The Zrc1 Is Involved in Zinc Transport System between Vacuole and Cytosol in Saccharomyces cerevisiae

doi:10.1006/bbrc.2001.4522

Biochemical and Biophysical Research Communications

Volume 282, Issue 1, 23 March 2001, Pages 79-83

https://doi.org/10.1006/bbrc.2001.4522 Get rights and content

Abstract

The ZRC1 gene encodes a multicopy suppressor of zinc toxicity in Saccharomyces cerevisiae; however, previously we found that the expression of ZRC1 was induced when the intracellular zinc level was decreased. Zrc1 has six putative transmembrane domains and we determined that a Zrc1-GFP fusion protein was localized to the vacuolar membrane. The steady state level of intracellular zinc in a zrc1Δ mutant cultured in the zinc-abundant medium was lower than that in wild type. No distinct difference was observed in the basal activity of glyoxalase I, which is a cytosolic enzyme requiring zinc for catalytic function and is used here as a marker for cytosolic zinc-availability, between wild type and zrc1Δ mutant, although the activity was decreased much greater extent in the zrc1Δ mutant if the cells were exposed to the metal-limited medium. Similarly, the basal expression level of ZRC1-lacZ reporter gene in zrc1Δ mutant was the same as that in wild type; however, the fold of induction of ZRC1-lacZ expression in zrc1Δ mutant under the zinc-limited conditions was higher than that in the wild type. Based on these results, we present a tentative model for the function of Zrc1 as a mechanism to maintain the zinc homeostasis in yeast.

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Madurella mycetomatis is the main cause of mycetoma, a chronic, granulomatous skin infection of the subcutaneous tissue. One of the main virulence factors is the formation of grains, which are difficult to treat with the currently available antifungal drugs. Studies have indicated that zinc homeostasis could be an important factor for grain formation. Therefore, in this review the mechanisms behind zinc homeostasis in other fungal species were summarized and an in silico analysis was performed to identify the components of zinc homeostasis in M. mycetomatis. Orthologues for many of the zinc homeostasis components found in other fungal species could also be identified in M. mycetomatis, including those components that have been identified to play a role in biofilm formation, a process which has some parallels with grain formation. Zinc homeostasis may well play an important role in the process of grain formation and, therefore, more knowledge on this subject in M. mycetomatis is required as it may lead to novel therapies to combat this debilitating disease.
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ZIP transport family also plays a role in the transport of toxic metals such as cadmium, arsenic and cobalt (Chen et al., 2008). ZRC1 was involved in the transport of Zn between vacuoles and cytoplasm in yeast, which may be related to the detoxification of excessive zinc ions in cells (Miyabe et al., 2001). In this study, ZIP transporter family members were highly expressed in the roots, which probably played the similar function at xylem loading.
Miscanthus sinensis is a C4 perennial grass species that is widely used as forage, ornamental grass and bioenergy crop due to its broad adaption and great biological traits. Recent studies indicated that M. sinensis could also grow in marginal lands which were contaminated with heavy metals, and exhibited important ecological restoration potential. In this study, transcriptome characterization of candidate genes related to chromium (Cr) uptake, transport and accumulation in M. sinensis were employed to investigate the molecular mechanism of plant tolerance to heavy metal stress. The result showed that following treatment of 200 mg/L of Cr, plant roots could accumulate most Cr and localize mainly in cell walls and soluble fractions, whereas Cr in stems and leaves was primarily in soluble fractions. A total of 83,645 differentially expressed genes (DEGs) were obtained after the treatment. Many genes involved in heavy metal transport, metal ion chelation and photosynthesis were found to be Cr-induced DEGs. Co-expression and weighted correlation network analysis revealed that Glutathion metabolism and ABC transporters pathways play an important role in Cr tolerance of M. sinensis. A hypothesis schematic diagram for the Cr uptake, transport and accumulation of M. sinensis cells were suggested, which could provide a molecular and genetic basis for future candidate genes validation and breeding of such crops.
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Plant metallochaperones are a class of specific metal-binding proteins that mediate metal distribution and homeostasis, but their biological functions and mechanisms underlying metal acquisition, translocation and detoxification are largely unknown. This study functionally characterized a novel heavy metal-associated isoprenylated plant protein (HIPP) subfamily member OsHIPP42 involved in rice tolerance to the toxic metal cadmium (Cd). The OsHIPP42 proteins were targeted to the nucleus and plasma membrane. It was transcriptionally expressed in nodes, basal stems and peduncles throughout the life span. OsHIPP42 was sufficiently induced in roots under excess Cd, zinc (Zn), manganese (Mn) and copper (Cu) stresses. The tolerant capability during Cd stress was demonstrated in the yeast cells expressing OsHIPP42. Transgenic rice overexpressing (OX) OsHIPP42 displayed improved plant elongation, biomass, and chlorophyll accumulation. In contrast, the OsHIPP42 knockout mutation due to the T-DNA insertion resulted in compromised growth response. Intriguingly, both Oshipp42-1 and OsHIPP42-1 mutant lines had a reduced Cd concentration in rice straw, particularly in the grains, with 38.6–44.6% lower than those of wild-type. Notably, the OsHIPP42 overexpression and mutant lines were not found to be involved in rice tolerance to excess Zn, Mn and Cu, suggesting that OsHIPP42 may work selectively on the Cd allocation and detoxification. This work highlights the significance of the genetically improved genotype materials that will potentially be used to minimize Cd accumulation in rice crop and reduce environmental risks to human health through food chains.
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Expressing the SaZIP4h gene and SaZIP4n gene in the yeast mutant ZHY3 restored the Zn uptake (Fig. 5a), which suggested that the SaZIP4h and SaZIP4n may be Zn transporters. In addition, the mutant strain Δzrc1 is a Zn/Cd sensitive mutant, lacking the vacuolar membrane Zn transporter (Miyabe et al., 2001). Expressing the SaZIP4h gene but not the SaZIP4n gene in the yeast mutant Δzrc1 increased the Cd sensitivity (Fig. 5b), which suggests that SaZIP4h may be also able to transport Cd.
Sedum alfredii Hance is a zinc (Zn)/cadmium (Cd) hyperaccumulator plant. However, the molecular mechanism of the Zn/Cd uptake of roots and shoots has not been thoroughly elucidated to date. In this work, two isoforms of the putative Zn/Cd transporter of S. alfredii Hance, SaZIP4, were investigated. SaZIP4h and SaZIP4n were cloned from the hyperaccumulating and non-hyperaccumulating ecotypes of S. alfredii, respectively. Transcriptional analysis, subcellular localization analysis, yeast functional complementation analysis and transgenic plants were used to characterize SaZIP4. The transcription levels of SaZIP4h are significantly and constitutively higher than those of SaZIP4n, and the expression levels of SaZIP4h and SaZIP4n in the roots were highly induced by Zn deficiency. A subcellular localization analysis indicated that both SaZIP4h and SaZIP4n are localized to the plasma membrane. In addition, expressing SaZIP4h and SaZIP4n in the yeast mutant ZHY3 can reverse the Zn uptake deficiency. However, expressing SaZIP4h alone in the yeast mutant Δzrc1 increased sensitivity to Cd. Transgenic Arabidopsis thaliana mutant zip4-2 expressing SaZIP4h reversed the Zn/Cd uptake defect, and wild-type A. thaliana ectopically overexpressing SaZIP4h displayed increased Zn accumulation both in roots and shoots. Together, these results suggest that SaZIP4 is an important Zn uptake transporter that takes up Zn in the roots and shoots of S. alfredii. These findings help to elucidate the molecular mechanisms of heavy metal and micronutrient uptake and accumulation in hyperaccumulator plants.
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Citation Excerpt :
ycf1Δ, cot1Δ and zrc1Δ strains were derived from the wild-type strain BY4741 (MATα, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) and the isogenic wild type strain DTY3 (cup1s) (MATα, leu2–3, 112his3Δ1, trp1–1, ura3–50, gal1, CUP1S) were used as positive controls. Thus Cd sensitive ycf1Δ lacks an ABC transporter gene YCF1 (Li et al., 1997) while deletion of the ZRC1, a gene encoding for transporter protein in zrc1Δ (Miyabe et al., 2001) and deletion of COT1 gene in cot1Δ (Conklin et al., 1992) has led to the hypersensitivity of these strains towards their respective metals. The copper-sensitive cup1Δ (DTY4) is lacking the copperthionein gene (Longo et al., 1996).
Release of heavy metals into the soil pose a significant threat to the environment and public health because of their toxicity accumulation in the food chain and persistence in nature. The potential of soil microbial diversity of cadmium (Cd) contaminated site was exploited through functional metatranscriptomics by construction of cDNA libraries A (0.1–0.5 kb), B (0.5–1.0 kb), and C (1-4 kb) of variable size, from the eukaryotic mRNA. The cDNA library B was further screened for cadmium tolerant transcripts through yeast complementation system. We are reporting one of the transformants ycf1^ΔPLBe1 capable of tolerating high concentrations of Cd (40 μM - 80 μM). Sequence analysis revealed that PLBe1 cDNA showed homology with ubiquitin domain containing protein fused with AN1 type zinc finger protein of Acanthameoba castellani. Further, this cDNA was tested for its tolerance towards other heavy metals such as copper (Cu), zinc (Zn) and cobalt (Co). Functional complementation assay of cDNA PLBe1 showed a range of tolerance towards copper (150 μM - 300 μM), zinc (10 mM - 12 mM) and cobalt (2 mM - 4 mM). This study promulgates PLBe1 as credible member of multi-metal tolerant gene in the eukaryotic soil microbial community and can be used as potential member to revitalise the heavy metal contaminated sites or can be used as a biomarker to detect heavy metal contamination in the soil environment.

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^☆: Abbreviations used: GFP, green fluorescent protein; ZRE, zinc-responsive element; CDF, cation diffusion facilitator; ZIP, ZRT, IRT-like protein.

¹: To whom correspondence should be addressed. Fax: +81-774-33-3004. E-mail: [email protected].

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Biochemical and Biophysical Research Communications

Abstract

References (28)

Zinc-induced inactivation of the yeast ZRT1 zinc transporter occurs through endocytosis and vacuolar degradation

J. Biol. Chem.

Expression of ZRC1 coding for suppressor of zinc toxicity is induced by zinc-starvation stress in Zap1-dependent fashion in Saccharomyces cerevisiae

Biochem. Biophys. Res. Commun.

Identification of the structural gene for glyoxalase I from Saccharomyces cerevisiae

J. Biol. Chem.

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding

Anal. Biochem.

Defects in the yeast high affinity iron transport system result in increased metal sensitivity because of the increased expression of transporters with a broad transition metal specificity

J. Biol. Chem.

Expression of the glyoxalase I gene of Saccharomyces cerevisiae is regulated by high osmolarity glycerol mitogen-activated protein kinase pathway in osmotic stress response

J. Biol. Chem.

Regulation of zinc homeostasis in yeast by binding of the ZAP1 transcriptional activator to zinc-responsive promoter elements

J. Biol. Chem.

Mammalian zinc transporters

J. Nutr.

The ZRT2 gene encodes the low affinity zinc transporter in Saccharomyces cerevisiae

J. Biol. Chem.

Active-site zinc ligands and activated H₂O of zinc enzymes

Proc. Natl. Acad. Sci. USA

The biochemical basis of zinc physiology

Physiol. Rev.

The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation

Proc. Natl. Acad. Sci. USA

Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter

Biochem. J.

Cited by (60)

A Pleiotropic Drug Resistance Family Protein Gene Is Required for Rice Growth, Seed Development and Zinc Homeostasis

The putative role of zinc homeostasis in grain formation by Madurella mycetomatis during mycetoma infection

Transcriptome characterization of candidate genes related to chromium uptake, transport and accumulation in Miscanthus sinensis

Functional characterization of a new metallochaperone for reducing cadmium concentration in rice crop

SaZIP4, an uptake transporter of Zn/Cd hyperaccumulator Sedum alfredii Hance

Isolation of multi-metal tolerant ubiquitin fusion protein from metal polluted soil by metatranscriptomic approach

Regular ArticleThe Zrc1 Is Involved in Zinc Transport System between Vacuole and Cytosol in Saccharomyces cerevisiae☆

Abstract

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Nutr.

J. Biol. Chem.

Active-site zinc ligands and activated H2O of zinc enzymes

Proc. Natl. Acad. Sci. USA

The biochemical basis of zinc physiology

Physiol. Rev.

The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation

Proc. Natl. Acad. Sci. USA

Zinc-regulated ubiquitin conjugation signals endocytosis of the yeast ZRT1 zinc transporter

Biochem. J.

Regular Article
The Zrc1 Is Involved in Zinc Transport System between Vacuole and Cytosol in Saccharomyces cerevisiae☆

Active-site zinc ligands and activated H₂O of zinc enzymes