A role for dZIP89B in Drosophila dietary zinc uptake reveals additional complexity in the zinc absorption process

doi:10.1016/j.biocel.2015.10.004

The International Journal of Biochemistry & Cell Biology

Volume 69, December 2015, Pages 11-19

https://doi.org/10.1016/j.biocel.2015.10.004 Get rights and content

Abstract

Dietary zinc is the principal source of zinc in eukaryotes, with its uptake and distribution controlled by a complex network of numerous membrane-spanning transport proteins. Dietary absorption is achieved by members of the SLC39A (ZIP) gene family, which encode proteins that are generally responsible for the movement of zinc into the cytosol. ZIP4 is thought to be the primary mammalian zinc uptake gene in the small intestine, with mutations in this gene causing the zinc deficiency disease Acrodermatitis enteropathica. In Drosophila, dual knockdown of the major dietary zinc uptake genes dZIP42C.1 (dZIP1) and dZIP42C.2 (dZIP2) results in a severe sensitivity to zinc-deficient media. However, the symptoms associated with ZIP4 loss can be reversed by zinc supplementation and dZIP42C.1 and 2 knockdown has minimal effect under normal dietary conditions, suggesting that additional pathways for zinc absorption exist in both mammals and flies. This study provides evidence that dZIP89B is an ideal candidate for this role in Drosophila, encoding a low-affinity zinc uptake transporter active in the posterior midgut. Flies lacking dZIP89B, while viable and apparently healthy, show indications of low midgut zinc levels, including reduced metallothionein B expression and compensatory up-regulation of dZIP42C.1 and 2. Furthermore dZIP89B mutants display a dramatic resistance to toxic dietary zinc levels which is abrogated by midgut-specific restoration of dZIP89B activity. We postulate that dZIP89B works in concert with the closely related dZIP42C.1 and 2 to ensure optimal zinc absorption under a range of dietary conditions.

Introduction

Zinc is an essential metal and performs numerous functions in a range of critical cellular processes. It is predicted that over 300 enzymes require zinc as a cofactor (Vallee and Auld, 1990). One key example of this is alkaline phosphatase, a ubiquitous, zinc-dependent enzyme whose activity decreases in response to low zinc levels (Suzuki et al., 2005). The role of zinc as a crucial structural cofactor is illustrated by the abundance of proteins containing zinc-finger motifs which are essential for their stabilisation and function.

A highly coordinated network of transport proteins is responsible for the movement of zinc across membranes and for the maintenance of zinc homeostasis in organelles, cells and tissues. Members of the ZIP/SLC39A family of proteins (14 in mammals and 10 in Drosophila) are generally responsible for the movement of zinc into the cytosol, while the ZNT/SLC30A family members (10 in mammals and 7 in Drosophila) act in the opposite direction, transporting zinc out of the cytosol (reviewed in Kambe et al., 2004).

Zinc is primarily acquired through dietary absorption and its uptake is regulated in response to dietary availability and physiological zinc requirements. ZIP4 has been characterised as the main dietary zinc uptake gene in the mammalian small intestine, with loss of function mutations in this gene causing the human zinc deficiency disease Acrodermatitis enteropathica (Dufner-Beattie et al., 2003, Kury et al., 2002). A reduction in systemic zinc levels in these patients leads to symptoms such as cognitive dysfunction, suppressed immune function, skin lesions and growth retardation (Danbolt, 1979).

Despite the requirement for ZIP4 under normal conditions, the phenotypes associated with its loss in humans and mice can be reversed by zinc supplementation, suggesting the presence of additional dietary zinc uptake pathways in mammals that can partially compensate for the absence of ZIP4 (Geiser et al., 2013b, Moynahan, 1974). However, to date no such alternative pathways have been identified. ZIP5 was hypothesised for this role, however it was found to have a basolateral membrane localisation in intestinal cells, suggesting a role in zinc sensing and excretion rather than dietary uptake in the enterocytes (Geiser et al., 2013a, Wang et al., 2004b).

dZIP42C.1 (dZIP1) and dZIP42C.2 (dZIP2) are two of four highly conserved Drosophila homologs of mammalian ZIP1, ZIP2 and ZIP3 and have been proposed to encode the chief dietary zinc uptake transporters in the fly midgut (Qin et al., 2013). The midgut-specific knockdown of dZIP42C.1 alone does not result in any phenotype, however knockdown of dZIP42C.2 results in a sensitivity to zinc-depleted media. The combinatorial knockdown of these two genes results in decreased alkaline phosphatase activity (a proxy measure of zinc status) and almost complete lethality on zinc-depleted media, highlighting the importance of these two genes in zinc-deficient conditions.

To date, only dZIP42C.1 and dZIP42C.2 have been implicated in dietary zinc uptake in Drosophila. However, the lethality that results from the dual knockdown of these two genes is only apparent under conditions of zinc deficiency, implying that alternative mechanisms are in place for dietary zinc uptake in Drosophila as well. Based on protein sequence homology, dZIP42C.1 and dZIP42C.2 cluster with dZIP89B and dZIP88E to form a highly conserved clade (Lye et al., 2012). This study explores the potential for dZIP89B to provide an additional zinc uptake pathway in Drosophila. Previous work on dZIP89B has demonstrated that it encodes a protein that when over expressed displays mostly apical membrane localisation and acts to increase cytosolic zinc levels (Dechen et al., 2015, Lye et al., 2013). The evidence presented in this study indicates that dZIP89B may be acting as a constitutive, low-affinity zinc transporter in the Drosophila intestine, working together with dZIP42C.1 and dZIP42C.2 to ensure adequate zinc supply under a range of dietary conditions.

Section snippets

Drosophila stocks

The following fly stocks were used: w¹¹¹⁸ (BL3605, Bloomington Stock Centre, IN, USA); GMR-GAL4 (P[longGMR-GAL4]3, BL8121); daughterless-GAL4 (P[GAL4-da.G32]2, BL55849); MEX-GAL4 (P[mex1-GAL4.2.1]) (Phillips and Thomas, 2006). RNA interference (RNAi) lines were obtained from the Vienna Drosophila RNAi Centre (VDRC). MtnB^eGFP was a gift from Walter Schaffner (University of Zurich, Switzerland). Microscopy utilised UAS-mCD8-eGFP to visualise reporter gene expression. A list of transgenic lines

Loss of dZIP89B confers resistance to very high dietary zinc levels

Previous work has shown that dZIP89B encodes a protein that displays a mostly apical membrane localisation when ectopically expressed in third instar larval salivary glands. Furthermore, analysis of interactions between dZIP89B and genetically induced cellular zinc toxicity phenotypes showed that it acts to increase cytosolic zinc levels (Dechen et al., 2015, Lye et al., 2013). To determine the endogenous function of the dZIP89B gene, a null mutant (herein referred to as ΔdZIP89B) was generated

Discussion

There is compelling evidence that alternative pathways for zinc absorption exist in both mammals and Drosophila. Here, we postulate that dietary zinc uptake in Drosophila is maintained by the complementary actions of dZIP89B, dZIP42C.1 and dZIP42C.2, in a fashion reminiscent of the functional relationship between ZRT1 and ZRT2 in yeast. ZRT2 has a low affinity for zinc and serves as a major zinc uptake transporter during zinc-replete conditions (Zhao and Eide, 1996b). In contrast, ZRT1 has a

Acknowledgments

This work was supported by Project Grant funding from the National Health and Medical Research Council of Australia (RB, Project Grant #606609). The Australian Drosophila Biomedical Research Support Facility assisted in the import and quarantine of fly strains used in this research. All transgenic Drosophila experiments carried out in this research were performed with the approval of the Monash University Institutional Biosafety Committee.

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A role for dZIP89B in Drosophila dietary zinc uptake reveals additional complexity in the zinc absorption process

Abstract

Introduction

Section snippets

Drosophila stocks

Loss of dZIP89B confers resistance to very high dietary zinc levels

Discussion

Acknowledgments

Int. J. Biochem. Cell Biol.

Int. J. Biochem. Cell Biol.

J. Biol. Chem.

J. Biol. Chem.

Lancet

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Acrodermatitis enteropathica

Br. J. Dermatol.

A family knockout of all four Drosophila metallothioneins reveals a central role in copper homeostasis and detoxification

Mol. Cell. Biol.

The zinc transporter Zip5 (Slc39a5) regulates intestinal zinc excretion and protects the pancreas against zinc toxicity

PLOS ONE

Clioquinol synergistically augments rescue by zinc supplementation in a mouse model of Acrodermatitis enteropathica

PLOS ONE