Zhao Yitong, Feresin Rafaela G, Falcon-Perez Juan M, Salazar Gloria
The Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL, USA.
Department of Dietetics and Nutrition, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Traffic. 2016 Mar;17(3):267-88. doi: 10.1111/tra.12371. Epub 2016 Feb 12.
The solute carrier 30A (SLC30A) family of zinc exporters transports zinc into the lumen of intracellular organelles in order to prevent zinc toxicity. We reported that formation of tyrosine dimers is required for ZnT3 (zinc transporter 3) zinc transport activity and targeting to synaptic-like microvesicles (SLMVs) in PC12 cells and the formation of ZnT3/ZnT10 heterodimers. Here, we focused on ZnT10 to determine the role of heterodimerization in the sorting of ZnTs in the endolysosomal pathway. Using cell fractionation, immunoprecipitation and immunofluorescence approaches, we found that ZnT10 resides in transferrin receptor and Rab5-positive endosomes and forms covalent heterodimers and oligomers with ZnT2, ZnT3 and ZnT4. The interaction of ZnT10 with ZnT3, mediated by dityrosine bonds, was unable to target ZnT10 into SLMVs in vitro or into synaptic vesicles isolated from mouse brain in vivo. However, ZnT3/ZnT10 heterodimers regulate epidermal growth factor receptor (EGF-R) signaling by increasing the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK1/2), but not EGF-R, C-Raf or Akt phosphorylation in response to EGF. Further, mutation of tyrosine 4 in ZnT10 reduced ZnT3/ZnT10 dityrosine-mediated heterodimerization and zinc transport, as well as MEK and ERK1/2 phosphorylation, which were also reduced by the zinc chelator TPEN. Phosphorylation of these kinases is likely to occur in the cytosol as no differences in phosphorylation were observed in membrane fractions of control and ZnT3/ZnT10-expressing cells. We propose that ZnT10 plays a role in signal transduction, which is mediated by homo and heterodimerization with other ZnTs.
锌输出体溶质载体30A（SLC30A）家族将锌转运到细胞内细胞器的管腔中，以防止锌中毒。我们曾报道，酪氨酸二聚体的形成是锌转运体3（ZnT3）锌转运活性以及其靶向PC12细胞中突触样微囊泡（SLMV）和形成ZnT3/ZnT10异源二聚体所必需的。在此，我们聚焦于ZnT10，以确定异源二聚化在内溶酶体途径中锌转运体分选过程中的作用。通过细胞分级分离、免疫沉淀和免疫荧光方法，我们发现ZnT10存在于转铁蛋白受体和Rab5阳性的内体中，并与ZnT2、ZnT3和ZnT4形成共价异源二聚体和寡聚体。由二酪氨酸键介导的ZnT10与ZnT3的相互作用，在体外无法将ZnT10靶向到SLMV中，在体内也无法将其靶向到从小鼠脑部分离出的突触小泡中。然而，ZnT3/ZnT10异源二聚体通过增加丝裂原活化蛋白激酶激酶（MEK）和细胞外信号调节激酶（ERK1/2）的磷酸化来调节表皮生长因子受体（EGF-R）信号传导，但对EGF刺激下的EGF-R、C-Raf或Akt磷酸化无影响。此外，ZnT10中酪氨酸4的突变减少了ZnT3/ZnT10二酪氨酸介导的异源二聚化和锌转运，以及MEK和ERK1/2的磷酸化，锌螯合剂TPEN也降低了这些磷酸化水平。这些激酶的磷酸化可能发生在细胞质中，因为在对照细胞和表达ZnT3/ZnT10的细胞的膜部分未观察到磷酸化差异。我们提出，ZnT10在信号转导中发挥作用，这是由其与其他锌转运体的同源和异源二聚化介导的。
