Biochemical and Biophysical Research Communications
Drosophila ZnT1 is essential in the intestine for dietary zinc absorption
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).
References (30)
The function of metallothionein
Neurochem. Int.
(1995)- et al.
Metallothioneins: new functional and structural insights
Curr. Opin. Chem. Biol.
(2000) - et al.
Zinc transporter 1 (ZNT1) expression on the cell surface is elaborately controlled by cellular zinc levels
J. Biol. Chem.
(2019) - et al.
Mammalian zinc transport, trafficking, and signals
J. Biol. Chem.
(2006) - et al.
The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene
J. Biol. Chem.
(2000) - et al.
The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene
J. Biol. Chem.
(2000) - et al.
The zebrafish Znt1a(sa17) mutant reveals roles of zinc transporter-1a in embryonic development
J. Trace Elem. Med. Biol.
(2020) - et al.
The galvanization of biology: a growing appreciation for the roles of zinc
Science
(1996) Zinc in human health: effect of zinc on immune cells
Mol. Med.
(2008)- et al.
The neurobiology of zinc in health and disease
Nat. Rev. Neurosci.
(2005)
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.
Cited by (8)
Metal ions in insect reproduction: a crosstalk between reproductive physiology and immunity
2022, Current Opinion in Insect ScienceCitation Excerpt :ZnT63C (also called dZnT1) is also important for zinc-dietary absorption in the midgut, and loss of its expression alters larvae and pupae development and reduces adult eclosion rates. Supplementation of zinc has been shown to partially rescue pupation rates, but the effects are more evident in the second generation, suggesting that maternal zinc absorption is important for subsequent generations [13•]. On the other hand, Zip99C (also called dZip13) is mainly important for iron homeostasis instead of zinc, and it is the only known iron exporter in Drosophila [14].
A comprehensive analysis on source-distribution-bioaccumulation-exposure risk of metal(loid)s in various vegetables in peri-urban areas of Shenzhen, China
2022, Environmental PollutionCitation Excerpt :These liberated ions could bind to metallothionein, enter the blood through the portal system, and then transport to various parts of the body with the carrier albumin (Roohani et al., 2013). It has been reported that the bioavailability of inorganic Zn is lower than that of organic Zn (Wang et al., 2020). There were several limitations in the current study.
Regulation and biological function of metal ions in Drosophila
2021, Current Opinion in Insect ScienceCitation Excerpt :Question marks in Table 1 reflect our present lack of knowledge how insects achieve manganese homeostasis. Consistent with its function in zinc absorption (reviewed in Refs. [17–19]), a null mutant of ZnT1 accumulates zinc in the iron cell region, posterior midgut, as well as gastric caeca and fails to grow beyond first instar larva [51]. The lethality is presumably a result of systemic zinc deficiency as it could be rescued by expressing a ZnT1 transgene exclusively in the intestine and to a lesser degree by zinc supplementation [51].
Advances in nutrient metabolism and emission of livestock and poultry dietary trace elements
2022, Kexue Tongbao/Chinese Science Bulletin
- 1
These authors contributed equally in this work.