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

Abstract

Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. Here, we report the generation of a metallothionein gene family knockout in Drosophila melanogaster by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, we find that different metallothioneins are expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's "copper cells." The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, we conclude that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. We conclude that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop.

FIG. 1.

Genetic map of Drosophila metallothioneins and knock-in strategy for MtnD. (A) A section of chromosome 3 with metallothioneins indicated in boldface type. The figure is adapted from the FlyBase database (http://flybase.bio.indiana.edu/). (B) Design of the MtnD knock-in allele. Arrows indicate the primers used for PCR. (C) Verification of the MtnD knock-in allele by PCR. Note that the shorter PCR wild-type product of about 0.5 kb is absent in homozygous MtnD^dsRed mutant flies.

FIG. 2.

Viability and metal content of Mtn and MTF-1 mutants and the wild type on normal or metal-supplemented food. (A) Absolute viability of different genotypes on normal food. Note that deviations between different strains are within the experimental error. (B) The bar diagrams depict the percentage of survival of mutant and wild-type (y w) embryos to adulthood. Survival on normal food of each genotype is set at 100%. Flies were allowed to deposit 150 to 300 eggs on food containing the indicated concentrations of metals, and eclosing adults were counted. Error bars represent standard deviations of several independent experiments, calculated from the number of flies in a total of 3 to 10 different tubes. (C) A single metallothionein transgene can partially rescue the phenotype of a metallothionein gene family knockout with the genotype qMtn*. Shown is the survival of transgenic embryos to adulthood on 500 μM copper (as a percentage of the survival of the isogenic MtnD* genotype). (D) Sensitivity of metallothionein mutants to copper shock. Approximately 30 third-instar larvae were transferred from and to the indicated type of food. The bars represent the percentage of eclosing adults. Note that there are no survivors of the qMtn^dsRed genotype. (E) Copper content of wild-type (wt) and metallothionein mutant flies. Flies were grown on the indicated type of food and analyzed by ICP-MS.

FIG. 3.

MREs are sufficient for tissue-specific expression of metallothioneins. Flies were raised on the type of food indicated. Shown is the fluorescence of reporter transgenes with the indicated promoters driving EYFP or dsRed. The anterior is shown to the left. copper, copper luminescence. (A) Expression patterns of MtnB and MtnD, two members of the MtnB subfamily, coincide completely. Shown is the fluorescence of the MtnB-EYFP reporter construct and the allele MtnD^dsRed near the midgut constriction (arrowhead). (B) Colocalization of MtnB promoter activity with copper cell luminescence on 1 mM copper. Shown is the expression of an MtnB-EYFP transgene. (C) A synthetic promoter with MREs derived from MtnB is sufficient for tissue-specific expression and for metal induction. Note the overlap of reporter transgenes with each other and with copper cell luminescence. (D) Overlap of expression near the midgut constriction of the MtnD^dsRed knock-in allele with a synthetic minipromoter composed of MREs of Ctr1B. Larvae were grown on normal food.

FIG. 4.

Metallothioneins form a luminescent complex with copper in copper cells of the larval midgut. Copper luminescence and DAPI staining (top) and expression of MtnD^dsRed (bottom) in the same gut is shown. The ubiquitous bluish fluorescence that partially masks DAPI staining is due to autofluorescence of midgut cells (18). Note that copper cell luminescence is reduced but not completely absent in both homozygous qMtn^dsRed and MTF-1 mutants. Homozygosity of the MtnD^dsRed locus leads to stronger dsRed expression than in heterozygotes. Larvae were raised on 50 μM copper until the third instar.

FIG. 5.

Metallothioneins negatively regulate their own expression. (A) MtnB-EYFP reporter expression in larvae heterozygous or homozygous for the qMtn* chromosome. The anterior of third-instar larvae is shown on the left. (B) Quantification of expression levels of MtnC in a wild-type (y w) and a triple-Mtn (tMtn) mutant background, carrying the alleles MtnA^ΔATG, MtnB^ΔATG, and MtnD*. Third-instar larvae were transferred for 6 h to 500 μM copper or 4 mM zinc, and transcript levels were assayed by S1 nuclease mapping. The bar diagram represents the quantification of the bands, normalized with the signal from actin5C transcripts. (C) Expression of a genomic Ctr1B-EGFP construct in a qMtn* or heterozygous wild-type larvae (qMtn*/wt). (D and E) Metallothionein transcript levels in wild-type MtnD (D) or MtnA (E) flies after a 6-h induction period or after constant growth on the indicated type of food. Quantification is relative to NF.

FIG. 6.

Life span of heterozygous wild-type (qMtn*/wt) and homozygous (qMtn*) metallothionein mutant flies raised at different copper concentrations. Day 0 represents the day of eclosion from pupae. Survivorship curves indicate the proportion of a population surviving at different ages. The life spans of heterozygous and homozygous qMtn mutant males (A) and females (B) are shown. (C) Life span of flies with copper depletion raised and kept in copper chelator BCS. This particular experiment was done at 26°C; all other experiments were done at 28°C. (D) SOD1 activity in flies raised on normal food or on low-copper food. (E) Metallothionein expression levels in males and females determined by S1 nuclease mapping. (F) Copper and zinc content of wild-type flies.