Lysosome remodeling mediates high zinc homeostasis

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.