Abstract
The trace element copper is required for normal growth and development, serving as an essential catalytic co-factor for enzymes involved in energy generation, oxidative stress protection, neuropeptide maturation, and other fundamental processes. In yeast and mammals copper acquisition occurs through the action of the Ctr1 family of high affinity copper transporters. Here we describe studies using Drosophila melanogaster to investigate the role of copper acquisition through Ctr1 in normal growth and development. Three distinct Drosophila Ctr1 genes (Ctr1A, Ctr1B, and Ctr1C) have been identified, which have unique expression patterns over the course of development. Interestingly, Ctr1B, which is expressed exclusively during the late embryonic and larval stages of development, is transcriptionally activated in response to nutritionally induced copper deprivation and down-regulated in response to copper adequacy. The generation of Ctr1B mutant flies results in decreased larval copper accumulation, marked body pigmentation defects that parallel defects in tyrosinase activity, and specific developmental arrest under conditions of both nutritional copper limitation and excess. These studies establish that copper acquisition through the Drosophila Ctr1B transporter is crucial for normal growth and in early and specific stages of metazoan development.
Highlights
The normal growth and development of eukaryotic cells and organisms require appropriate spatial and temporal patterns of gene expression, signal transduction, and biosynthetic and metabolic reactions
The trace element copper is required for normal growth and development, serving as an essential catalytic co-factor for enzymes involved in energy generation, oxidative stress protection, neuropeptide maturation, and other fundamental processes
The Ctr1 proteins are a family of high affinity copper transporters, characterized in fungi, plants, amphibians, and mammals, that are predominantly localized to the plasma membrane and that are thought to transport reduced copper (Cu(I)) [1, 12,13,14,15]
Summary
Cloning and the Expression of Drosophila Ctr Genes in Saccharomyces cerevisiae—Three potential copper transporter (Ctr1) genes were identified from the D. melanogaster genome by a BLAST search at the Flybase web site (flybase.bio.indiana.edu/) using human Ctr and Schizosaccharomyces pombe Ctr protein sequences as probes; the three genes were designated Ctr1A, Ctr1B, and Ctr1C. CG7459) was cloned from a genetic screen in the S. cerevisiae ctr1⌬ctr3⌬ strain MPY17, which is deficient in high affinity copper transport and, cannot grow on nonfermentable carbon sources [29] This strain was transformed with a Drosophila cDNA library expressed from the constitutive yeast ADH1 promoter [30], and transformants were identified that complement the respiratory growth defect of ctr1⌬ctr3⌬ cells. The EP[3]833 line, which harbors a single P element ϳ800 bp upstream of the Ctr1B transcription start site, was obtained from the Bloomington Stock Center (Bloomington, IN) This strain was crossed to a transposase-expressing ⌬2–3 Sb/TM6 line, and the Sb/p[wϩ] male progeny were crossed to TM3/TM6 females. Males in the pools that gave shorter products than predicted precise excision were separated and individually crossed to TM3/TM6 females; their progeny were again characterized by the PCR method described above, and the DNA sequence in the Ctr1B region determined. Total protein was extracted from late pupae or young adults and the in-gel tyrosinase assay was performed as previously described [23]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
