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 coppermediated 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 metalresponsive 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.

Characterization of MtnE, the fifth metallothionein member in Drosophila

“…The expression patterns of MtnD and MtnE mostly overlap in the Drosophila larval gut, but there are some differences: MtnE is more widely expressed than MtnD and MtnB, the latter of which is strongly expressed in a sub-region of the middle midgut that contains "copper cells" [11]. Copper cells are known to accumulate metal ions following copper or cadmium intoxication [11,47,48]. As indicated by blue arrows, copper also boosts the expression of MtnD in copper cells, while MtnE remains less induced.…”

“…Like that of other Drosophila metallothioneins, MtnE expression was observed in the intestine of larvae on normal food (Figure 6a) and was boosted by copper-containing food ( Figure 3, Figure 6b). The expression patterns of MtnD and MtnE mostly overlap in the Drosophila larval gut, but there are some differences: MtnE is more widely expressed than MtnD and MtnB, the latter of which is strongly expressed in a sub-region of the middle midgut that contains "copper cells" [11]. Copper cells are known to accumulate metal ions following copper or cadmium intoxication [11,47,48].…”

“…It encodes a protein that is highly similar to the other Drosophila metallothioneins, especially MtnB, C and D [11,30], and was named metallothionein E (MtnE) (Figure 1a and b). CG42872 (MtnE) was discovered by targeted cDNA screening of gene predictions as part of the modENCODE transcriptome project [43].…”

“…Drosophila was thought to have four MTs (MtnA-D) expressed mainly in the digestive tract [11]. S. cerevisiae has two MTs, CUP1 and CRS5 [12,13].…”

Huntington disease arises from a combinatory toxicity of polyglutamine and copper binding

“…Metallothioneins (Mtn), as metal detoxifiers in the cells (24), are transcriptionally induced by copper and are considered sensitive indicators of intracellular copper levels (19,24). Ctr1B-RNAi and DmATP7-OE flies showed a significant reduction of both MtnA and MtnB, whereas DmATP7-RNAi flies displayed increased levels of MtnA expression ( Fig.…”