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SLC30A3 (ZnT3) oligomerization by dityrosine bonds regulates its subcellular localization and metal transport capacity.

Salazar G, Falcon-Perez JM, Harrison R, Faundez V - PLoS ONE (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Divison of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. gsalaza@emory.edu

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.

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ZnT3 amino terminal domain regulates tyrosine dimerization.A) ZnT3 mouse and human chimeras, hmZnT3-HA and mhZnT3-myc, in which amino terminal domains (1–75) were exchanged were incubated with or without H2O2 or MG132 (B and C). Oligomerization was then compared with wild type mouse and human ZnT3. B) The amino terminal domain of mouse ZnT3 decreases human transporter's oligomerization. C) Human amino terminal increases mouse ZnT3 tyrosine oligomerization.
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pone-0005896-g008: ZnT3 amino terminal domain regulates tyrosine dimerization.A) ZnT3 mouse and human chimeras, hmZnT3-HA and mhZnT3-myc, in which amino terminal domains (1–75) were exchanged were incubated with or without H2O2 or MG132 (B and C). Oligomerization was then compared with wild type mouse and human ZnT3. B) The amino terminal domain of mouse ZnT3 decreases human transporter's oligomerization. C) Human amino terminal increases mouse ZnT3 tyrosine oligomerization.

Mentions: Mouse and human ZnT3 dimer formation differs dramatically despite the fact that both ZnT3 orthologs are 86% identical. This led us to hypothesize that additional structural elements could modulate the ability of tyrosine residues to form covalent bonds. To test this hypothesis, we asked whether the amino terminal domain of human ZnT3 could confer sensitivity to oxidative stress-induce oligomerization to the mouse ZnT3 and vice verse. To this end, we constructed a mouse ZnT3-HA chimeras containing the amino terminal of human ZnT3 (hmZnT3-HA) and a human ZnT3-myc chimera containing the amino terminal of mouse ZnT3 (mhZnT3-myc) (Fig. 8A). Constructs were transfected in PC12 cells and cells incubated with H2O2 or MG-132 (Fig. 8B and C). Mouse ZnT3 amino terminal decreased the H2O2-induced dimerization of the human chimera mhZnT3-myc (Fig. 8B). In contrast, the human amino terminal domain increases dimerization of the mouse ZnT3 chimera (hmZnT3-HA) in response to H2O2 and oligomerization-induced by MG-132. Therefore, the amino terminal domain influences the ability of carboxy-terminal tyrosines to form covalent tyrosine bonds.


SLC30A3 (ZnT3) oligomerization by dityrosine bonds regulates its subcellular localization and metal transport capacity.

Salazar G, Falcon-Perez JM, Harrison R, Faundez V - PLoS ONE (2009)

ZnT3 amino terminal domain regulates tyrosine dimerization.A) ZnT3 mouse and human chimeras, hmZnT3-HA and mhZnT3-myc, in which amino terminal domains (1–75) were exchanged were incubated with or without H2O2 or MG132 (B and C). Oligomerization was then compared with wild type mouse and human ZnT3. B) The amino terminal domain of mouse ZnT3 decreases human transporter's oligomerization. C) Human amino terminal increases mouse ZnT3 tyrosine oligomerization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2690824&req=5

pone-0005896-g008: ZnT3 amino terminal domain regulates tyrosine dimerization.A) ZnT3 mouse and human chimeras, hmZnT3-HA and mhZnT3-myc, in which amino terminal domains (1–75) were exchanged were incubated with or without H2O2 or MG132 (B and C). Oligomerization was then compared with wild type mouse and human ZnT3. B) The amino terminal domain of mouse ZnT3 decreases human transporter's oligomerization. C) Human amino terminal increases mouse ZnT3 tyrosine oligomerization.
Mentions: Mouse and human ZnT3 dimer formation differs dramatically despite the fact that both ZnT3 orthologs are 86% identical. This led us to hypothesize that additional structural elements could modulate the ability of tyrosine residues to form covalent bonds. To test this hypothesis, we asked whether the amino terminal domain of human ZnT3 could confer sensitivity to oxidative stress-induce oligomerization to the mouse ZnT3 and vice verse. To this end, we constructed a mouse ZnT3-HA chimeras containing the amino terminal of human ZnT3 (hmZnT3-HA) and a human ZnT3-myc chimera containing the amino terminal of mouse ZnT3 (mhZnT3-myc) (Fig. 8A). Constructs were transfected in PC12 cells and cells incubated with H2O2 or MG-132 (Fig. 8B and C). Mouse ZnT3 amino terminal decreased the H2O2-induced dimerization of the human chimera mhZnT3-myc (Fig. 8B). In contrast, the human amino terminal domain increases dimerization of the mouse ZnT3 chimera (hmZnT3-HA) in response to H2O2 and oligomerization-induced by MG-132. Therefore, the amino terminal domain influences the ability of carboxy-terminal tyrosines to form covalent tyrosine bonds.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Divison of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. gsalaza@emory.edu

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.

Show MeSH
Related in: MedlinePlus