Live Cells Using Bimolecular Fluorescence Complementation

“…Recently, co-immunoprecipitation and Western blot analysis revealed that ZnT3 forms covalent homodimers mediated by intermolecular dityrosine bonds, which are induced by oxidative stress (22); these covalent dityrosine bonds were found to modulate subcellular localization and zinc transport activity of ZnT3 (22). Herein we showed that homodimers of ZnT3 localize at intracellular vesicles, consistent with its vesicular localization in the brain (45).…”

“…(b) Consistently, this C-terminal region of ZnT5 is important for recognizing ZnT6 as a partner for heterodimerization (23). (c) The corresponding region of ZnT3 appears to regulate intracellular trafficking and zinc transport activity by forming a covalent dityrosine bond in response to oxidative stress (22). (d) The C-terminal region was further found to be important for proteinprotein interactions between ZnT1 and the protein kinase Raf-1 (44).…”

“…Although dimerization of the bacterial YiiP has a critical role in modulating zinc transport activity (20), little is known about homodimerization and heterodimerization of human ZnTs. In this respect, only a few ZnTs, including ZnT2 and ZnT3, were studied to some extent and were suggested to form homodimers (16,17,22), whereas ZnT5 and ZnT6 were found to undergo heterodimerization (23). We recently identified a novel heterozygous G87R mutation in ZnT2 (16), in two Ashkenazi Jewish mothers; this mutation resulted in the production of zinc-deficient milk, leading to the development of TNZD in their exclusively breast-fed infants.…”

Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway

“…This enabled us to perform detailed analysis of the functions of ZnTs in the supply of zinc to zinc-requiring enzymes such as TNAP. Our findings revealed that over 95% of TNAP protein is activated by ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers and that the other ZnTs barely contribute, despite having zinc transport activity in the secretory pathway when exogenously expressed (25,36,37). Treatment with zinc pyrithione increased the zinc content in the early secretory pathway and, consistently, increased TNAP activity via other minor pathways, but this effect was not comparable in magnitude with that of ZnT5/ZnT6 heterodimers and/or ZnT7 homo-oligomers.…”

The Physiological, Biochemical, and Molecular Roles of Zinc Transporters in Zinc Homeostasis and Metabolism

“…ZnT transporters are predicted to have six TM helices with the exception that ZnT5 has a long NH 2 -terminal portion with nine putative TM helices. ZnT transporters form homodimers (114,180,230,231,291,353,391), again with the exception that ZnT5 forms heterodimers with ZnT6 (114,195,230). The cytosolic COOH-terminal portion, which is important for their dimerization (114,353), contributes to other proteinprotein interactions and enables ZnT transporters to control the activity of counterpart proteins or to be controlled by counterpart proteins (114,186,189,355).…”