ZnT2 is an electroneutral proton-coupled vesicular antiporter displaying an apparent stoichiometry of two protons per zinc ion
ZnT2 is an electroneutral proton-coupled vesicular antiporter displaying an apparent stoichiometry of two protons per zinc ion
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DOI：
10.1371/journal.pcbi.1006882
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发表时间：
2019-03-01
影响因子：
4.3
通讯作者：
Assaraf, Yehuda G.
中科院分区：
文献类型：
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作者：
Golan, Yarden;Alhadeff, Raphael;Assaraf, Yehuda G.
锌是一种重要的微量元素，对大约3000种细胞蛋白质的正常功能至关重要。具体地说，锌在核酸代谢、基因表达调控、信号转导、细胞分裂、免疫和神经系统功能、伤口愈合和细胞凋亡等关键生理过程中都是必不可少的。因此，锌稳态的损害扰乱了关键的细胞功能，导致了各种人类疾病。哺乳动物锌的转运通过两个转运蛋白家族ZNT和ZIP进行。然而，在哺乳过程中，负责囊泡锌积累和锌分泌到母乳中的锌的详细作用机制目前尚不清楚。此外，尽管已经提出了酸性囊泡中锌的转运与质子梯度之间的耦合假设，但这一假设尚未得到确凿的证实。在这里，我们模拟了ZnT2的作用机制，并利用功能锌转运分析从计算和实验两个方面证明了ZnT2确实是一个质子偶联的锌逆向转运蛋白。Bafilmycin A1是一种特异性的液泡型质子ATPase(V-ATPase)抑制剂，它可以碱性酸性小泡，阻止依赖于锌向细胞内小泡的锌运输。此外，使用LysoTracker Red和Lyso-pHluorin，我们进一步表明，当细胞内小泡瞬时过表达ZnT2并加入外源锌时，泡囊的pH发生了碱性变化，这可能是由于质子-锌反向连接；而在特异性锌螯合剂TPEN的存在下，这一现象被逆转。最后，在计算能量的基础上，我们提出了以2H(+)/Zn~(2+)离子为化学计量比的反向转运蛋白。因此，ZnT2是一种质子驱动力驱动的电子中和型囊泡锌交换器，以质子向细胞质挤出为代价，将锌集中在酸性囊泡中。锌对母乳中锌的积累是必不可少的，因此具有重要的医学意义。在我们以前研究的基础上，我们在这里提出了能量计算，表明ZnT2作为质子/锌逆向转运体发挥作用。我们的计算包括静电计算和pK(A)计算以及锌结合自由能曲线。综合我们的计算结果，我们得出结论：作为一种具有2H(+)/Zn2+化学计量比的逆向转运蛋白，我们构建了一个蒙特卡罗模型来检验这种模式的转运活性，并用活体人乳腺上皮细胞对我们的计算结果进行了验证。这些功能实验表明，在没有质子的情况下，ZnT2不能发挥作用，这表明它是底物诱导的交替访问转运蛋白，表现出明显的2H(+)/Zn2+化学计量比。
Zinc is a vital trace element crucial for the proper function of some 3,000 cellular proteins. Specifically, zinc is essential for key physiological processes including nucleic acid metabolism, regulation of gene expression, signal transduction, cell division, immune- and nervous system functions, wound healing, and apoptosis. Consequently, impairment of zinc homeostasis disrupts key cellular functions resulting in various human pathologies. Mammalian zinc transport proceeds via two transporter families ZnT and ZIP. However, the detailed mechanism of action of ZnT2, which is responsible for vesicular zinc accumulation and zinc secretion into breast milk during lactation, is currently unknown. Moreover, although the putative coupling of zinc transport to the proton gradient in acidic vesicles has been suggested, it has not been conclusively established. Herein we modeled the mechanism of action of ZnT2 and demonstrated both computationally and experimentally, using functional zinc transport assays, that ZnT2 is indeed a proton-coupled zinc antiporter. Bafilomycin A1, a specific inhibitor of vacuolar-type proton ATPase (V-ATPase) which alkalizes acidic vesicles, abolished ZnT2-dependent zinc transport into intracellular vesicles. Moreover, using LysoTracker Red and Lyso-pHluorin, we further showed that upon transient ZnT2 overexpression in intracellular vesicles and addition of exogenous zinc, the vesicular pH underwent alkalization, presumably due to a proton-zinc antiport; this phenomenon was reversed in the presence of TPEN, a specific zinc chelator. Finally, based on computational energy calculations, we propose that ZnT2 functions as an antiporter with a stoichiometry of 2H(+)/Zn2+ ion. Hence, ZnT2 is a proton motive force-driven, electroneutral vesicular zinc exchanger, concentrating zinc in acidic vesicles on the expense of proton extrusion to the cytoplasm.Author summary Herein we explored the mechanism of action of the human ZnT2 zinc transporter. ZnT2 is essential for zinc accumulation in breast milk and is therefore of paramount medical significance. Expanding on our previous study, we herein present energy calculations suggesting that ZnT2 functions as a proton/zinc antiporter. Our calculations consist of electrostatic and pK(a) calculations as well as zinc binding free-energy curves. Upon integration of our calculation results, we conclude that ZnT2 functions as an antiporter with a 2H(+)/Zn2+ stoichiometry, construct a Monte Carlo model to test this mode of ZnT2 transport activity, and validate our computational results experimentally using live human breast epithelial cells. These functional experiments reveal that ZnT2 cannot function in the absence of protons suggesting that it operates as a substrate-induced alternating-access transporter, displaying an apparent 2H(+)/Zn2+ stoichiometry.