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Molecular and Cellular Biology, March 2006, p. 2286-2296, Vol. 26, No. 6
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.6.2286-2296.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Family Knockout of All Four Drosophila Metallothioneins Reveals a Central Role in Copper Homeostasis and Detoxification{dagger}

Dieter Egli,1 Hasmik Yepiskoposyan,1 Anand Selvaraj,1 Kuppusamy Balamurugan,1 Rama Rajaram,1 Andreas Simons,2 Gerd Multhaup,2 Simone Mettler,1 Alla Vardanyan,1 Oleg Georgiev,1 and Walter Schaffner1*

Institute of Molecular Biology, University of Zurich, CH-8057 Zurich, Switzerland,1 Freie Universität Berlin, Institut für Chemie/Biochemie, Thielallee 63, D-14195 Berlin, Germany2

Received 2 August 2005/ Returned for modification 3 October 2005/ Accepted 8 December 2005

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.

* Corresponding author. Mailing address: IMB Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Phone: 41 1 6353150. Fax: 41 1 6356830. E-mail: walter.schaffner{at}molbio.unizh.ch.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, March 2006, p. 2286-2296, Vol. 26, No. 6
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.6.2286-2296.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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