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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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Dityrosine-dependent dimerization in SLC30A family members.A) Carboxy terminal primary sequences of ZnTs 1–8 containing tyrosine residues are compared with ZnT3 tyrosines 330, 357 and 372. Negatively (E/D) and positively (R/K) charge residues are depicted in green and blue, respectively. ZnT3Y372 and ZnT4Y355 and 413 share a conserved YXXE sequence. NT denotes not tested. B) HEK293 cells were transiently transfected with plasmids encoding myc-tagged versions of human ZnT1, 3, 4, 5 and 7. Cell extracts were analyzed by immunoblot with myc antibodies. All zinc transporters tested, except ZnT7 showed covalently modified species resistant to SDS and reducing agents in the presence of H2O2. C) HEK293 cells transiently transfected with myc-tagged versions of wild type ZnT4 and Y355F, Y404F and Y413F mutants were incubated with the indicated concentrations of H2O2 during 30 min. Samples were analyzed by western blots with myc and SUMO2/3 antibodies. All three ZnT4 tyrosine mediated covalent tyrosine dimers.
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pone-0005896-g007: Dityrosine-dependent dimerization in SLC30A family members.A) Carboxy terminal primary sequences of ZnTs 1–8 containing tyrosine residues are compared with ZnT3 tyrosines 330, 357 and 372. Negatively (E/D) and positively (R/K) charge residues are depicted in green and blue, respectively. ZnT3Y372 and ZnT4Y355 and 413 share a conserved YXXE sequence. NT denotes not tested. B) HEK293 cells were transiently transfected with plasmids encoding myc-tagged versions of human ZnT1, 3, 4, 5 and 7. Cell extracts were analyzed by immunoblot with myc antibodies. All zinc transporters tested, except ZnT7 showed covalently modified species resistant to SDS and reducing agents in the presence of H2O2. C) HEK293 cells transiently transfected with myc-tagged versions of wild type ZnT4 and Y355F, Y404F and Y413F mutants were incubated with the indicated concentrations of H2O2 during 30 min. Samples were analyzed by western blots with myc and SUMO2/3 antibodies. All three ZnT4 tyrosine mediated covalent tyrosine dimers.

Mentions: ZnTs 1 to 8 possess C-terminal tyrosines that could mediate dityrosine bonding, as it is the case of human ZnT3 (Fig. 7A). To explore whether dityrosine bonds could be a common structural element in other SLC30A family members, we first examined whether myc-tagged versions of human ZnTs form covalent dimers in response to H2O2 (Fig. 7B). Similar to the human ZnT3, H2O2 induced covalently modified species in myc-tagged versions of human ZnT1, ZnT4 and ZnT5, but not ZnT7 (Fig. 7B).


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)

Dityrosine-dependent dimerization in SLC30A family members.A) Carboxy terminal primary sequences of ZnTs 1–8 containing tyrosine residues are compared with ZnT3 tyrosines 330, 357 and 372. Negatively (E/D) and positively (R/K) charge residues are depicted in green and blue, respectively. ZnT3Y372 and ZnT4Y355 and 413 share a conserved YXXE sequence. NT denotes not tested. B) HEK293 cells were transiently transfected with plasmids encoding myc-tagged versions of human ZnT1, 3, 4, 5 and 7. Cell extracts were analyzed by immunoblot with myc antibodies. All zinc transporters tested, except ZnT7 showed covalently modified species resistant to SDS and reducing agents in the presence of H2O2. C) HEK293 cells transiently transfected with myc-tagged versions of wild type ZnT4 and Y355F, Y404F and Y413F mutants were incubated with the indicated concentrations of H2O2 during 30 min. Samples were analyzed by western blots with myc and SUMO2/3 antibodies. All three ZnT4 tyrosine mediated covalent tyrosine dimers.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005896-g007: Dityrosine-dependent dimerization in SLC30A family members.A) Carboxy terminal primary sequences of ZnTs 1–8 containing tyrosine residues are compared with ZnT3 tyrosines 330, 357 and 372. Negatively (E/D) and positively (R/K) charge residues are depicted in green and blue, respectively. ZnT3Y372 and ZnT4Y355 and 413 share a conserved YXXE sequence. NT denotes not tested. B) HEK293 cells were transiently transfected with plasmids encoding myc-tagged versions of human ZnT1, 3, 4, 5 and 7. Cell extracts were analyzed by immunoblot with myc antibodies. All zinc transporters tested, except ZnT7 showed covalently modified species resistant to SDS and reducing agents in the presence of H2O2. C) HEK293 cells transiently transfected with myc-tagged versions of wild type ZnT4 and Y355F, Y404F and Y413F mutants were incubated with the indicated concentrations of H2O2 during 30 min. Samples were analyzed by western blots with myc and SUMO2/3 antibodies. All three ZnT4 tyrosine mediated covalent tyrosine dimers.
Mentions: ZnTs 1 to 8 possess C-terminal tyrosines that could mediate dityrosine bonding, as it is the case of human ZnT3 (Fig. 7A). To explore whether dityrosine bonds could be a common structural element in other SLC30A family members, we first examined whether myc-tagged versions of human ZnTs form covalent dimers in response to H2O2 (Fig. 7B). Similar to the human ZnT3, H2O2 induced covalently modified species in myc-tagged versions of human ZnT1, ZnT4 and ZnT5, but not ZnT7 (Fig. 7B).

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