Drosophila ZnT1 is essential in the intestine for dietary zinc absorption

doi:10.1016/j.bbrc.2020.09.077

Biochemical and Biophysical Research Communications

Volume 533, Issue 4, 17 December 2020, Pages 1004-1011

https://doi.org/10.1016/j.bbrc.2020.09.077 Get rights and content

Highlights

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A genetic mutant of dZnT1were built by CRISPER-Cas9 system.
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The mutation resulted in early lethality of Drosophila, similar to that in the mouse studies.
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Gut expression of ZnT1 (dZnT1 and mammalian ZnT1 alike) largely rescued dZnT1 mutant.
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Supplementation of zinc together with clioquinol or hinokitiol conferred a limited but observable rescue upon dZnT1 loss.

Abstract

Zinc is an essential trace element and participates in a variety of biological processes. ZnT (SLC30) family members are generally responsible for zinc efflux across the membrane regulating zinc homeostasis. In mammals, the only predominantly plasma membrane resident ZnT has been reported to be ZnT1, and ZnT1^-/ZnT1^- mice die at the embryonic stage. In Drosophila, knock down of ZnT1 homologue (dZnT1//ZnT63C/CG17723) results in growth arrest under zinc-limiting conditions. To investigate the essentiality of dZnT1 for zinc homeostasis, as well as its role in dietary zinc uptake especially under normal physiological conditions, we generated dZnT1 mutants by the CRISPER/Cas9 method. Homozygous mutant dZnT1 is lethal, with substantial zinc accumulation in the iron cell region, posterior midgut as well as gastric caeca. Expression of human ZnT1 (hZnT1), in the whole body or in the entire midgut, fully rescued the dZnT1 mutant lethality, whereas tissue-specific expression of hZnT1 in the iron cell region and posterior midgut partially rescued the developmental defect of the dZnT1 mutant. Supplementation of zinc together with clioquinol or hinokitiol conferred a limited but observable rescue upon dZnT1 loss. Our work demonstrated the absolute requirement of dZnT1 in Drosophila survival and indicated that the most essential role of dZnT1 is in the gut.

Introduction

Zinc is an indispensable micronutrient which serves as an essential component of numerous enzymes and other proteins. The maintenance of zinc homeostasis is thus vital for living organisms [1]. On the one hand, low zinc levels cause pleiotropic problems; On the other hand, overt zinc accumulation also results in physiologic abnormalities [2,3].

Zinc transporters are categorized into two major families, the zinc transporter (ZnT) family (SLC30A) and the ZRT- and IRT-like protein (Zip) family (SLC39A) [4]. In general, ZnT proteins function in transporting zinc into extra- or intracellular vesicles from the cytoplasm [5], while Zip proteins transport zinc into the cytoplasm. In addition to these transporters, non-specific metal-binding proteins termed metallothioneins (MTs) can bind and regulate labile zinc levels in the cytoplasm [6]. These proteins and other regulators work together to regulate homeostasis of zinc and other metals, as well as to mitigate heavy metal poisoning or stress [7].

ZnT1 is the first identified member of zinc transporters in humans [8], which is ubiquitously expressed and is the only ZnT protein primarily localized in the plasma membrane in mammalian cells [9]. In intestinal epithelial cells, ZnT1 is usually located in the basolateral membrane releasing zinc into the portal vein [10]. In exocrine pancreatic cells, ZnT1 locates in the apical membrane involved in the secretion of zinc [11]. It is also proposed to play an important role in the reabsorption of zinc in the kidney [12]. The mRNA level of ZnT1 is upregulated by the increase of intracellular zinc concentration [13]. ZnT1 knockout mice exhibited early embryonic lethality [14], indicating the essential role of ZnT1 in the early fetus development likely as a result of lacking zinc absorption from the mother.

Drosophila melanogaster is an ideal model for studying metal homeostasis. Recent adaptation of the CRISPER/Cas system to the fly further facilitated genetic amiability of this organism [15]. The zinc transport and regulation machinery discovered in yeast and mammals are conserved in the fruit fly. The fly’s genome encodes 10 Zip and 7 ZnT proteins. In comparison, mammalian zinc transporters consist of 14 Zip members and 10 ZnT members [11]. Flies also have five MT genes, and knockout mutants of Mtns displayed sensitivity to copper, cadmium, and zinc [7]. Systematic analyses via over-expression and RNA interference of Zip/ZnT proteins in Drosophila were conducted to study the molecular and cellular functions by the UAS/GAL4 system [16]. Their involvement in zinc absorption and excretion was also investigated in detail [17,18].

ZnT63C (CG17723) is the homologue of human ZnT1 in Drosophila, also called dZnT1. In our previous work, ubiquitous knockdown of dZnT1 resulted in non-obvious phenotype under normal growth conditions, but developmental retardation and growth arrest when dietary zinc is restricted. dZnT1 overexpression in the whole-body of Drosophila resulted hypersensitivity to zinc repletion [19]. In addition to the involvement of dietary zinc absorption, dZnT1 was localized to the basolateral membrane of Malpighian tubule cells and functions in zinc reabsorption to recover zinc from excretion [18]. Another dZnT1 homologue, CG5130 (dZnT77C), has also been shown to be a player in dietary zinc absorption, complicating the scenario of zinc uptake and efflux in the intestine [17,20].

In this study, we focused on generating genetic mutants of dZnT1 to delineate its role under normal growth conditions. We discovered that dZnT1 is absolutely required for Drosophila development, and its most critical role appears to be in acquiring zinc from the diet. Our study may shed light on future higher organism studies.

Section snippets

Generating Drosophila ZnT1 (ZnT63C) mutant by CRISPR/Cas9

Drosophila protein encoded by CG17723 (ZnT63C) shares the highest homology with hZnT1 (Fig. 1A). Some conserved features of the ZnT family, such as the 6 transmembrane domains and the histidine-rich long ring structure, the possible zinc ion binding motif between the fourth and fifth transmembrane regions, are present in CG17723 protein. Our previous work showed that CG17723 was the ZnT1 orthologue in Drosophila melanogaster, and was named dZnT1 [19].

Past functional analysis of dZnT1 largely

Discussion

Aided by the CRISPR/Cas9 method, two different lines of ZnT63C/dZnT1 mutant were generated containing frame-shift mutations at the start of the predicted TM6. Both mutations would remove TM6 and the C terminus, about half of the dZIP13 protein. dZnT1 mutations are lethal, similar to that of the mouse ZnT1 knockout. Expressing hZnT1could effectively suppress the lethality associated with the generated mutations, confirming dZnT1 is functionally analogous to hZnT1 and the observed lethality

Fly stocks, culture media, and transgenic

Fly stocks were raised at 18 °C, and all the experiments were carried out at 25 °C on standard cornmeal food. Fly stocks are da-GAL4, MtnB-EYFP, NP3084-GAL4, NP2375-GAL4, UAS-EGFP and drm-GAL4/TM3 were mentioned before [18,23]; Mutant dZnT1 flies were generated by the lab of Dr. Renjie Jiao (Guangzhou Medical University, China). CQ (Sigma, USA) and hinokitiol (Selleck, USA) were both dissolved in DMSO at 100 mM as the stocks.

RNA isolation and quantitative real-time PCR

The protocol has been mentioned before [18]. The primers used for PCR

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We greatly appreciate the kind gifts of fly stocks from Dr. Walter Schaffner (University of Zurich, Zurich, Switzerland) and Dr. Renjie Jiao (Guangzhou Medical University, China). Other fly stocks were obtained from the Bloomington Stock Center and the Drosophila Genetic Resource Center at the Kyoto Institute of Technology. This study was supported by the National Key Research and Development Program of China (2018YFA0900100) and National Natural Science Foundation of China (31971087).

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¹: These authors contributed equally in this work.

View full text

Drosophila ZnT1 is essential in the intestine for dietary zinc absorption

Highlights

Abstract

Introduction

Section snippets

Generating Drosophila ZnT1 (ZnT63C) mutant by CRISPR/Cas9

Discussion

Fly stocks, culture media, and transgenic

RNA isolation and quantitative real-time PCR

Declaration of competing interest

Acknowledgments

Neurochem. Int.

Curr. Opin. Chem. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Trace Elem. Med. Biol.

The galvanization of biology: a growing appreciation for the roles of zinc

Science

Zinc in human health: effect of zinc on immune cells

Mol. Med.

The neurobiology of zinc in health and disease

Nat. Rev. Neurosci.

Physiological roles of zinc transporters: molecular and genetic importance in zinc homeostasis

J. Physiol. Sci.

The physiological, biochemical, and molecular roles of zinc transporters in zinc homeostasis and metabolism

Physiol. Rev.

Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc

EMBO J.

Overview of and update on the physiological functions of mammalian zinc transporters

Nihonseigaku Zasshi Japanese Journal of Hygiene

Mammalian zinc transporters: nutritional and physiologic regulation

Annu. Rev. Nutr.