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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 homology modeling with the bacteria Yiip zinc transporter.Human ZnT3 pairs or ZnT3 pairs bridged in trans by dityrosine bonds between tyrosine residues 357 and 372 were modeled using AMMP and visualized with Pymol. Modeling coordinates were obtained from the crystal structure of YiiP bound to zinc atoms. A) Depicts lateral views of ZnT3 pair surface models. Gray lines represent the middle of the lipid bilayer. Zinc atoms are depicted as green spheres. Tyrosines 357 and 372 are depicted either single or bonded in yellow. B) Depicts diaphanized surface models to highlight the position of zinc atoms (green spheres). C) Cytoplasmic views of the solvent exposed areas in the absence or presence of dityrosine bonding. Blue depicts the negative and red the positive potentials. D) Cytoplasmic view of zinc atoms arrangement in the cytosolic domain of ZnT3 pairs either in the absence of dityrosine bonds (green spheres) or in the presence of dityrosines (yellow spheres). Dityrosines bonds are represented as a reference point.
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pone-0005896-g006: ZnT3 homology modeling with the bacteria Yiip zinc transporter.Human ZnT3 pairs or ZnT3 pairs bridged in trans by dityrosine bonds between tyrosine residues 357 and 372 were modeled using AMMP and visualized with Pymol. Modeling coordinates were obtained from the crystal structure of YiiP bound to zinc atoms. A) Depicts lateral views of ZnT3 pair surface models. Gray lines represent the middle of the lipid bilayer. Zinc atoms are depicted as green spheres. Tyrosines 357 and 372 are depicted either single or bonded in yellow. B) Depicts diaphanized surface models to highlight the position of zinc atoms (green spheres). C) Cytoplasmic views of the solvent exposed areas in the absence or presence of dityrosine bonding. Blue depicts the negative and red the positive potentials. D) Cytoplasmic view of zinc atoms arrangement in the cytosolic domain of ZnT3 pairs either in the absence of dityrosine bonds (green spheres) or in the presence of dityrosines (yellow spheres). Dityrosines bonds are represented as a reference point.

Mentions: 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 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 (Fig. 6) using as a backbone the crystal structure coordinates of the bacterial ZnT3 homologue YiiP bound to zinc atoms [33]. ZnT3 dimers lacking dityrosine bonds closely resembled the crystal structure of YiiP (Fig. 6A, ZnT3). 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 (Fig. 6A, ZnT3 diY357–372). The major conformational change involved in the 357–372 dityrosine dimer depended on the motion of the two cytoplasmic loops containing these two tyrosines. Since tyrosine 372 is on the C-terminus and therefore has few conformational restrictions, it moves more than tyrosine 357 (Fig. 6A). The modeling method was unlikely to produce major conformation changes as it used conjugate gradients, which is a local optimizer. The distribution of charges, as reflected in the electrostatic field calculated from the model in the presence of dielectric and counter ions showed few differences between the undimerized and dimerized tyrosine residues (Fig. 6C). The arrangement of zinc atoms was further explored by projecting cytosolic zinc atoms into the dityrosine bonds (Fig. 6D). Zinc atoms moved closer to the unexposed surface of the C-terminal domain of ZnT3 in dimers carrying 357–372 dityrosine bonds (Fig. 6D, compare yellow and green dots). Models made for other pairs of possible dityrosine states, such as 330–357 or 330–372 did not alter the zinc binding sites. The rearrangement seen with the 357–372 dityrosine bridge appears to alter the zinc binding sites and open up buried binding sites that are not present in molecules lacking dityrosine bonds (Fig. 6B). Zinc atoms not associated with this binding site are buried in both the tyrosine and dityrosine models. After formation of the dityrosine bond a complete set of well-formed zinc binding sites is generated. This set of sites spans the whole length of the molecule suggesting that dityrosine formation facilitates zinc transport by forming the shielded binding pathway for the C-terminal part of the transporter. This modeling supports the notion that ZnT3 domains involved in zinc binding undergo structural rearrangements in the presence of dityrosine 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 homology modeling with the bacteria Yiip zinc transporter.Human ZnT3 pairs or ZnT3 pairs bridged in trans by dityrosine bonds between tyrosine residues 357 and 372 were modeled using AMMP and visualized with Pymol. Modeling coordinates were obtained from the crystal structure of YiiP bound to zinc atoms. A) Depicts lateral views of ZnT3 pair surface models. Gray lines represent the middle of the lipid bilayer. Zinc atoms are depicted as green spheres. Tyrosines 357 and 372 are depicted either single or bonded in yellow. B) Depicts diaphanized surface models to highlight the position of zinc atoms (green spheres). C) Cytoplasmic views of the solvent exposed areas in the absence or presence of dityrosine bonding. Blue depicts the negative and red the positive potentials. D) Cytoplasmic view of zinc atoms arrangement in the cytosolic domain of ZnT3 pairs either in the absence of dityrosine bonds (green spheres) or in the presence of dityrosines (yellow spheres). Dityrosines bonds are represented as a reference point.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005896-g006: ZnT3 homology modeling with the bacteria Yiip zinc transporter.Human ZnT3 pairs or ZnT3 pairs bridged in trans by dityrosine bonds between tyrosine residues 357 and 372 were modeled using AMMP and visualized with Pymol. Modeling coordinates were obtained from the crystal structure of YiiP bound to zinc atoms. A) Depicts lateral views of ZnT3 pair surface models. Gray lines represent the middle of the lipid bilayer. Zinc atoms are depicted as green spheres. Tyrosines 357 and 372 are depicted either single or bonded in yellow. B) Depicts diaphanized surface models to highlight the position of zinc atoms (green spheres). C) Cytoplasmic views of the solvent exposed areas in the absence or presence of dityrosine bonding. Blue depicts the negative and red the positive potentials. D) Cytoplasmic view of zinc atoms arrangement in the cytosolic domain of ZnT3 pairs either in the absence of dityrosine bonds (green spheres) or in the presence of dityrosines (yellow spheres). Dityrosines bonds are represented as a reference point.
Mentions: 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 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 (Fig. 6) using as a backbone the crystal structure coordinates of the bacterial ZnT3 homologue YiiP bound to zinc atoms [33]. ZnT3 dimers lacking dityrosine bonds closely resembled the crystal structure of YiiP (Fig. 6A, ZnT3). 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 (Fig. 6A, ZnT3 diY357–372). The major conformational change involved in the 357–372 dityrosine dimer depended on the motion of the two cytoplasmic loops containing these two tyrosines. Since tyrosine 372 is on the C-terminus and therefore has few conformational restrictions, it moves more than tyrosine 357 (Fig. 6A). The modeling method was unlikely to produce major conformation changes as it used conjugate gradients, which is a local optimizer. The distribution of charges, as reflected in the electrostatic field calculated from the model in the presence of dielectric and counter ions showed few differences between the undimerized and dimerized tyrosine residues (Fig. 6C). The arrangement of zinc atoms was further explored by projecting cytosolic zinc atoms into the dityrosine bonds (Fig. 6D). Zinc atoms moved closer to the unexposed surface of the C-terminal domain of ZnT3 in dimers carrying 357–372 dityrosine bonds (Fig. 6D, compare yellow and green dots). Models made for other pairs of possible dityrosine states, such as 330–357 or 330–372 did not alter the zinc binding sites. The rearrangement seen with the 357–372 dityrosine bridge appears to alter the zinc binding sites and open up buried binding sites that are not present in molecules lacking dityrosine bonds (Fig. 6B). Zinc atoms not associated with this binding site are buried in both the tyrosine and dityrosine models. After formation of the dityrosine bond a complete set of well-formed zinc binding sites is generated. This set of sites spans the whole length of the molecule suggesting that dityrosine formation facilitates zinc transport by forming the shielded binding pathway for the C-terminal part of the transporter. This modeling supports the notion that ZnT3 domains involved in zinc binding undergo structural rearrangements in the presence of dityrosine 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