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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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Identification of ZnT3 oligomeric states.A) Triton soluble extracts of PC12 cells (450 µg) co-expressing ZnT3-HA and ZnT3-myc, treated with and without cross-linker (DSP), were immunoprecipitated with HA (lanes 1 and 2), myc (lanes 4 and 5) or synaptophysin (Sphysin, lanes 7 and 8) antibodies. Western blots were probed with myc, HA and synaptophysin and ZnT3 antibodies, respectively. Control immunoprecipitation with synaptophysin antibodies fail to isolate ZnT3. Input 10 µg. B) 1.5 mg of Triton-X100 soluble supernatant of ZnT3-myc expressing cells treated in the presence of vehicle (DMSO) or DSP (+DSP) were separated by sucrose sedimentation. Fractions were collected from the bottom and analyzed by immunoblot with myc antibodies. An 80 kDa and high molecular weight forms of ZnT3 were observed, together with the monomeric 40 kDa species.
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pone-0005896-g001: Identification of ZnT3 oligomeric states.A) Triton soluble extracts of PC12 cells (450 µg) co-expressing ZnT3-HA and ZnT3-myc, treated with and without cross-linker (DSP), were immunoprecipitated with HA (lanes 1 and 2), myc (lanes 4 and 5) or synaptophysin (Sphysin, lanes 7 and 8) antibodies. Western blots were probed with myc, HA and synaptophysin and ZnT3 antibodies, respectively. Control immunoprecipitation with synaptophysin antibodies fail to isolate ZnT3. Input 10 µg. B) 1.5 mg of Triton-X100 soluble supernatant of ZnT3-myc expressing cells treated in the presence of vehicle (DMSO) or DSP (+DSP) were separated by sucrose sedimentation. Fractions were collected from the bottom and analyzed by immunoblot with myc antibodies. An 80 kDa and high molecular weight forms of ZnT3 were observed, together with the monomeric 40 kDa species.

Mentions: We examined the formation of ZnT3 oligomeric species by co-immunoprecipitation of ZnT3 carrying different tags in their carboxy terminal domains. In order to facilitate detection of low affinity ZnT3 oligomers (dimers and/or high molecular weight species) we used dithiobis–succinimidylpropionate (DSP). DSP is a homobifunctional cell permeable cross-linker. This agent contains a disulfide bond, which allows complete cleavage of cross-linked products under reducing conditions [38]. PC12 cells co-expressing ZnT3-HA and ZnT3-myc, treated with and without DSP, were lysed and detergent soluble supernatants immunoprecipitated with magnetic beads coated with HA or myc antibodies (Fig. 1A). Immune complexes were analyzed by immunoblot with antibodies against myc or HA.


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)

Identification of ZnT3 oligomeric states.A) Triton soluble extracts of PC12 cells (450 µg) co-expressing ZnT3-HA and ZnT3-myc, treated with and without cross-linker (DSP), were immunoprecipitated with HA (lanes 1 and 2), myc (lanes 4 and 5) or synaptophysin (Sphysin, lanes 7 and 8) antibodies. Western blots were probed with myc, HA and synaptophysin and ZnT3 antibodies, respectively. Control immunoprecipitation with synaptophysin antibodies fail to isolate ZnT3. Input 10 µg. B) 1.5 mg of Triton-X100 soluble supernatant of ZnT3-myc expressing cells treated in the presence of vehicle (DMSO) or DSP (+DSP) were separated by sucrose sedimentation. Fractions were collected from the bottom and analyzed by immunoblot with myc antibodies. An 80 kDa and high molecular weight forms of ZnT3 were observed, together with the monomeric 40 kDa species.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005896-g001: Identification of ZnT3 oligomeric states.A) Triton soluble extracts of PC12 cells (450 µg) co-expressing ZnT3-HA and ZnT3-myc, treated with and without cross-linker (DSP), were immunoprecipitated with HA (lanes 1 and 2), myc (lanes 4 and 5) or synaptophysin (Sphysin, lanes 7 and 8) antibodies. Western blots were probed with myc, HA and synaptophysin and ZnT3 antibodies, respectively. Control immunoprecipitation with synaptophysin antibodies fail to isolate ZnT3. Input 10 µg. B) 1.5 mg of Triton-X100 soluble supernatant of ZnT3-myc expressing cells treated in the presence of vehicle (DMSO) or DSP (+DSP) were separated by sucrose sedimentation. Fractions were collected from the bottom and analyzed by immunoblot with myc antibodies. An 80 kDa and high molecular weight forms of ZnT3 were observed, together with the monomeric 40 kDa species.
Mentions: We examined the formation of ZnT3 oligomeric species by co-immunoprecipitation of ZnT3 carrying different tags in their carboxy terminal domains. In order to facilitate detection of low affinity ZnT3 oligomers (dimers and/or high molecular weight species) we used dithiobis–succinimidylpropionate (DSP). DSP is a homobifunctional cell permeable cross-linker. This agent contains a disulfide bond, which allows complete cleavage of cross-linked products under reducing conditions [38]. PC12 cells co-expressing ZnT3-HA and ZnT3-myc, treated with and without DSP, were lysed and detergent soluble supernatants immunoprecipitated with magnetic beads coated with HA or myc antibodies (Fig. 1A). Immune complexes were analyzed by immunoblot with antibodies against myc or HA.

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