Lysosome remodeling mediates high zinc homeostasis

摘要 锌是一种对人类健康有深远影响的必需营养物质，因为约有10%的蛋白质组与锌结合。 锌的动态平衡的一个关键是在锌过剩时储存，在锌缺乏时释放。 溶酶体在大分子的降解中具有公认的作用，它正在作为一种 保存的锌储存地点。溶酶体如何整合锌储存和降解的双重功能 定义不明确。我们的数据表明，溶酶体在不同的隔室中调节这些双重功能。 我们使用线虫来证明过量的锌存储在肠道细胞的溶酶体中；CDF-2(ZnT2 in 哺乳动物)是SLC30家族的转运蛋白，将锌装载到溶酶体中，而Zipt-2.3是SLC39家族 释放锌的运输者。CDF-2和Zipt-2.3的相互调节调节锌的流向； 在过量的锌中，CDF-2上调以增加储存，而Zipt-2.3下调以减少释放。 溶酶体是如何迅速改变其表面转运蛋白的组成的？使用超分辨率 显微镜下，我们发现溶酶体有一个膨胀室连接到酸化的中央 车厢。膨胀室在锌充足的条件下收缩，但在 对高锌的反应。我们的总体假设是，扩展隔间允许快速输送 CDF-2到溶酶体促进锌稳态--而不干扰酸化过程中的降解过程 车厢。这一假设是创新的，因为我们只观察到具有 最近出现了超分辨率显微镜。为了确定这一机制是否保守，我们研究了 哺乳动物细胞中溶酶体的动力学；锌转运蛋白改变其在溶酶体上的定位 对高锌的反应，与溶酶体重塑一致。为了测试我们模型的预测，我们将 鉴定隔室的分子性质，确定组装和转录过程中的变化 拆解，并在哺乳动物细胞中验证我们的发现。 具体目标1，首先，我们将使用透射电子显微镜来确定溶酶体是否有第二个膜结合 装有CDF-2的隔间。第二，我们将使用X射线荧光显微镜来测试锌是否 被隔离在这个结构里。 具体目标2，我们将把这些发现扩展到哺乳动物细胞。我们将测试ZnT2转运体是否 需要储存锌，确定释放锌的ZIP蛋白，并测试这些转运蛋白是否 相互调节以重塑溶酶体。 具体目的3，我们将分析溶酶体重构的调节。我们证明了溶酶体 生物发生调节剂HLH-30(哺乳动物中的TFEB)是重塑所必需的。我们将决定如何 对高锌的转录反应介导溶酶体重塑。这些研究将在#年产生重大影响 定义了溶酶体生物学的一个新方面，这对锌的动态平衡至关重要。

Abstract Zinc is an essential nutrient that profoundly affects human health, since ~10% of the proteome binds zinc. A key to zinc homeostasis is storage during times of excess and release during periods of deficiency. Lysosomes, which have a well-established role in the degradation of macromolecules, are emerging as a conserved site of zinc storage. How lysosomes integrate the dual functions of zinc storage and degradation is not well defined. Our data indicate that lysosomes mediate these dual functions in separate compartments. We used C. elegans to demonstrate excess zinc is stored in lysosomes of intestinal cells; CDF-2 (ZnT2 in mammals) is the SLC30 family transporter that loads zinc into lysosomes, and ZIPT-2.3 is the SLC39 family transporter that releases zinc. Reciprocal regulation of CDF-2 and ZIPT-2.3 regulates the direction of zinc flow; in excess zinc, CDF-2 is upregulated to increase storage and ZIPT-2.3 is downregulated to decrease release. How do lysosomes rapidly change the composition of transporters on their surface? Using super-resolution microscopy, we discovered that lysosomes have an expansion compartment connected to the acidified central compartment. The expansion compartment is contracted in zinc-replete conditions, but grows dramatically in response to high zinc. Our overall hypothesis is that the expansion compartment allows the rapid delivery of CDF-2 to lysosomes to promote zinc homeostasis –without disturbing degradative processes in the acidified compartment. This hypothesis is innovative, since we only observed the expansion compartment with the recent availability of super-resolution microscopy. To determine if this mechanism is conserved, we examined lysosome dynamics in mammalian cells; the ZnT2 zinc transporter alters its localization on lysosomes in response to high zinc, consistent with lysosome remodeling. To test the predictions of our model, we will characterize the molecular nature of the compartment, identify transcriptional changes during assembly and disassembly, and validate our findings in mammalian cells. Specific Aim 1, First, we will use TEM to determine whether lysosomes have a second membrane-bound compartment that contains CDF-2. Second, we will use X-ray fluorescence microscopy to test whether zinc is sequestered in this structure. Specific Aim 2, we will extend these findings to mammalian cells. We will test whether the ZnT2 transporter is required to store zinc, identify the ZIP protein that releases zinc, and test whether these transporters are reciprocally regulated to remodel lysosomes. Specific Aim 3, we will analyze the regulation of lysosome remodeling. We demonstrated that the lysosome biogenesis regulator HLH-30 (TFEB in mammals) is required for remodeling. We will determine how the transcriptional response to high zinc mediates lysosome remodeling. These studies will have a major impact by defining a new aspect of lysosome biology that is critical for zinc homeostasis.